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Current Topics in Developmental Biology

Volume 56

Series Editor Gerald P. Schatten Director, PITTSBURGH DEVELOPMENTAL CENTER Deputy Director, Magee-Women’s Research Institute Professor and Vice-Chair of Ob-Gyn-Reproductive Sci. & Cell Biol.-Physiology University of Pittsburgh School of Medicine Pittsburgh, PA 15213

Editorial Board Peter Gru¨ss Max-Planck-Institute of Biophysical Chemistry Go¨ttingen, Germany

Philip Ingham University of Sheffield, United Kingdom

Mary Lou King University of Miami, Florida

Story C. Landis National Institutes of Health National Institute of Neurological Disorders and Stroke Bethesda, Maryland

David R. McClay Duke University, Durham, North Carolina

Yoshitaka Nagahama National Institute for Basic Biology, Okazaki, Japan

Susan Strome Indiana University, Bloomington, Indiana

Virginia Walbot Stanford University, Palo Alto, California

Founding Editors A. A. Moscona Alberto Monroy

Current Topics in Developmental Biology Volume 56 Edited by

Gerald P. Schatten Director, PITTSBURGH DEVELOPMENTAL CENTER Deputy Director, Magee-Women’s Research Institute Professor and Vice-Chair of Ob-Gyn-Reproductive Sci. & Cell Biol.-Physiology University of Pittsburgh School of Medicine Pittsburgh, PA 15213

This book is printed on acid-free paper. Copyright ß 2003, Elsevier Inc.

All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher. The appearance of the code at the bottom of the first page of a chapter in this book indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. (222 Rosewood Drive, Danvers, Massachusetts 01923), for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-2003 chapters are as shown on the title pages. If no fee code appears on the title page, the copy fee is the same as for current chapters. 0070-2153/2003 $35.00 Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone: (+44) 1865 843830, fax: (+44) 1865 853333, e-mail: [email protected]. You may also complete your request on-line via the Elsevier homepage (http://elsevier.com), by selecting ‘‘Customer Support’’ and then ‘‘Obtaining Permissions.’’

Academic Press An Elsevier Imprint. 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http://www.academicpress.com

Academic Press 84 Theobald’s Road, London WC1X 8RR, UK http://www.academicpress.com International Standard Book Number: 0-12-153156-2 PRINTED IN THE UNITED STATES OF AMERICA 03 04 05 06 07 08 9 8 7 6 5 4 3 2 1

Contents

Contributors

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1 Selfishness in Moderation: Evolutionary Success of the Yeast Plasmid Soundarapandian Velmurugan, Shwetal Mehta, and Makkuni Jayaram I. II. III. IV. V. VI. VII. VIII.

Introduction 2 The Origins and Evolution of Selfishness 2 Selfish Nucleic Acids in Prokaryotes and Eukaryotes 3 The Yeast Plasmids 5 Organization and Regulation of the 2-m Circle Genome The Plasmid Partitioning and Amplification Systems 6 Regulation of Plasmid Gene Expression 7 The Yeast Cohesin Complex Interacts Specifically with the Rep-STB System 15 IX. Models for Cohesin-Mediated Plasmid Segregation 17 X. Summary and Perspectives 18 XI. Open Questions for Future Research 19 References 21

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2 Nongenomic Actions of Androgen in Sertoli Cells William H. Walker I. II. III. IV.

Introduction 26 Hormonal Regulation of Spermatogenesis 27 Nongenomic Actions of Androgen 30 The Regulation of Female and Male Germ Cell Development by Nongenomic Actions of Androgen

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Contents V. Summary 46 Acknowledgments References 46

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3 Regulation of Chromatin Structure and Gene Activity by Poly(ADP-Ribose) Polymerases Alexei Tulin, Yurii Chinenov, and Allan Spradling I. II. III. IV. V. VI. VII. VIII.

Introduction 56 Background 56 Mechanism of Action 63 Poly(ADP-Ribose) Polymerase and Transcription 66 Multiple Routes to Poly(ADP-Ribose) Polymerase Activation Specificity of Transcriptional Activation 72 Poly(ADP-Ribose) Polymerase and Other Chromatin Processes Concluding Thoughts 75 Acknowledgments 76 References 76

4 Centrosomes and Kinetochores, Who Needs 'Em? The Role of Noncentromeric Chromatin in Spindle Assembly Priya Prakash Budde and Rebecca Heald I. Introduction 85 II. Role of Centrosomes and Kinetochores in Spindle Assembly 86 III. Role of Chromatin in Spindle Assembly 88 IV. How Does Chromatin Stabilize Microtubules? 91 V. Chromatin-Associated Kinases and Microtubules 91 VI. Chromatin-Associated Phosphatases and Microtubule Dynamics 94 VII. The Elusive Chromatin Signal: RanGTP 95

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Contents VIII. IX. X. XI. XII. XIII.

RCC1, the Chromatin Regulator of Microtubules 96 The RanGTP Gradient 97 Ran and Microtubule Stabalization and Organization 98 Ran and Microtubule Nucleation 101 Ran and Chromosome Condensation 102 Chromatin-Associated Microtubule-Based Motor Proteins and Spindle Assembly 102 XIV. Centrosomes and Kinetochores Revisited 105 XV. Conclusions 106 Acknowledgments 107 References 107

5 Modeling Cardiogenesis: The Challenges and Promises of 3D Reconstruction Jeffrey O. Pentecost, Claudio Silva, Maurice Pesticelli, Jr., and Kent L. Thornburg I. II. III. IV. V. VI.

Introduction 115 Embryonic Heart Development 117 Reconstruction and Modeling Techniques The Embyronic Human Heart 128 Recent Developments 132 Summary and Future Directions 138 References 140

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6 Plasmid and Chromosome Traffic Control: How ParA and ParB Drive Partition Jennifer A. Surtees and Barbara E. Funnell I. Introduction: Chromosome Dynamics in Bacterial Cells 145 II. P1 ParA and ParB 146

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Contents III. Other Plasmid Partition Systems IV. Bacterial ParA and ParB Proteins V. Concluding Remarks 173 Acknowledgments 174 References 174

Index 181 Contents of Previous Volumes

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163 168

Contributors Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Priya Prakash Budde (85), Department of Molecular and Cell Biology, University of California, Berkeley, California 94720 Yurii Chinenov (55), HHMI Laboratories, Carnegie Institution of Washington, Baltimore, Maryland 21210 Barbara E. Funnell (145), Department of Medical Genetics and Microbiology, University at Toronto, Toronto, Ontario M5S 1A8, Canada Rebecca Heald (85), Department of Molecular and Cell Biology, University of California, Berkeley, California 94720 Makkuni Jayaram (1), Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, Texas 78712 Shwetal Mehta (1), Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, Texas 78712 Jeffrey O. Pentecost (115), Department of Medical Informatics and Outcomes Research, Oregon Health and Science University, Portland, Oregon 97201 Maurice Pesticelli, Jr. (115), Departments of Anatomy, Cell Biology, and Surgery, University of Illinois, Chicago, Illinois 60612 Claudio Silva (115), OGI School of Science and Engineering, Oregon Health and Science University, Beaverton, Oregon 97006 Allan Spradling (55), HHMI Laboratories, Carnegie Institution of Washington, Baltimore, Maryland 21210 Jennifer A. Surtees (145), Department of Medical Genetics and Microbiology, University at Toronto, Toronto, Ontario M5S 1A8, Canada Kent L. Thornburg (115), Department of Physiology/Pharmacology, Oregon Health and Science University, Portland, Oregon 97201 Alexei Tulin (55), HHMI Laboratories, Carnegie Institution of Washington, Baltimore, Maryland 21210 Soundarapandian Velmurugan (1), Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, Texas 78712 William H. Walker (25), Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261 ix

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Selfishness in Moderation: Evolutionary Success of the Yeast Plasmid Soundarapandian Velmurugan, Shwetal Mehta, and Makkuni Jayaram Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, Texas 78712

I. II. III. IV. V. VI. VII.

Introduction The Origins and Evolution of Selfishness Selfish Nucleic Acids in Prokaryotes and Eukaryotes The Yeast Plasmids Organization and Regulation of the 2-mm Circle Genome The Plasmid Partitioning and Amplification Systems Regulation of Plasmid Gene Expression A. Mechanisms for Plasmid Partitioning B. Plasmid Organization, Localization, and Dynamics in the Yeast Nucleus C. Interactions among the Rep Proteins and the STB DNA in Plasmid Partitioning D. Host Factors Required for 2-mm Circle Partitioning E. A Potential Role for the Yeast Cohesin Complex in Plasmid Partitioning

VIII. IX. X. XI.

The Yeast Cohesin Complex Interacts Specifically with the Rep–STB System Models for Cohesin-Mediated Plasmid Segregation Summary and Perspectives Open Questions for Future Research Acknowledgments References

The yeast plasmid 2-mm circle is an extrachromosomal selfish DNA element whose genetic endowments are devoted to its stable, high copy propagation. The mean steady state plasmid copy number of approximately 60 per cell appears to be evolutionarily optimized at its permissible maximum value. A plasmid-encoded negative regulatory mechanism prevents a rise in copy number that might imperil normal host metabolism and thus indirectly reduce plasmid fitness. The plasmid utilizes the host replication machinery for its own duplication. A plasmid-encoded partitioning system mediates even distribution of the replicated molecules to daughter cells, apparently by feeding into the chromosome segregation pathway. The plasmid also harbors an amplification system as a potential safeguard against a fall in copy number due to an occasional missegregation event. The 2-mm circle provides a model for how moderation of selfishness can ensure the successful persistence of an extrachromosomal element without compromising the fitness of its host. Current Topics in Developmental Biology, Vol. 56 Copyright 2003, Elsevier Inc. All rights reserved. 0070-2153/03 $35.00

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I. Introduction The interplay of conflict and cooperation, of selfishness and altruism, has a strong bearing on the inclusive fitness and overall success of social populations (Dawkins, 1990; Sundstrom and Boomsma, 2001; Thomas, 2000). Insofar as a living cell is made up of societies of interacting individual molecules, the rules of direct fitness gains through selfishness and indirect, collective fitness gains through altruism are generally applicable at the molecular level. In this review, we shall first outline general considerations regarding the evolution, spread, and establishment of selfish nucleic acid elements in prokaryotes and eukaryotes. They may exist as autonomously replicating extrachromosomal entities (plasmids, for example) or as chromosomally integrated moieties (lysogenic phage, bacterial insertion sequences, and repeat families in eukaryotes). Several of these elements, say, plasmids or transposons harboring antibiotic resistance, provide their hosts with some advantage, at least under certain environmental challenges. Yet others rely on the hosts for their survival, but apparently oVer nothing in return. Such elements are truly selfish. The evolutionary success of a selfish genome depends on how well it can adjust its degree of selfishness to the biochemical resources available to it. Unfettered avarice could be self-destructive by the rapid depletion of these resources. The long-term persistence of an element would be better served by regulating its metabolic needs so as to not jeopardize the well-being of its host. In this context, we shall focus on the 2-mm plasmid of yeast to illustrate some of the strategies that shape the character of a model selfish DNA element.

II. The Origins and Evolution of Selfishness The primordial RNA world likely provided the breeding ground for the earliest selfish genetic elements in the form self-replicating oligoribonucleotide entities (Doudna and Cech, 2002; Joyce, 2002). The naturally occurring ribozymes in present day life forms that assist in RNA processing, facilitate the replication of certain viral genomes, or catalyze peptide bond formation are perhaps the evolutionary vestiges from this lost world. On the other hand, in vitro evolved ribozymes that can synthesize nucleotides and coenzymes or form amide bonds are likely proxies for some of the missing links in the transition from RNA-based to protein-based biocatalysis. Strikingly, in vitro evolution can recapitulate the fortuitous

1. Selfishness in Moderation

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emergence of selfish RNA molecules called RNA Z (Breaker and Joyce, 1994). By the appropriation of a promoter sequence, purely by accident, they become preferred substrates for amplification under the selection pressures imposed by the experiment.

III. Selfish Nucleic Acids in Prokaryotes and Eukaryotes DNA/RNA elements that fit the description of ‘‘selfish genomes’’ are widespread among prokaryotes and eukaryotes. They may, however, diVer considerably in the guile and sophistication of their selfishness. Classically, bacterial plasmids have provided the paradigm for successful extrachromosomal elements (Thomas, 2000). They utilize a fairly limited set of biochemical strategies for replication yet incorporate mechanisms for controlling copy number and coordinating replication with cell growth. The problem of propagating the replicated molecules to daughter cells is dealt with in one of two ways. Provided the steady state copy number of the plasmid is reasonably high (and there are no constraints to free diVusion), the chances of a plasmid-free cell arising at any given generation would be quite low. However, as a rule of thumb, a low copy number would be favored by reducing the metabolic burden on the host. The potential disadvantage of plasmid loss by missegregation is circumvented by the acquisition of an active partitioning system. Finally, the horizontal spread and establishment of plasmids among bacteria may be promoted or accelerated by the evolution of functions for mobilization and conjugative transfer. Similar molecular themes or their variations are revealed by eukaryotic selfish genetic elements as well. The satellite RNA/DNA molecules associated with adenovirus or the tobacco ringspot virus have little or no coding capacity but have established themselves stably by virtue of their active replication potential (Kado, 1998). The large Ti plasmids of Agrobacterium (that transpose a segment of their DNA to the plant genome) have not only manipulated the host bacterial cells for their survival but can induce higher plant cells to produce biochemical environments conducive for their host and hostile for those of competitors. Several mammalian viruses that exist primarily as nonintegrated episomes (papilloma and Epstein-Barr, for example) ensure their stable propagation by attaching themselves to the host chromosomes using protein tethers (Ilves et al., 1999; Lehman and Botchan, 1998). The ensemble of repeated DNA families found in eukaryotic genomes represent, in one sense, the limit case of chromosomal attachment, namely, covalent integration. However, the load of such integrated elements is tightly controlled, as exemplified by the limits to the integration of multiple forms of retrotransposons in mammalian cells.

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Figure 1 Organization of the 2-mm plasmid and recombination-mediated copy number control. (a) In the standard dumbbell representation of the double-stranded circular plasmid, the parallel lines (the handle of the dumbbell) indicate the inverted repeats (IRs) of the plasmid. The open reading frames are highlighted, with the arrowheads pointing in the direction of their transcription. The cis-acting DNA elements in the plasmid are the replication origin (ORI ), the partitioning locus (STB), and the Flp recombination target sites (FRT ). The STB element can be subdivided into two regions: ‘‘proximal’’ and ‘‘distal’’ with respect to ORI. STB-proximal contains the tandem array of 5–6 copies of a 62-bp consensus sequence and is central to plasmid partitioning. STB-distal is important in maintaining the ‘‘active configuration’’ of STBproximal, which is subject to context eVects. Forms A and B of the plasmid are generated by Flp-mediated recombination at the FRT sites. (b) A recombination-mediated amplification


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IV. The Yeast Plasmids Circular, double-stranded DNA plasmids found in Saccharomyces cerevisiae and other yeasts have been viewed as evolutionarily optimized ‘‘benign’’ parasitic elements (Broach and Volkert 1991). They do not provide any obvious benefit to their hosts. Yet, they persist in cell populations at relatively high copy numbers and exhibit almost chromosome-like stability. They exploit the genetic endowments of the host for their replication and segregation but adjust their degree of selfishness to avoid overtaxing the host’s metabolic resources. For example, sophisticated regulatory mechanisms guard against large upward or downward deviations from the optimized ‘‘high’’ steady state plasmid copy number. The 2-mm circle, the most well-characterized member of this class of plasmids, is representative of the genetic organization and functional strategies that underlie the success of the yeast plasmid family as selfish DNA elements.

V. Organization and Regulation of the 2-mm Circle Genome The 2-mm circle is almost ubiquitously present in strains of Saccharomyces yeasts at an average copy number of 40–60 per cell. The high copy propagation of the plasmid is achieved through an eYcient partitioning system and a copy number amplification system. The structural and functional design of the entire plasmid genome appears to be devoted solely to this end. In the standard dumbbell representation of the plasmid (Fig. 1a), the parallel lines represent two copies of a 599-bp sequence in inverted orientation. The plasmid codes for a site-specific recombinase Flp; two partitioning proteins, (Rep1p and Rep2p) and a presumed positive regulator for Flp expression (Raf1p) The cis-acting elements important in the physiology of the plasmid are the replication origin (ORI ), the targets for site-specific recombination (FRT = Flp recombination target) within the inverted repeats, and

mechanism proposed by Futcher (1986). Bidirectional replication starting at the origin in a plasmid molecule (a) duplicates the proximal FRT site before the distal one (b). An Flpmediated inversion (c) results in two replication forks oriented in the same direction (d). Movement of the two forks around the circular template amplifies copy number (e). A second recombination event (f ) restores bidirectional fork movement (g). The products of replication are a template copy (i) and an amplified moiety containing multiple tandem copies of the plasmid (h). The tandem multimer can be resolved by Flp recombination (or even homologous recombination) into plasmid monomers ( j, k). The diagram of the Futcher model shown here follows its representation by Broach and Volkert (1991). (c) Flp-mediated recombination between the repeats numbered 1 of a replicating 2-mm circle and a nonreplicating one (left) results in a replicating plasmid dimer (middle). A second intramolecular recombination between the repeats numbered 2 produces plasmid monomers joined by the replication forks (right).

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the stability-conferring partitioning locus (STB). Yeast cells harbor an equilibrium mixture of the plasmid forms A and B, formed as a result of Flp–FRT recombination and present in roughly equal abundance. A carefully timed site-specific recombination reaction carried out during plasmid replication is believed to be the critical event in copy number amplification.

VI. The Plasmid Partitioning and Amplification Systems The natural stability of the 2-mm circle approaches that of the yeast chromosomes (a loss rate of approximately 10 5 to 10 4 per generation) and is mediated by the partitioning system consisting of the two Rep proteins and the STB locus. Contrary to what their names might suggest, the Rep proteins have nothing to do with plasmid replication, which is carried out by the host replication machinery. Each of the 60 or so molecules duplicates only once during each cell cycle (Zakian et al., 1979), and the Rep–STB system ensures that the replicated molecules are partitioned nearly equally to the daughter cells (Velmurugan et al., 2000). The details of the partitioning mechanism are only beginning to emerge. The amplification system is brought into action only when there is a drop in copy number due to an accidental missegregation event. The Futcher model for amplification (Fig. 1b; Futcher, 1986) is predicated on the asymmetric location of the replication origin with respect to the FRT sites. This unequal spacing is retained in all the 2-mm–like plasmids, suggesting the conservation of a common recombination-based amplification mechanism during plasmid evolution. Normally, replication of the 2-mm plasmid proceeds bidirectionally from the origin and terminates at the opposite end by convergence of the forks. In the amplification mode (induced by low plasmid levels), a recombination event between a copy of the duplicated ORI-proximal FRT site and the unreplicated distal one causes the inversion of one of the two forks. They now chase each other on the circular template to spin out multiple tandem copies of the plasmid. A second recombination event and restoration of the bidirectional forks may terminate amplification. The tandem array of 2-mm plasmids in the amplicon can be resolved into unit size molecules by Flp-mediated resolution or via homologous recombination. Consistent with the prediction of the Futcher model, plasmid amplification cannot occur when Flp recombination is abolished (Reynolds et al., 1987; Volkert and Broach, 1986; Volkert et al., 1986). However, there is no direct proof that amplification proceeds through the intermediates diagrammed in Fig. 1b. In fact, potential amplifying moieties called pince-nez (PN) structures that are made up of linked double rolling circles (Fig. 1c) have been identified by electron microscopy (Petes and Williamson, 1994). They can be produced by intermolecular recombination between a


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replicating plasmid and a nonreplicating one, followed by resolution of the plasmid into monomers. Each circle of the PN has a constant size corresponding to that of a plasmid monomer (2-mm), whereas the size of the tether linking them is variable.

VII. Regulation of Plasmid Gene Expression Early genetic experiments, together with rather sparse biochemical data, have led to a model in which the Rep1 and Rep2 proteins form a bipartite regulator that negatively controls the expression of the FLP, REP1, and RAF1 loci (Fig. 2; Murray et al., 1987; Som et al., 1988). The REP2 gene is apparently not subject to this negative regulation; therefore the Rep2 protein is not limiting for the assembly of the Rep1p–Rep2p repressor. At the steady state copy number, the level of Rep1p is high enough to establish the critical concentration of the repressor required to turn oV FLP and RAF1 and thus keep the amplification system under check. A drop in copy number causes the repressor to fall below threshold, thus turning on FLP and RAF1. The Raf1p is believed to antagonize the repressor, thereby accelerating the amplification response. As copy number builds up, Rep1p and the repressor levels are boosted, and eventually the steady state is restored. Overall, the regulatory circuit is designed to rapidly commission and

Figure 2 Positive and negative controls of gene expression in the 2-mm plasmid. The schematic diagram depicting 2-mm circle gene regulation is adapted from Som et al. (1988). The putative bipartite regulator Rep1p–Rep2p (R1–R2) negatively controls expression of the FLP (Flp), RAF1 (D) and REP1 (R1). As a result, the level of the R1–R2 repressor is controlled as a function of the copy number, and at steady state the amplification system is essentially turned oV. The product of the RAF1 gene (D) antagonizes R1–R2, permitting rapid triggering of recombination-mediated amplification when plasmid copy number needs a boost. The REP2 locus appears to be free from repression by R1–R2. Aside from their role in controlling plasmid gene expression, the Rep1 and Rep2 proteins interact with the STB DNA to bring about equal segregation of the plasmid molecules at cell division.

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decommission the amplification machinery as demanded by the copy number status. The copy number, in turn, is indirectly read out as a function of Rep1p or the Rep1p–Rep2p repressor. The Rep proteins, in concert with the STB locus, are also required for plasmid partitioning. It is not clear whether the Rep1p–Rep2p repressor is also the active entity in partitioning, or whether the repressor and partitioning functions can be uncoupled from each other.

A. Mechanisms for Plasmid Partitioning Why does a plasmid that has a copy number of 60 utilize a partitioning system? Why not rely on random segregation, as is the norm with high copy bacterial plasmids? In principle, the amplification system can readily make the adjustments to rectify copy number deficits resulting from this segregation mode. Autonomously replicating yeast plasmids that lack the Rep– STB system (ARS plasmids) tend to show a finite segregation bias toward the mother during cell division (Murray and Szostak, 1983). The 2-mm circle partitioning system might overcome this bias in one of two ways. It could either (1) promote random segregation by causing the plasmids to be freely diVusible or (2) mediate active segregation, say, by attaching plasmids to a nuclear moiety that is equally partitioned between mother and daughter. One argument against random segregation is the lack of evidence for the continuous operation of the amplification system during cell growth. In density shift experiments, during one generation, essentially all of the plasmid fraction sediments at the intermediate density, suggesting a single round of replication for each molecule (Zakian et al., 1979). Low amplification levels (that would be consistent with the high plasmid copy number) could have been easily missed in this assay. However, as described in the following section, more recent evidence favors the active partitioning model.

B. Plasmid Organization, Localization, and Dynamics in the Yeast Nucleus Fluorescence tagging of STB-containing plasmids via GFP–LacI/LacO interaction in live cells or by immunostaining in fixed cells indicates that they are localized in the nucleus as a compact cluster in association with the Rep1 and Rep2 proteins (rows 1 and 2 in Fig. 3a; Velmurugan et al., 2000). An ARS plasmid (lacking STB) is not confined to the Rep protein zone (row 3 of Fig. 3a). The Rep proteins, and therefore the 2-mm plasmid, are almost always seen at or close to the spindle poles (Fig. 3b). The compactness of the plasmid cluster, measured as the width of the plasmid residence zone


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Figure 3 Organization and localization of the 2-mm plasmid in the yeast nucleus. (a) Plasmids are visualized by immunostaining using antibodies to the Lac repressor bound at the Lac operator repeats harbored by them. Rep1 and Rep2 proteins are visualized by using antibodies to the native proteins. The STB plasmid is contained within the Rep1p/Rep2p staining region within the larger DAPI staining area (top two rows). The ARS plasmid dots are often seen lying outside of the Rep1p/Rep2p zone (bottom row). (b) The localization of the Rep1 protein with respect to the mitotic spindle is shown in yeast cells at diVerent stages of the cell cycle. Nearly all of the Rep1 protein (with the associated 2-mm plasmid) is concentrated at or close to the spindle poles. The same pattern is obtained with Rep2 protein as well. The spindle is displayed using antibodies to tubulin. (c) An STB-containing plasmid appears as a compact cluster when examined by Z-series sectioning using confocal fluorescence microscopy in a [cirþ] host strain providing the Rep1 and Rep2 proteins from the native 2-mm plasmid (top row). In a [cir0] strain, lacking the Rep proteins, the plasmid cluster is less cohesive (middle row). A similar loosening of cohesion is observed when the cells are treated with the microtubule depolymerizing drug nocodazole (bottom row). The plasmid residence zone (PRZ) for each cell examined is expressed as the ratio of the widths of the plasmid and DAPI fluorescence patches. The values are averaged from 20 cells for each experimental group. (See Color Insert.)

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(PRZ in Fig. 3c) in the nucleus, is considerably loosened when the Rep system is inactive (in a host strain without native 2-mm circles, [cir0], and therefore lacking the Rep1 and Rep2 proteins; row 2 of Fig. 3c) or when the spindle is depolymerized with nocodazole (row 3 of Fig. 3c). The values at the right in Fig. 3c are normalized by dividing the number of sections containing green fluorescence (size of the plasmid cluster) into the number of sections containing blue DAPI fluorescence (size of the nucleus). Timelapse microscopy of cells released from -factor–induced G1-arrest reveals a rough doubling of the plasmid fluorescence (corresponding to plasmid replication) during bud growth, followed by separation of the cluster into two in large-budded cells and the rapid migration of each cluster toward opposite poles (Fig. 4a). These observations suggest that it is a high-order plasmid– protein complex that is the partitioning entity. As such, the copy number

Figure 4 (a) Segregation kinetics of the 2-mm plasmid in a wild type host; plasmid association with chromosome spreads. The fluorescence-tagged 2-mm circle reporter plasmid is followed from the point of bud emergence (time zero) through one full division cycle. The plasmid fluorescence is doubled in the 6- to 18-minute period (early S phase) and plasmid partitioning occurs in the 42- to 48-minute interval (G2/M). The observed timing of segregation is quite similar to that of a fluorescence-tagged chromosome. (b) In yeast chromosome spreads, the Rep1 protein and the STB containing reporter plasmid (immunostained with antibodies to bound Lac repressor) are colocalized within the DAPI staining area. (See Color Insert.)

1. Selfishness in Moderation Figure 5 Segregation patterns of the 2-mm plasmid segregation in mutant hosts that missegregate chromosomes at the nonpermissive temperature. In the mutant hosts, two types of plasmid segregation patterns are seen in relation to chromosome missegregation. In type ‘‘a’’ cells, the plasmid missegregates in tandem with the bulk of the chromosomes. In type ‘‘b’’ cells, the plasmid segregation is largely independent of that of the chromosomes. The percentage of cells that show the ‘‘a’’ or the ‘‘b’’ phenotypes for two reporter plasmids, one harboring STB and the other lacking it, is listed for the six mutant strains tested. All strains are [cirþ] and hence provide the Rep proteins in trans. The results shown here represent the segregation profile at the nonpermissive temperature. At the permissive temperature, chromosome segregation is normal. (See Color Insert.)

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of the plasmid is eVectively unity, and the need for an eYcient partitioning mechanism is justified. An STB plasmid can be detected in chromosome spreads prepared from yeast cells, but only if both Rep1 and Rep2 proteins are expressed in them (Fig. 4b; Mehta et al., 2002). Either Rep1p alone or Rep2p alone is not retained in the spreads. Together, the two proteins associate with the spreads even in the absence of an STB plasmid. Thus, it is the Rep proteins, acting in concert, that are responsible for the specific localization of the plasmid. Because of limited resolution, the chromosome spread assays cannot distinguish direct plasmid–chromosome association from independent localization of the plasmid and certain chromosomal domains common to nuclear sites. In synchronously dividing yeast cells, there is a striking similarity in the segregation kinetics of a fluorescence-tagged plasmid with a similarly tagged chromosome (Velmurugan et al., 2000). Furthermore, in several mutant yeast strains that missegregate chromosomes at the nonpermissive temperature, an STB plasmid also missegregates, and most often does so in tandem with the bulk of the chromosomes. This missegregation pattern is represented by the type ‘‘a’’ cells in Fig. 5. When STB is removed from the plasmid or when either Rep protein is absent, the comissegregation of the plasmid with the chromosomes is no longer observed. This behavior is denoted by the large increase in the type ‘‘b’’ cells (compare the ARS plasmid with the STB plasmid in each mutant host strain) in Fig. 5. Taken together, the data are consistent with the plasmid and chromosome segregation pathways being interlinked or coordinately regulated. Perhaps the plasmid is tethered to the chromosomes. Or, it may exploit components of the host mitotic machinery for its own equal segregation. Or, the partitioning system may provide a checkpoint to prevent plasmid entry into a cell that lacks a full chromosome complement.

C. Interactions among the Rep Proteins and the STB DNA in Plasmid Partitioning The long-held notion that the interactions of the Rep proteins with STB are important for plasmid stability has received experimental support only recently. The colocalization of the Rep proteins in the yeast nucleus with plasmids harboring the STB DNA, as well as their presence in chromosome spreads in a strictly partner-dependent manner, is consistent with this idea. By in vivo dihybrid assays and in vitro aYnity trapping assays, self- and cross-interactions of the Rep proteins have now been demonstrated (Ahn et al., 1997; Scott-Drew and Murray, 1998; Sengupta et al., 2001; Velmurugan et al., 1998). Evidence for STB binding by the Rep proteins has been more diYcult to establish. Partially pure Rep proteins fail to associate with


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STB when probed by standard gel mobility shift methods (Y. T. Ahn and M. Jayaram, unpublished data). Hadfield et al. (1995) showed that ureasolubilized yeast cell extracts containing Rep1p and Rep2p can bind STB, suggesting the potential involvement of host protein(s) in binding. By using a southwestern assay, Sengupta et al. (2001) have shown that the carboxyl– terminal portion of Rep2p has DNA binding activity. A detailed mutational analysis of the Rep1 protein (X. M. Yang and M. Jayaram, unpublished data) supports the functional relevance of Rep1p–Rep2p interaction and Rep1p–STB interaction in plasmid partitioning. Rep1p variants containing point mutations that disrupt either of the two interactions (or both) are not able to support normal plasmid maintenance.

D. Host Factors Required for 2-mm Circle Partitioning The prospect that a simple tripartite system, consisting of two proteins and a relatively short stretch of DNA, can confer chromosome-like stability on the 2-mm plasmid would seem highly unlikely. A search by yeast dihybrid and monohybrid assays for host proteins that interact with the Rep1p/Rep2p or the STB locus has revealed several candidates, among which at least three are particularly interesting: the products of BRN1, FUN30, and CST6/SHF1 (Velmurugan et al., 1998; X. M. Yang and M. Jayaram, unpublished data). Whereas BRN1 is an essential gene, both FUN30 and SHF1 are not. The Brn1 protein is a component of the yeast condensin complex, which plays a central role in proper chromosome segregation (Lavoie et al., 2000; Ouspenski et al., 2000). Brn1p appears to interact with Rep1p independent of Rep2p, and vice versa. Whether these interactions mirror the requirement of the condensin complex in 2-mm circle partitioning needs to be verified. Fun30p interacts with Rep1p directly but interacts only indirectly with Rep2p, presumably through Rep1p. Both Brn1p and Fun30p can associate with STB in a Rep-protein–dependent manner. The Shf1 protein binds to STB directly, as suggested by in vivo monohybrid results and by in vitro mobility retardation assays (Velmurugan et al., 1998). In the Shf1 background, there is a modest drop in the stability of a 2-mm circle-derived test plasmid. Interestingly, independent genetic assays have implicated FUN30 as well as SHF1 (CST6) as being important for chromosome partitioning (Ouspenski et al., 1999). The Fun30 protein contains peptide motifs characteristic of the SNF2 family of transcriptional regulators with potential chromatin remodeling activity (SGD database). The Shf1 protein appears to belong to the ATF/ bZIP class of transcription factors and harbors a consensus CREB motif (Velmurugan et al., 1998). It is likely that chromatin organization at the STB locus and/or its transcriptional status may aVect its eYciency in

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plasmid partitioning. Consistent with this notion, a recent study demonstrates the requirement of the Rsc2 protein, which forms part of a chromatin remodeling complex in yeast, for the normal stability of the 2-mm circle (Wong et al., 2002). The nucleosome pattern at STB is altered, and the association between Rep1p and STB is aVected by the absence of Rsc2p. It is noteworthy that the region of STB proximal to the 2-mm circle origin, consisting of approximately six copies of a 62-bp repeat unit, is kept free of transcription by a termination signal located in the ‘‘distal’’ STB segment (Sutton and Broach, 1985). Also, a 24-bp silencer element, capable of suppressing the activity of a nearby promoter in an orientation-independent manner, has been identified within the distal STB (Murray and Cesareni, 1986). Rather surprisingly, the 2-mm origin itself has been shown to function as a silencer whose activity is dependent on the Sir proteins, the origin recognition complex (ORC), and the Hst3 protein, a Sir2 histone acetylase homolog (Grunweller and Ehrenhofer-Murray, 2002). It is known that transcription through eukaryotic replication origins and centromeres, as well as the partitioning loci of certain bacterial plasmids, can adversely aVect their function (Rodionov et al., 1999). It is likely that the STB locus is also under a similar constraint, and its native location appears to have been selected to place the repeat units in a transcription-free zone. From earlier work we know that the stability of yeast plasmids (lacking the Rep–STB system) can be enhanced by the presence in cis of yeast telomere–associated sequences or the silencing element E associated with the unexpressed yeast mating-type locus HMRa (Ansari and Gartenberg, 1997; Kimmerly and Rine, 1987; Longtine et al., 1992; Longtine et al., 1993). The partitioning activity of the subtelomeric repeats is absolutely dependent on the Rap1 protein, whereas that by the E element is mediated through the Sir1-4 proteins. The underlying common theme in both types of plasmid stabilization appears to be the organization of a silent chromatin domain. It has been suggested that the silencing complex anchors plasmids to a nuclear component that is symmetrically divided between daughter cells. The old and new results can be accommodated by a model in which the plasmid replication is spatially restricted to a nuclear locale that facilitates the subsequent partitioning event.

E. A Potential Role for the Yeast Cohesin Complex in Plasmid Partitioning The plausible connection between chromosome segregation and 2-mm circle segregation is strengthened cumulatively by several pieces of circumstantial evidence summarized in the earlier sections. They include (1) cell biological observations of plasmid dynamics during segregation, (2) similar eVects of


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host mutations on chromosome and plasmid partitioning, and (3) the interaction between host proteins required for chromosome segregation and the plasmid partitioning system. Because of the interaction between Brn1p and the Rep proteins, our immediate attention was centered on the yeast condensin complex. We found, in our collection of Rep1p mutants, one that cannot interact with Brn1p and fails to support plasmid partitioning (X. M. Yang and M. Jayaram, unpublished data). The same mutant shows normal interaction with Rep2p and STB in dihybrid and monohybrid assays, respectively. However, the analysis of condensin in plasmid partitioning is impeded by its nonspecific association with DNA. The yeast cohesin complex then became the object of our interest because of its relatedness to condensin in a subset of its subunits and because of the cooperative roles that these complexes play during the segregation of sister chromatids during mitosis. In addition, the binding of cohesin to chromosomal locales is highly discriminatory (Laloraya et al., 2000). As indicated by the results summarized in the following section, the shift in experimental strategy has paid oV.

VIII. The Yeast Cohesin Complex Interacts Specifically with the Rep–STB System As noted earlier, the duplication of the 2-mm plasmid cluster followed by the segregation of the two clusters into daughter cells is reminiscent of the duplication and segregation of sister chromatids. Cohesin plays a central role in chromosome segregation by establishing sister chromatid pairing during the S phase and maintaining it until chromosomes are ready to be separated during anaphase (Carson and Christman, 2001; Cohen-Fix, 2001; Nasmyth, 2001; Nasmyth et al., 2000; Skibbens et al., 1999; Toth et al., 1999; Uhlmann and Nasmyth, 1998; Uhlmann et al., 1999; Uhlmann et al., 2000; Wang et al., 2000). Cleavage of the integral cohesin component Mcd1p/Scc1p by the Esp1 protease dissolves the cohesin bridge, and the sisters, bipolarly attached to the spindle, are rapidly pulled apart. A segregation mechanism based on cohesin-mediated pairing and unpairing of plasmid clusters would be expected to mimic chromosome segregation in its timing, as has been observed (Velmurugan et al., 2000). In chromatin immunoprecipitation assays, the Mcd1 protein associates specifically with the STB DNA in a Rep1p- and Rep2p-dependent manner (Mehta et al., 2002). Other regions of the plasmid, including the replication origin, are not occupied by Mcd1p. A similar association of STB is observed with other cohesin components as well, e.g., Smc1p and Smc3p. Furthermore, inactivation of Smc1p or Smc3p by Ts mutations disrupts Mcd1p–STB association at the nonpermissive temperature. These results are consistent with the preassembled cohesin complex being recruited

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Figure 6 Association of the cohesin complex with the 2-mm plasmid assayed by chromatin immunoprecipitation; noncleavable Mcd1p in cohesin blocks the separation of duplicated plasmid clusters. (a) Cells arrested in G1 by factor are released from pheromone arrest at time zero and followed by chromatin immunoprecipitation (using antibodies to the cohesin component Mcd1p), light microscopy (DIC), and FACS analysis. During each cell cycle, association of cohesin with the STB element occurs early in S phase and lasts until late G2/M. Note the nearly perfect synchrony between the chromosomes (as indicated by the presence of a cohesin binding site on chromosome V in the immunoprecipitate) and the plasmid in cohesin association and dissociation. ‘‘WCE’’ refers to whole cell extract. (b) Small budded cells


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by the plasmid partitioning system. The timing and periodicity of cohesin recruitment to the plasmid during the yeast cell cycle match nearly perfectly those of cohesin recruitment to the chromosome (Fig. 6a). Moreover, when cohesin disassembly during anaphase is blocked, the duplicated plasmid clusters mimic sister chromatids in failing to separate (Fig. 6b). It should be emphasized that the mechanism of cohesin association with STB is clearly distinct from that of cohesin binding to chromosomal loci. There is no apparent sequence similarity between STB and cohesin binding sites on the chromosomes, and obviously the Rep proteins are not required for the chromosomal recruitment of cohesin. Yet the timing of cohesin association and dissociation are well synchronized between the chromosomes and the 2-mm plasmid. Thus, the Rep–STB system appears to be clever molecular trickery evolved by the plasmid to feed into the temporal program that its host has established for the cycle of cohesin association–dissociation on chromosomes.

IX. Models for Cohesin-Mediated Plasmid Segregation Assuming that the yeast cohesin complex plays fundamentally similar roles in the partitioning of yeast chromosomes and the 2-mm plasmid, one or more segregation models can be considered. It is possible that cohesin facilitates pairing between the two duplicated plasmid clusters that, in turn, are tethered to a pair of sister chromatids. The coincident dissolution of the cohesin bridge between the sister chromatids and the plasmid clusters would dispatch each cluster in opposite directions in association with the chromosomes. The plasmid-chromosome attachment could be mediated by cohesin itself or through other factors. If cohesin is the tethering agent, there must be some mechanism to postpone Mcd1p cleavage within this tether until after segregation has been completed. Another possibility is that the two postreplication plasmid clusters are bridged by the cohesin complex but are not tethered to chromosomes. Upon disassembly of cohesin, each unpaired plasmid cluster moves to opposite cell poles without assistance from the

harboring a copy of the native MCD1 gene and one of the noncleavable versions under GAL promoter are transferred from dextrose to galactose at time zero. They are followed for 150 minutes by time lapse fluorescence microscopy to monitor a tagged chromosome (top two rows), an STB reporter plasmid (central two rows), or an ARS plasmid (bottom two rows). Of the 10 cells examined in each case (and arrested at the large budded state), the fractions exhibiting one chromosomal dot vs two dots and one plasmid cluster vs two clusters are indicated.

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chromosomes. This movement may be mediated by spindle attachment (a spindle-associated motor protein could be involved), by an active transport system unrelated to the spindle, or by association with a subcellular entity that is evenly partitioned at cell division.

X. Summary and Perspectives From the various lines of evidence presented here, the 2-mm plasmid emerges as a minimalist yet carefully optimized structural design for a selfish nucleic acid. By harboring a replication origin that is functionally equivalent to the chromosomal origins, the plasmid enjoys duplication by the host replication machinery. By pilfering host factors using components of its stability system, the plasmid apparently gains access to a sophisticated partitioning mechanism, and by preserving a recombination-mediated amplification system in readiness, the plasmid ensures that its copy number is maintained at the steady state value. The apparent conservation of the basic 2-mm circle paradigm by the other yeast plasmids speaks to its eVectiveness and evolutionary durability as a strategy for benign parasitism through moderation of selfishness. Why does yeast still maintain a high copy extrachromosomal element that apparently makes no contribution to its fitness? The built-in sophistication of the strategies for plasmid maintenance suggests that the plasmid at one time might have conferred a significant selective advantage on its host, and paradoxically, this very sophistication may make it diYcult and slow for yeast to get rid of the plasmid now. From one evolutionary perspective, the progenitor of the present day 2-mm family of yeast plasmids might have been an infectious agent (similar to episomal viral genomes) that established itself in an ancestral host by its ability to attach to the mitotic spindle or to the chromosomes. An early partitioning system, from which the Rep–STB system evolved, could have assisted plasmid propagation during cell division. Maintenance of the element would have been determined by the balance between occasional loss and reinfection. Later acquisition of Flp, perhaps by an integrative transposition event, accompanied by the loss of infective coding capacity, might have quarantined the plasmid in the lineage leading to the yeast strains that currently possess the 2-mm circle and its relatives. A viable alternative view is that the high copy plasmid segregated by a random mechanism during its early evolutionary history, relying on the amplification system for copy number adjustments. The Rep–STB system may have originated more recently in response to a reduction in the eVective copy number as a result of plasmid clustering.


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XI. Open Questions for Future Research The regulatory scheme outlined in Fig. 2 no doubt neatly fits into the normal physiology of the 2-mm plasmid, namely, equal segregation at steady state with provisions for upregulation in copy number, if required. Nevertheless, the model has its limitations. Although the FLP promoter can be repressed by overexpression of Rep1p and Rep2p from an inducible promoter, the magnitude of the eVect under physiological levels of the Rep proteins is almost imperceptible. The possibility that high Rep protein levels might titrate out host proteins, including transcription factors, to manifest secondary eVects on FLP expression cannot be ruled out. Little attention has been paid thus far to potential contributions by the host to the copy number regulation of a DNA element that it stably shelters. For example, are there cell cycle controls on the expression and/or steady state levels of the 2-mm circle proteins? Are the target DNA sites for these proteins diVerentially occupied as a function of the cell cycle? Answers to these and related questions would be central to understanding how the stability and amplification systems communicate with each other to establish homeostasis in plasmid partitioning and copy number maintenance. The conceptual simplicity and mechanistic parsimony of the Futcher model for plasmid amplification notwithstanding, the proof for its operation is incomplete. There is no doubt that plasmid amplification cannot occur when Flp recombination is abolished (Volkert and Broach, 1986). Yet, there is no direct evidence that amplification proceeds through the intermediates diagrammed in Fig. 1b. As pointed out earlier, potential amplifying moieties called pince-nez (PN) structures (Petes and Williamson, 1994; Fig. 1c) raise the specter of intermolecular amplification that falls outside the purview of the Futcher model. Although the need to amplify plasmid in the event of missegregation has been given considerable thought, the other side of the issue has been virtually ignored. What happens to a cell that receives more than its fair share of plasmids? Is plasmid replication in such a cell dampened to readjust the copy number to the steady state value? We do not know the answer, and experimental designs to address this issue have been lacking. What we do know is that very high plasmid copy numbers, imposed by inducing FLP expression from a strong promoter, are not relished by host cells (Murray et al., 1987; Reynolds et al., 1987). Such cells divide very slowly or not at all. In a growing population, cells with high plasmid load (if they do arise) will be quickly overtaken by those with normal plasmid density. A chromosomal mutation, nib1, causes clonal lethality in yeast in a 2-mm-circle– dependent manner, giving rise to colonies with a nibbled morphology (Holm, 1982a; Holm, 1982b). It is possible that the cells destined to die are

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the ones in which the plasmid load has crossed a critical value either by amplification or as a result of missegregation. Because of the selective disadvantage of the nib1 [cirþ] genotype, plasmid-free cells arising in a mutant population have a strong growth advantage and tend to establish their lineage rather rapidly. While cohesin-mediated partitioning is attractive in explaining the chromosome-like stability of the 2-mm plasmid, evidence in support of it is almost all circumstantial. Whereas the recruitment of cohesin by the Rep–STB system has received strong experimental support, the same cannot be said for the bridging of plasmid clusters via cohesin. The only evidence that favors such bridging is negative, namely, the failure of plasmid clusters to separate when the cohesin complex is assembled using the noncleavable version of Mcd1p. Because cohesin is essential for chromosome segregation, one major technical stumbling block is to selectively aVect plasmid segregation without simultaneously aVecting chromosome segregation. Perhaps mutations or experimental conditions that specifically disrupt either plasmid–cohesin association or chromosome–cohesin association, but not both, can help overcome this impediment. The replication-dependent loading of cohesin is critical for segregating two sister chromosomes into opposite cell compartments before cytokinesis. For a diploid organism, the cohesin-mediated pairing avoids the potential problem of distinguishing homologues from sisters following DNA replication. By contrast, the plasmid does not face this dilemma. If cohesin is indeed required for plasmid segregation, it is interesting to ask whether cohesinassisted plasmid pairing is mediated only concomitant with replication or may occur independent of replication. Assuming that cohesin provides a counting mechanism, are the plasmids counted approximately by the pairing of two clusters containing roughly equal numbers, or are they counted exactly by the pairing of each molecule with its sister in the duplicated cluster? It is now possible to tackle this issue experimentally. One may place inside the same cell 2-mm plasmids tagged with two colors, say, yellow and cyan, and follow them through sequential division cycles. In the precise counting scheme, the ratio of yellow to cyan will remain constant at each cell division; in the imprecise scheme, this ratio will change perceptibly over a set of divisions. Our current thinking on the mechanisms for plasmid stability is based on the notion that the Rep proteins serve to recruit chromosomally encoded partitioning factors to the STB locus. Yet, we still know little about how the Rep proteins themselves associate with STB. Currently available evidence argues against direct association between either of the Rep proteins and STB and suggests the involvement of one or more host proteins in this process. If such accessory proteins do exist, their identities need to be revealed. In addition, how does the partitioning system interact with a


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variety of host proteins, whose list seems to grow steadily? One possibility is that the Rep proteins and their host, encoded partner proteins polymerize along the STB locus to form a supramolecular partitioning complex not unlike the kinetochore complex. An alternative, but not mutually exclusive, possibility is that the Rep proteins may switch their partners as a function of the cell cycle to best suit their partitioning needs. As we have pointed out here, the plasmid cluster is normally positioned in the proximity of the spindle pole, and depolymerization of microtubules by nocodazole has a measurable adverse eVect on the compactness of the cluster. Recent unpublished results that extend these observations suggest that the mitotic spindle itself may directly or indirectly contribute to plasmid partitioning. A concerted role for both cohesin and the spindle in plasmid segregation, if upheld, would lend further credence to the suspected mechanistic connection between chromosome and plasmid partitioning. The yeast plasmid has provided us with the first glimpse of how an apparently rudimentary partitioning system might raise its level of sophistication by taking advantage of the mitotic segregation pathway of its host. Understanding the finer details of this hitherto unsuspected molecular poaching must await further work.

Acknowledgments Work in the Jayaram laboratory on the recombination and partitioning systems of the yeast plasmid has been supported over the years by funds from the National Institutes of Health, the National Science Foundation, the Robert F. Welch Foundation, the Council for Tobacco Research, the Texas Higher Education Coordinating Board, and the Human Frontiers in Science Program.

References Ahn, Y. T., Wu, X. L., Biswal, S., Velmurugan, S., Volkert, F. C., and Jayaram, M. (1997). The 2 micron-plasmid-encoded Rep1 and Rep2 proteins interact with each other and colocalize to the Saccharomyces cerevisiae nucleus. J. Bacteriol. 179, 7497–7506. Ansari, A., and Gartenberg, M. R. (1997). The yeast silent information regulator Sir4p anchors and partitions plasmids. Mol. Cell. Biol. 17, 7061–7068. Breaker, R. R., and Joyce, G. F. (1994). Emergence of a replicating species from an in vitro RNA evolution reaction. Proc. Natl. Acad. Sci. USA 91, 6093–6097. Broach, J. R., and Volkert, F. C. (1991). ‘‘Circular DNA Plasmids in Yeasts.’’ Cold Spring Harbor Press, Cold Spring Harbor, NY. Carson, D. R., and Christman, M. F. (2001). Evidence that replication fork components catalyze establishment of cohesion between sister chromatids. Proc. Natl. Acad. Sci. USA 98, 8270–8275.

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Cohen-Fix, O. (2001). The making and breaking of sister chromatid cohesion. Cell 106, 137–140. Dawkins, R. (1990). ‘‘The Selfish Gene.’’ Oxford University Press, Oxford, UK. Doudna, J. A., and Cech, T. R. (2002). The chemical repertoire of natural ribozymes. Nature 418, 222–228. Futcher, A. B. (1986). Copy number amplification of the 2 micron circle plasmid of Saccharomyces cerevisiae. J. Theor. Biol. 119, 197–204. Grunweller, A., and Ehrenhofer-Murray, A. E. (2002). A novel yeast silencer: The 2mu origin of Saccharomyces cerevisiae has HST3-, MIG1- and SIR-dependent silencing activity. Genetics 162, 59–71. Hadfield, C., Mount, R. C., and Cashmore, A. M. (1995). Protein binding interactions at the STB locus of the yeast 2 micron plasmid. Nucleic Acids Res. 23, 995–1002. Holm, C. (1982a). Clonal lethality caused by the yeast plasmid 2 micron DNA. Cell 29, 585–594. Holm, C. (1982b). Sensitivity to the yeast plasmid 2mu DNA is conferred by the nuclear allele nibl. Mol. Cell. Biol. 2, 985–992. Ilves, I., Kivi, S., and Ustav, M. (1999). Long-term episomal maintenance of bovine papillomavirus type 1 plasmids is determined by attachment to host chromosomes, which is mediated by the viral E2 protein and its binding sites. J. Virol. 73, 4404–4412. Joyce, G. F. (2002). The antiquity of RNA-based evolution. Nature 418, 214–221. Kado, C. I. (1998). Origin and evolution of plasmids. Antonie Van Leeuwenhoek 73, 117–126. Kimmerly, W. J., and Rine, J. (1987). Replication and segregation of plasmids containing cisacting regulatory sites of silent mating-type genes in Saccharomyces cerevisiae are controlled by the SIR genes. Mol. Cell. Biol. 7, 4225–4237. Laloraya, S., Guacci, V., and Koshland, D. (2000). Chromosomal addresses of the cohesin component Mcd1p. J. Cell Biol. 151, 1047–1056. Lavoie, B. D., Tuffo, K. M., Oh, S., Koshland, D., and Holm, C. (2000). Mitotic chromosome condensation requires Brn1p, the yeast homologue of Barren. Mol. Biol. Cell 11, 1293–1304. Lehman, C. W., and Botchan, M. R. (1998). Segregation of viral plasmids depends on tethering to chromosomes and is regulated by phosphorylation. Proc. Natl. Acad. Sci. USA 95, 4338–4343. Longtine, M. S., Enomoto, S., Finstad, S. L., and Berman, J. (1992). Yeast telomere repeat sequence (TRS) improves circular plasmid segregation, and TRS plasmid segregation involves the RAP1 gene product. Mol. Cell. Biol. 12, 1997–2009. Longtine, M. S., Enomoto, S., Finstad, S. L., and Berman, J. (1993). Telomere-mediated plasmid segregation in Saccharomyces cerevisiae involves gene products required for transcriptional repression at silencers and telomeres. Genetics 133, 171–182. Mehta, S., Yang, X. M., Chan, C. S., Dobson, M. J., Jayaram, M., and Velmurugan, S. (2002). The 2 micron plasmid purloins the yeast cohesion complex: A mechanism for coupling plasmid partitioning and chromosome segregation? J. Cell. Biol. 158, 625–637. Murray, A. W., and Szostak, J. W. (1983). Construction of artificial chromosomes in yeast. Nature 305, 189–193. Murray, J. A., and Cesareni, G. (1986). Functional analysis of the yeast plasmid partitioning locus. STB. EMBO J. 5, 3391–3400. Murray, J. A., Scarpa, M., Rossi, N., and Cesareni, G. (1987). Antagonistic controls regulate copy number of the yeast 2 micron plasmid. EMBO J. 6, 4205–4212. Nasmyth, K. (2001). Disseminating the genome: Joining, resolving, and separating sister chromatids during mitosis and meiosis. Annu. Rev. Genet. 35, 673–745. Nasmyth, K., Peters, J. M., and Uhlmann, F. (2000). Splitting the chromosome: Cutting the ties that bind sister chromatids. Science 288, 1379–1385.


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Ouspenski, II, Cabello, O. A., and Brinkley, B. R. (2000). Chromosome condensation factor Brn1p is required for chromatid separation in mitosis. Mol. Biol. Cell 11, 1305–1313. Ouspenski, II, Elledge, S. J., and Brinkley, B. R. (1999). New yeast genes important for chromosome integrity and segregation identified by dosage effects on genome stability. Nucleic. Acids Res. 27, 3001–3008. Petes, T. D., and Williamson, D. H. (1994). A novel structural form of the 2 micron plasmid of the yeast Saccharomyces cerevisiae. Yeast 10, 1341–1345. Reynolds, A. E., Murray, A. W., and Szostak, J. W. (1987). Roles of the 2 micron gene products in stable maintenance of the 2 microns plasmid of Saccharomyces cerevisiae. Mol. Cell. Biol. 7, 3566–3573. Rodionov, O., Lobocka, M., and Yarmolinksy, M. (1999). Silencing of genes flanking the P1 plasmid centromere. Science 283, 546–549. Scott-Drew, S., and Murray, J. A. (1998). Localisation and interaction of the protein components of the yeast 2 mm circle plasmid partitioning system suggest a mechanism for plasmid inheritance. J. Cell. Sci. 111, 1779–1789. Sengupta, A., Blomqvist, K., Pickett, A. J., Zhang, Y., Chew, J. S., and Dobson, M. J. (2001). Functional domains of yeast plasmid-encoded Rep proteins. J. Bacteriol. 183, 2306–2315. Skibbens, R. V., Corson, L. B., Koshland, D., and Hieter, P. (1999). Ctf7p is essential for sister chromatid cohesion and links mitotic chromosome structure to the DNA replication machinery. Genes Dev. 13, 307–319. Som, T., Armstrong, K. A. F., Volkert, C., and Broach, J. R. (1988). Autoregulation of 2 micron circle gene expression provides a model for maintenance of stable plasmid copy levels. Cell 52, 27–37. Sundstrom, L., and Boomsma, J. J. (2001). Conflicts and alliances in insect families. Heredity 86, 515–521. Sutton, A., and Broach, J. R. (1985). Signals for transcription initiation and termination in the Saccharomyces cerevisiae plasmid 2 micron circle. Mol. Cell. Biol. 5, 2770–2780. Thomas, C. M. (2000). Paradigms of plasmid organization. Mol. Microbiol. 37, 485–491. Toth, A., Ciosk, R., Uhlmann, F., Galova, M., Schleiffer, A., and Nasmyth, K. (1999). Yeast cohesin complex requires a conserved protein, Eco1p(Ctf7), to establish cohesion between sister chromatids during DNA replication. Genes Dev. 13, 320–333. Uhlmann, F., Lottspeich, F., and Nasmyth, K. (1999). Sister-chromatid separation at anaphase onset is promoted by cleavage of the cohesin subunit Scc1. Nature 400, 37–42. Uhlmann, F., and Nasmyth, K. (1998). Cohesion between sister chromatids must be established during DNA replication. Curr. Biol. 8, 1095–1101. Uhlmann, F., Wernic, D., Poupart, M. A., Koonin, E. V., and Nasmyth, K. (2000). Cleavage of cohesin by the CD clan protease separin triggers anaphase in yeast. Cell 103, 375–386. Velmurugan, S., Ahn, Y. T., Yang, X. M., Wu, X. L., and Jayaram, M. (1998). The 2 micrometer plasmid stability system: Analyses of the interactions among plasmid- and hostencoded components. Mol. Cell. Biol. 18, 7466–7477. Velmurugan, S., Yang, X. M., Chan, C. S., Dobson, M., and Jayaram, M. (2000). Partitioning of the 2-micron circle plasmid of Saccharomyces cerevisiae: Functional coordination with chromosome segregation and plasmid-encoded rep protein distribution. J. Cell. Biol. 149, 553–566. Volkert, F. C., and Broach, J. R. (1986). Site-specific recombination promotes plasmid amplification in yeast. Cell 46, 541–550. Volkert, F. C., Wu, L. C., Fisher, P. A., and Broach, J. R. (1986). Survival strategies of the yeast plasmid two-micron circle. Basic Life Sci. 40, 375–396. Wang, Z., Castano, I. B., De Las Penas, A., Adams, C., and Christman, M. F. (2000). Pol kappa: A DNA polymerase required for sister chromatid cohesion. Science 289, 774–779.

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Wong, M. C., Scott-Drew, S. R., Hayes, M. J., Howard, P. J., and Murray, J. A. (2002). RSC2, encoding a component of the RSC nucleosome remodeling complex, is essential for 2 micron plasmid maintenance in Saccharomyces cerevisiae. Mol. Cell. Biol. 22, 4218–4229. Zakian, V. A., Brewer, B. J., and Fangman, W. L. (1979). Replication of each copy of the yeast 2 micron DNA plasmid occurs during the S phase. Cell 17, 923–934.

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Nongenomic Actions of Androgen in Sertoli Cells William H. Walker Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

I. Introduction II. Hormonal Regulation of Spermatogenesis A. The Process of Spermatogenesis and Support of Spermatogenesis by Sertoli Cells B. Regulation of Spermatogenesis by Follicle-Stimulating Hormone and Testosterone C. The Testosterone Paradox III. Nongenomic Actions of Androgen A. Androgen Receptor–Independent Responses to Androgen B. Candidate Surface Receptors for Androgen C. Androgen Receptor–Dependent Actions of Androgen D. Evidence that Receptors for Androgen Are Associated with the Plasma Membrane E. Activation of the Mitogen-Activated Protein Kinase Signaling Pathway by Androgens IV. The Regulation of Female and Male Germ Cell Development by Nongenomic Actions of Androgen A. Induction of Oocyte Maturation B. Testosterone Elevates Intracellular Ca2þ Levels in Sertoli Cells C. Testosterone Activates Mitogen-Activated Protein Kinase and the CREB Transcription Factor in Sertoli Cells V. Summary Acknowledgments References

The steroid hormone testosterone is essential for the production of spermatozoa and, therefore, male fertility. However, the molecular mechanisms by which testosterone supports spermatogenesis are not well characterized. Previously, testosterone was thought to act solely by stimulating androgen receptor (AR) interactions with specific gene promoter elements to stimulate gene expression in target Sertoli cells that support developing germ cells. New evidence suggests that testosterone actions that are independent of AR–DNA interactions may contribute to the support of germ cell development. In this review, studies of these nongenomic actions of testosterone in Sertoli cells and other androgen target tissues are summarized. In addition, potential receptors mediating nongenomic androgen-induced signaling, as well as the secondary messengers and signaling pathways that are activated by testosterone, are discussed. Finally, a novel signaling pathway that is induced by androgen in Sertoli cells is outlined that results in the activation Current Topics in Developmental Biology, Vol. 56 Copyright 2003, Elsevier Inc. All rights reserved. 0070-2153/03 $35.00

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of the mitogen-activated protein (MAP) kinase signaling pathway, the CREB transcription factor, and CREB-mediated transcription. It is proposed that this novel pathway of testosterone action may complement classical genomic mechanisms to regulate Sertoli cell processes required to support spermatogenesis.

I. Introduction Androgens are a class of steroid hormones that are required for normal male sexual development and the maintenance of the male phenotype. The principal physiologically relevant androgens are testosterone and its metabolite 5-dihydrotestosterone (DHT). Potential target tissues for androgens include brain, heart, muscle, bone, skin, prostate, external genitalia, reproductive tract, and testis. The classical mechanism by which androgens and other steroid hormones exert their eVects is initiated with the diVusion of the hormone into a target cell through the plasma membrane (Fig. 1). The hormones then bind with high aYnity to specific intracellular receptor proteins that are present in the cytoplasm and/or nucleus. The binding of the steroid to its receptor produces conformational changes that result in the formation of a ‘‘transformed’’ or activated receptor that has high aYnity for specific DNA-binding sites (Tsai and O’Malley, 1994). Once the steroid-receptor complex is formed, it acts as a ligand-inducible transcription factor that is able to recruit coactivator proteins and stimulate gene transcription (Bagchi et al., 1992). The entire process required to initiate gene expression via this classical mechanism takes at least 30–45 minutes (Shang et al., 2000; Shang et al., 2002), and the length of time required to produce significant levels of nascent proteins is in the order of hours. During the past several years, a number of steroid actions, including those for androgen, as well as progesterone, estrogen, corticosterone, aldosterone, and vitamin D, have been reported that cannot be accounted for via the direct activation of gene expression by steroid receptors (reviewed in Cato et al., 2002; Falkenstein et al., 2000; Losel and Wehling, 2003; Revelli et al., 1998; Wehling, 1997). Instead of the delayed responses involved in regulating AR-mediated gene expression, in many instances steroid hormones have been found to rapidly alter other cellular processes within seconds or minutes. Often various second messengers, including phospholipase C, diaclyglycerol (DAG), cAMP, and calcium, are mobilized, resulting in the activation of protein kinases and cell signaling cascades. Because these responses do not require the synthesis of new mRNAs or proteins and are insensitive to inhibitors of transcription or translation, they have been classified as nongenomic. These alternative nongenomic actions increase the potential cell regulatory options for steroid hormones, and many of the physiological

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2. Nongenomic Actions of Androgen in Sertoli Cells Steroid hormone

hsp 90 hsp 70 SR

SR SR

Coactivators

RNA pol II

SR SR Target gene

HRE

AAAAA Altered cellular function

Protein

Figure 1 The classical mechanism of steroid action. Steroid hormones diVuse passively into the cell and combine with their cognate receptors in either the cytoplasm or the nucleus. In the cytoplasm, the binding of steroid to the receptor causes conformational changes in the receptor, allowing it to be released from heat shock proteins. The receptors then dimerize and migrate to the nucleus. Once in the nucleus, the steroid-bound receptor binds to specific hormone response elements (HREs) in the promoters of genes and recruits coactivator proteins that in turn alter chromatin structure and recruit RNA polymerase to the transcription initiation site. As a result, mRNA and proteins are produced that regulate cellular functions. Adapted from Onate, 2001, with permission from Humana Press, Inc.

eVects of nongenomic steroid actions are only now being discovered. In this chapter the nongenomic actions of androgen are discussed, with a special focus on the potential importance of androgen nongenomic signals in regulating Sertoli cell processes that support male germ cell development.

II. Hormonal Regulation of Spermatogenesis A. The Process of Spermatogenesis and Support of Spermatogenesis by Sertoli Cells Spermatogenesis is the multi-step process by which spermatogonial germ cells diVerentiate into mature spermatozoa within the seminiferous tubules of the testis (Fig. 2). The process of spermatogenesis begins with a

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Spermatozoa

Round spermatid

Spermatocyte

Tight junctions

Spermatogonium Sertoli cell nucleus Basement membrane

Leydig cell

Blood vessel

Interstitial tissue

Figure 2 A cross section through a part of the testis showing the progression of germ cell development and supporting somatic cells. Three seminiferous tubules (one with detail provided) and the interstitial tissue between the tubules are shown. Within the interstitial tissue are Leydig cells that produce testosterone. Blood vessels and extensive lymphatic spaces are also present. Within a seminiferous tubule, two Sertoli cells are shown. Also present is a diploid spermatogonium that undergoes a series of mitotic divisions and moves oV of the basement membrane and through Sertoli cell tight junctions to become a spermatocyte. The spermatocyte progresses through meiosis, resulting in haploid round spermatids. The spermatids then diVerentiate to become mature spermatozoa that are released into the lumen of the seminiferous tubule.

2. Nongenomic Actions of Androgen in Sertoli Cells

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spermatogonium going through a series of mitotic divisions. A final mitotic division results in spermatocyte germ cells that undergo meiosis. After the completion of meiosis, the resulting haploid spermatid germ cells undergo extensive diVerentiation to become spermatozoa, including condensation of the nucleus, termination of gene expression, elimination of much of the cytoplasm, and cell elongation. In addition, the spermatids develop flagellum and the acrosome that are critical for fertilization. Spermatogenesis is completed with the release of spermatozoa into the lumen of the seminiferous tubule. In addition to germ cells at various developmental stages, the seminiferous tubules contain somatic Sertoli cells that extend from the outer basement membrane to the inner lumen of the tubule. The Sertoli cells surround the developing germ cells and form special occluding tight junctions that prevent direct transport of factors larger than 1 kD to developing germ cells that are beyond the spermatogonial stage of development. This blood–testis barrier provides a defined, protected microenvironment for the developing germ cells but requires that Sertoli cells provide all the nutrients and growth factors required by germ cells (Skinner, 1991). Sertoli cell functions, in turn, are controlled by the major hormonal regulators of spermatogenesis, including testosterone that is produced by Leydig cells in the interstitial tissue surrounding the seminiferous tubules and follicle-stimulating hormone (FSH) produced by the anterior pituitary. In the seminiferous tubules, these hormonal signals act exclusively on the somatic Sertoli cells to regulate the production of factors required by germ cells (Plant and Marshall, 2001; Sharpe, 1994).

B. Regulation of Spermatogenesis by Follicle-Stimulating Hormone and Testosterone FSH binding to the FSH receptor, a specific G-protein coupled receptor (GPCR) on the surface of Sertoli cells, results in the elevation of cAMP and intracellular calcium ([Ca2þ]i) levels. During the prepubertal period FSH stimulates Sertoli cell proliferation, whereas during and after puberty the major role for FSH appears to be stimulating the production of factors such as transferrin and lactate that are required by germ cells. FSH is an important facilitator of spermatogenesis, but it does not appear to be essential in maintaining germ cell development. In the absence of FSH or the FSH receptor, spermatogenesis proceeds, although the numbers of spermatozoa and their fertilization capabilities are reduced (reviewed in Plant and Marshall, 2001). In contrast to FSH, androgen is absolutely essential for the maintenance of spermatogenesis. It is generally believed that testosterone, not DHT, is the androgen that regulates spermatogenesis. Unless relatively high levels of testosterone (>70 nM) are present, spermatogenesis is halted before the

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completion of meiosis so that few if any spermatids are produced (reviewed in Rommerts, 1988; Sharpe, 1994). The molecular mechanisms by which testosterone supports spermatogenesis have not been well characterized, but it has been established that within the seminiferous tubules testosterone acts on the 110 kD androgen receptor (AR) in the Sertoli cells (reviewed in Sharpe, 1994). AR is known to be essential for spermatogenesis because naturally occurring mutations in AR that cause the elimination of AR activity result in the testicular feminization (tfm) phenotype or androgen insensitivity syndrome (AIS) and the absence of mature germ cells (Quigley et al., 1995).

C. The Testosterone Paradox Testosterone actions in Sertoli cells present an interesting paradox in that numerous genes and proteins are up-regulated in response to hormonal stimulation (Cheng et al., 1986; Kokontis and Liao, 1999; Roberts and Griswold, 1989; Sharpe, 1994), but thus far in Sertoli cells only the two genes that encode the Myc and Pem transcription factors are known to be induced by androgens through the classical mechanism of AR binding to specific promoter elements (Lim et al., 1994; Lindsey and Wilkinson, 1996). In contrast, at least two observations support the hypothesis that testosterone may act through alternative mechanisms to complement classical AR actions in Sertoli cells. First, Sertoli cells require testicular testosterone levels greater than 70 nM to support spermatogenesis even though testosterone binding to AR and gene expression responses to testosterone are saturated at 1 nM (Rommerts, 1988; Sharpe, 1994; Veldscholte et al., 1992). Second, [Ca2þ]i levels are elevated in primary Sertoli cells within seconds of androgen stimulation and thus cannot be dependent on AR–DNA interactions and initiation of gene expression (Gorczynska and Handelsman, 1995; Lyng et al., 2000; Steinsapir et al., 1991). Together, these observations suggest that testosterone acts in Sertoli cells through nongenomic pathways in addition to classical mechanisms to regulate spermatogenesis.

III. Nongenomic Actions of Androgen Until recently, nongenomic actions of testosterone had not been extensively investigated in Sertoli cells. However, nongenomic actions have been studied in cells from other androgen target tissues including brain, heart, muscle, bone, and prostate, as well as immune cells. These studies have provided important information regarding secondary messengers and signaling pathways that can be activated in response to androgen stimulation. Therefore, the data acquired from more established systems will be reviewed initially


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to gain perspective for later discussions of new evidence for nongenomic actions of androgen in Sertoli cells and their potential importance for the regulation of spermatogenesis. Previous studies in nontestis tissues have been useful for characterizing the receptors that initiate nongenomic signaling pathways stimulated by androgen. At least two mechanisms are used to initiate and relay nonclassical actions of androgens: those that are inhibited by AR antagonists, such as hydroxyflutamide, and those that are insensitive to AR antagonists (outlined in the following section). These findings suggest that in some cases AR or AR-like factors are required to mediate nongenomic signaling, whereas novel ARs are hypothesized to facilitate androgen actions in other instances.

A. Androgen Receptor–Independent Responses to Androgen Androgen target cells that are insensitive to AR antagonists include osteoblast cells that display sex-specific nongenomic responses to steroids. Testosterone (10 pM–10 nM) elevated [Ca2þ]i as well as induced IP3 and DAG formation within 10 seconds only in rat osteoblasts isolated from males (Lieberherr and Grosse, 1994). 17 -Estradiol (estradiol) stimulated similar responses only in female osteoblasts (Lieberherr et al., 1993). In male osteoblasts, neomycin and pertussis toxin abolished the eVects of testosterone, suggesting that the androgen acts through membrane receptors coupled to phospholipase C via a pertussis toxin-sensitive G-protein (Lieberherr et al., 1993). Further evidence for nongenomic steroid actions in osteoblasts was provided in an in vivo study employing a synthetic steroid that reproduces the nongenomic eVects of estradiol and androgen without promoting classical steroid hormone receptor-mediated transcription. In this study, the synthetic steroid increased bone mass and strength in ovariectomized female and orchidectomized male mice (Kousteni et al., 2002). Another example of androgen target cells that are insensitive to AR antagonists is human granulosa cells. The addition of the androgen androstenedione (AD) (0.1 nM–mM) to granulosa cells elevated [Ca2þ]i levels within 2–5 seconds, resulting from both transmembrane influx and mobilization of Ca2þ from the endoplasmic reticulum (Machelon et al., 1998). AD is thought to act via a pertussis toxin-sensitive G-protein–coupled receptor (GPCR) to activate voltage-dependent Ca2þ channels in the plasma membrane and phospholipase C. Epithelia from the eVerent duct and epididymis that are used to transport spermatozoa from the termini of the seminiferous tubules to the vas deferens are also regulated by nongenomic testosterone actions that apparently act independently of AR. Testosterone and, to a lesser extent, DHT at concentrations as low as 1–10 mM reduced chloride secretion from cultured eVerent

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ducts and epididymis within 10–20 seconds. This testosterone action is physiologically significant because chloride secretion has been shown to be required for the fluid reabsorption that takes place when spermatozoa acquire their fertilizing capability. Cyproterone acetate, a steroidal antiandrogen, and flutamide, a nonsteroidal antiandrogen, did not block testosterone actions, suggesting that classical AR was not required for the observed responses to androgen. Testosterone was also found to cause a dose-dependent inhibition in forskolin-induced cAMP levels in eVerent duct and epididymis epithelia. Furthermore, pertussis toxin inhibited testosterone-stimulated chloride secretion, suggesting that the receptor for androgen is coupled to adenylate cyclase via an inhibitory G-protein (Leung et al., 2001). Perhaps the best argument for AR-independent androgen actions are IC-21 macrophages that do not express AR and splenic T cells in which the AR present does not have detectable androgen binding activity and does not migrate to the nucleus in response to androgen. Testosterone at physiological concentrations (1–10 nM) mediated Ca2þ influx within 5 seconds through non–voltage-gated, Ni2þ-blockable Ca2þ channels in the plasma membrane of splenic T-cells (Benten et al., 1999b). IC-21 macrophages also responded to similar levels of testosterone with a 100 nM increase in [Ca2þ]i within seconds. However, unlike T-cells, the increase in [Ca2þ]i originated from intracellular stores (Benten et al., 1999a). Further studies of the IC-21 macrophages demonstrated that the testosterone-induced increase in [Ca2þ]i was not aVected by the AR blockers cyproterone or flutamide. Ca2þ mobilization was inhibited by the phospholipase inhibitor U73122 and the Gi antagonist pertussis toxin. Binding sites for testosterone were detected on the surface of the macrophage cells, and upon stimulation with testosterone the binding sites became selectively internalized. Internalization was dependent upon adenosine triphosphate (ATP), cytoskeletal elements, and phospholipase C and G proteins (Benten et al., 1999a). Together, these data suggest that GPCRs for testosterone can be localized to plasma membranes.

B. Candidate Surface Receptors for Androgen The rapid and AR-independent actions of androgens suggest that a receptor on the plasma membrane may initiate signaling pathways in some cell types. One candidate surface receptor that may mediate androgen signaling is the sex hormone–binding globulin (SHBG) receptor, RSHBG. Thus far, RSHBG is defined as a functional membrane binding activity for SHBG because it has not yet been cloned and little is known about the protein. In the serum, 44% of testosterone is tightly bound to SHBG, 54% is bound weakly to albumin, and 2% is free (Dunn et al., 1981). Estradiol is also able to bind


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to SHBG, but with lower aYnity than androgens (Mendel, 1989). In the absence of steroid, SHBG binds with high aYnity to RSHBG; however, SHBG already complexed with steroid will not bind to the receptor (reviewed in Rosner et al., 1999). The SHBG-receptor complex is activated within minutes by estradiol or androgen binding to SHBG and is thought to be coupled to a G-protein or associated with a GPCR to account for the observed increases in cAMP production and induction of cAMP-dependent transcription (Fig. 3, pathway 1) (Nakhla et al., 1990; Nakhla et al., 1999). Another candidate surface receptor for androgen is a receptor such as GPR30 that has been shown to transduce signals from the gonadal steroids estradiol and progesterone. From the deduced 375 amino acid sequence, the receptor is predicted to exhibit a serpentine, heptahelical structure that is characteristic of the GPCR family. By structural homology, GPR30 most closely resembles receptors for angiotensin II and chemokines (Filardo, 2002). Although androgen interactions with GPR30 or similar receptors have not yet been demonstrated, it is possible that in some cell types androgen could act by a mechanism similar to that used by estradiol and progesterone (Fig. 3, pathway 2). GPR30 has been found to transduce estradiol and progesterone signals that can stimulate or inhibit the proliferation of breast carcinoma cells (Ahola et al., 2002; Filardo et al., 2000; Filardo et al., 2002). Estradiol elicits growth-inhibitory eVects by stimulating GPR30 and causing the dissociation of receptor-coupled G and G G-proteins. G then activates adenylate cyclase, thus elevating cAMP levels and resulting in the activation of protein kinase A (PKA) that inhibits Raf-1, one of the initial kinases in the MAP kinase cascade that promotes cell proliferation (Filardo et al., 2002). Activation of the MAP kinase pathway and promotion of cell proliferation by GPR30 occurs by a second mechanism. As a result of estradiol–GPR30 interactions, liberated G causes the activation of Src-related tyrosine kinases, leading to activation of the MAP kinase signaling pathway. However, activation of the MAP kinase pathway occurs via an indirect route that is dependent on the transactivation of the epidermal growth factor receptor (EGFR). The activated Src kinase stimulates a transmembrane matrix metaloprotease (MMP) that releases heparin-bound epidermal growth factor (HB–EGF) from the surface of the stimulated cell. The free EGF then binds to the EGFR and triggers the activation of the MAP kinase signaling pathway (Filardo et al., 2000). It is important to note that in other studies of breast cancer cells an estradiol-dependent pathway nearly identical to that initiated by GPR30 has been shown to require membrane-associated estrogen receptor (ER) (Fig. 3, pathway 3). These studies demonstrated that ER, when bound by estradiol, independently triggered Gq-, Gi-, and G -dependent stimulation of Src that activated MMP and EGFR (Razandi et al., 2003). The

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Androgen

GPR30-like GPRC

HBEGF

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HBEGF

MMP

SHBG AR

RSHBG

a

AC a

a

a b g

Src

a b g

Raf

EGFR

b g

cAMP

ATP

MEK PKA

ERK

Figure 3 Signaling pathways leading from candidate surface receptors for androgen. Three potential signaling pathways leading from surface ARs are proposed. In the first pathway (1) the SHBG receptor RSHBG binds SHBG, but remains inactive. Upon binding of androgen to SHBG, the receptor is activated and adenylate cyclase is stimulated via a coupled G-protein or associated GPCR. cAMP levels then increase and PKA activity is induced. In the second pathway (2) studies performed with estradiol suggest that the GPR30 receptor or a similar receptor could bind androgen in the presence or absence of associated proteins. As a result, the receptor-coupled heterotrimeric G-protein complex dissociates into G and G subunits. G then activates adenylate cyclase (AC) that catalyzes the formation of cAMP. PKA is activated by cAMP and can inhibit Raf-1 and the MAP kinase signaling cascade. The liberation of G causes the activation of Src tyrosine kinase that then stimulates the transmembrane matrix metaloprotease MMP to release heparin-bound epidermal growth factor (HB-EGF) from the cell surface. HBEGF then binds to the EGF receptor (EGFR) and induces the MAP kinase cascade, resulting in the activation of ERK and other MAP kinases. Alternatively, in a third pathway (3) androgen may bind to membrane-associated AR to independently activate G-proteins and initiate a similar pathway to that described for GPR30.

identification of this ER-dependent mechanism used to propagate estradiol signals raises the possibility that androgen may act directly though AR to elicit nongenomic actions.

C. Androgen Receptor–Dependent Actions of Androgen The first examples of androgen acting through AR to elicit nongenomic responses were LNCaP human prostate cancer cells that displayed hydroxyflutamide-sensitive increases in [Ca2þ]i within 2 minutes of mibolerone (dimethylnortestosterone) or DHT addition (Steinsapir et al., 1991). Later studies of prostate cells and human genital skin fibroblasts indicated that AR was required for DHT-mediated activation of Erk-1 and Erk-2 MAP kinases, as well as the PI3-kinase and protein kinase C pathways (Peterziel et al., 1999). A hydroxyflutamide-sensitive increase in [Ca2þ]i was also observed in freshly isolated immature Sertoli cells in response to testosterone or DHT (Gorczynska and Handelsman, 1995). Furthermore,


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in studies of primary Sertoli cells cultured for 1–4 days, testosterone or the synthetic androgen agonist R1881 (methyltrienolone), at levels of 1 pM to 1 nM, induced transient increases (2–3 minutes) in [Ca2þ]i within 20 seconds of addition, whereas higher concentrations of testosterone or R1881 (>10 nM) caused rapid and sustained increases (>15 minutes) in [Ca2þ]i (Lyng et al., 2000). Together, these studies argue that in some cells the classical receptor is used to initiate nongenomic eVects of androgens.

D. Evidence that Receptors for Androgen Are Associated with the Plasma Membrane Whether androgens act through AR, nonclassical receptors, or a combination of the two species, there is evidence to suggest that the receptors associated with rapid androgen actions are located in or near the plasma membrane. Much of the evidence for membrane-associated ARs has been derived from the use of testosterone–BSA conjugates that, due to their large size, are not able to cross the plasma membrane by diVusion. Fluorescently labeled conjugates have been used to visualize specific testosterone binding to the membranes of testosterone-responsive cells. For example, in one collection of studies, testosterone–BSA conjugates were shown to bind to the surfaces of T-cells, macrophages, osteroblasts, and Sertoli cells (Benten et al., 1999a; Lieberherr and Grosse, 1994; Lyng et al., 2000; Wunderlich et al., 2002). In these same studies the conjugates were used to argue that rapid responses to testosterone such as increased [Ca2þ]i and IP3 levels, as well as activation of MAP kinase and elevated DAG formation, resulted from testosterone interactions with receptors at the plasma membrane. The conjugate studies have generated some controversy. To elicit nongenomic eVects, levels of testosterone–BSA often must be at least 10- to 100-fold higher than those of free androgen. Furthermore, arguments have been made that, without prior filtration and purification treatments, free steroid is carried with the conjugate. There is also evidence that, even after filtration, free steroid is continually disassociated from the complex. These technical diYculties raise the possibility that free steroid from the conjugates diVuses through the membrane to elicit the observed nongenomic eVects (Stevis et al., 1999). Thus far no immunohistochemistry, cell fractionation, or biochemical studies have been reported for mammalian cells that localize classical AR to the cell membrane. In contrast, due to successful implementation of these techniques, it is now accepted that in some cells estrogen can interact with a pool of classical ER (ER or ER ) localized to the plasma membrane (mER) to activate intracellular signaling pathways (Chambliss et al., 2002; Norfleet et al., 1999; Pappas et al., 1995; Ramirez et al., 1996; Razandi

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et al., 1999; Russell et al., 2000; Towle and Sze, 1983). Steady state levels of ER and ER associated with the membrane were estimated to be 2–3% of the total cellular receptor pool (Razandi et al., 1999). However, it is possible that the levels of mER may transiently increase because in some cases estrogen can cause ER translocation to the membrane (Razandi et al., 2002). The challenges that must be overcome to identify ER associated with the membrane and the characteristics of cells expressing mER have been summarized (Campbell et al., 2002; Watson et al., 2002). Based on the parallels observed between ER and AR nongenomic actions, the following lessons learned from ER studies may be applied to characterizing membraneassociated AR. First, for immunohistochemistry studies, specific fixation and permeabilization protocols had to be developed to preserve the membrane structure but allow identification of ER epitopes. Second, there is evidence that cells containing mER are more likely to incur estrogen-mediated changes in the interactions of cellular attachment proteins with their substrates and other cells. As a result, cells containing mER can detach from the plate within 3 minutes of estrogen stimulation. Third, mER is short lived because it is rapidly eliminated after knockdown of ER expression. Fourth, mER is expressed at higher overall levels when cells are first plated, but the percentage of cells expressing mER later drops to 10–20%. One explanation for this observation is that mER may be regulated with the cell cycle such that mER levels are highest in G1 phase. Finally, as cells reach densities that put them in contact with one another, mER levels decrease, and only cells plated at low density or at the periphery of an expanding group of cells contain high levels of mER. Although AR has not been directly localized to the membrane, both AR and ER have been found to be associated in a ligand-dependent manner with caveolin, a 22-kD transmembrane phosphoprotein that serves as a scaVolding protein for many signaling molecules, including PI3-kinase, Ras, and Src (Kim et al., 1999; Lu et al., 2001; Okamoto et al., 1998; Schlegel et al., 1999). Interactions with caveolin facilitated the translocation of ER to the plasma membrane (Razandi et al., 2002). Similarly, in LNCaP prostate cells, increased AR localization to caveolin-containing membrane fractions was ligand dependent and occurred within 10 minutes of DHT stimulation, suggesting that androgen may stimulate AR to move to the plasma membrane (Lu et al., 2001). These studies raise the possibility that AR may be transiently associated with the plasma membrane. However, further direct proof for AR localization to the plasma membrane is needed because 10–15% of caveolin is located in the cytoplasm and is associated with the same subset of chaperone proteins that is involved in the classical mechanisms for androgen activation of AR (transformation) and nuclear translocation (Pratt and Toft, 1997; Pratt et al., 1993; Uittenbogaard et al., 1998).


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E. Activation of the Mitogen-Activated Protein Kinase Signaling Pathway by Androgens Increasing evidence has been gathered demonstrating that androgen can directly activate cellular signaling pathways independent of AR binding to DNA. Particularly relevant is the activation of the MAP kinase pathway by androgen. Androgen can induce a number of factors that have been implicated in regulating the MAP kinase cascade, including PKA, calmodulin, phospholipase C, protein kinase C (PKC), and guanine nucleotide exchange factors (GEFs) (Finkbeiner and Greenberg, 1996). All these factors have been found to be capable of initiating the MAP kinase cascade by stimulating a Ras or a Ras-like protein to activate a Raf MAP kinase kinase kinase (MAPKKK) (Pearson et al., 2001). Evidence for androgen stimulation of the MAP kinase pathway includes the finding that the nonhydrolyzable androgen agonist R1881 (1 nM) activated the Erk MAP kinase in human PMC42 breast cancer cells (Zhu et al., 1999). Similarly, DHT rapidly and transiently (2–60 minutes) increased Erk phosphorylation in primary prostate stroma cells. Erk phosphorylation was also elevated by DHT concentrations as low as 0.1 nM in LNCaP cells that contain AR and in PC3 cells stably transfected with AR, but not in wild-type PC3 cells that are AR deficient (Peterziel et al., 1999). There is evidence that, in some cell types, androgen binding to AR and estradiol binding to ER can stimulate the MAP kinase pathway through the Src tyrosine kinase. Normally, Src is localized to the inner surface of the plasma membrane and is inactive due to interactions between the tyrosine kinase, SH2, and SH3 domains of the protein. In LNCaP cells, treatment with R1881 or estradiol triggered direct interactions between AR and the SH3 domain of Src or ER and the SH2 domain of Src that released inhibition of the tyrosine kinase activity. These steroid receptor interactions with Src caused the activation of Src within 1 minute and the subsequent stimulation of the MAP kinase cascade within 5 minutes (Migliaccio et al., 2000). Further proof of AR and ER acting through Src was shown in Src-deficient embryonic fibroblasts from srl /src mice that did not show any Src kinase activity in response to androgen or estradiol (Kousteni et al., 2001). In studies of osteoblasts, osteocytes, and embryonic fibroblasts, androgenAR-Src interactions recruited the Shc adaptor protein to the complex (Kousteni et al., 2001). Src activation of Shc is known to lead to the activation of Ras or Ras-like GTPase proteins and the subsequent stimulation of the Raf-1 MAPKKK (Rusanescu et al., 1995). Studies of ER-mediated activation of MAP kinase have shown that ER can act directly through the binding of Shc, therefore raising the possibility of functionally important direct AR–Shc interactions (Song et al., 2002).

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A receptor-interacting scaVold protein called MNAR (modulator of nongenomic activity of ER) has been found to facilitate ER interactions with the Src family of tyrosine kinases, activate the MAPK pathway, and ultimately stimulate ER-mediated gene expression. MNAR may also facilitate the activation of Src kinases and MAPK by androgen because this adaptor protein also interacts with AR (Wong et al., 2002).

IV. The Regulation of Female and Male Germ Cell Development by Nongenomic Actions of Androgen A. Induction of Oocyte Maturation The maturation of Xenopus oocytes, an event that does not require transcription or even the presence of nuclei, has been used as an example of nongenomic steroid actions for over 30 years (Masui and Markert, 1971; Smith and Ecker, 1971). During growth, oocytes are arrested at the G2/prophase border of meiosis I. After progesterone stimulation, the oocyte resumes meiosis I and maturation, including germinal vesicle breakdown (GVBD), before halting again in metaphase of meiosis II. These ‘‘mature’’ oocytes are then competent for ovulation and subsequent fertilization, after which the final stages of meiosis are completed (Maller and Krebs, 1980). Initially, only nongenomic actions of progesterone were thought to regulate oocyte maturation. However, now androgens are thought to play a significant role in the maturation process. Early studies suggested that progesterone acted upon membraneassociated receptors because the hormone is more eVective in inducing maturation when applied to the outside of the oocyte than it is when microinjected into the oocyte (Masui and Markert, 1971; Smith and Ecker, 1971). Although analyses of Xenopus progesterone receptors (XPRs) demonstrated that XPR is predominately cytoplasmic, 5% of receptors were found to be associated with the plasma membrane and available to bind progesterone (Bagowski et al., 2001). Progesterone has been known for some time to inhibit adenylate cyclase, resulting in lower intracellular cAMP concentrations within minutes (Maller et al., 1979; Sadler and Maller, 1985). Decreases in cAMP and the subsequent decline in protein kinase A (PKA) have been observed to occur during progression to GVBD (Huchon et al., 1981; Maller and Krebs, 1977). More recently, G scavengers have been used to identify these G-protein subunits as required factors for progesterone-induced maturation (Lutz et al., 2000; Sheng et al., 2001). Progesterone also activates the MAP kinase pathway by an indirect mechanism that results in the


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stabilization of c-mos RNA, thus increasing the translational eYciency of this activator of the MEK MAPKK (reviewed in Ferrell, 1999; Maller, 2001). Several pieces of evidence have been presented suggesting that androgens may be responsible for inducing Xenopus oocyte maturation. First, the potent progesterone antagonist RU486 does not inhibit maturation (Sadler et al., 1985). Second, the androgens testosterone and androstenedione (AD) promote maturation and activation of the MAPK signaling cascade as well as or better than equal concentrations of progesterone (Lutz et al., 2001). Third, Xenopus oocytes express an unusually active isoform of the steroidogenic enzyme CYP17 that converts progesterone into AD and likely accounts for the virtually undetectable levels of progesterone and the 10-fold higher levels of AD and testosterone that are present in the serum and ovary of frogs injected with hCG (Yang et al., 2003). Fourth, the AR antagonist flutamide reduced testosteroneand AD-mediated maturation, suggesting that AR facilitates maturation (Lutz et al., 2001). Finally, immunohistochemical and biochemical evidence suggest that the classical AR is present in the oocyte plasma membrane (S. Hammes, personal communication). Taken in total, studies of Xenopus oocytes suggest that both progesterone and androgens are capable of promoting oocyte maturation via their classical receptors in vitro; however, only androgens appear to be present at high enough concentrations in vivo to initiate signaling pathways necessary for oocyte maturation (Lutz et al., 2001). It should be noted that presently there is no direct evidence to suggest that a nongenomic mechanism for androgen regulation of oocytes maturation is conserved in mammals. However, the AD rapidly (5–60 seconds) elevated [Ca2þ]i in human granulosa cells that support the oocytes. The use of specific inhibitors suggested that a GPCR linked to phospholipase C mediates the androgen eVects in granulosa cells (Machelon et al., 1996). Furthermore, testosterone and DHT have been reported to stimulate granulosa cell proliferation and the growth of follicles in the primate ovary via an unknown mechanism (Vendola et al., 1998). Administration of testosterone and DHT also reportedly increased expression of insulin-like growth factor 1 (IGF-1) and IGF-1 receptor mRNAs in granulosa cells, theca cells, and oocytes in the primate follicle (Vendola et al., 1999a; Vendola et al., 1999b). Additional studies revealed that testosterone and DHT elevated the levels of FSH receptor mRNA in granulosa cells (Weil et al., 1999). Although further study is required, these results raise the possibility that nongenomic actions of androgens contribute to follicular growth and may account for the increased number of follicles and enhanced responsiveness to FSH stimulation that has been noted in women with elevated androgen levels and polycystic ovary syndrome (Weil et al., 1999).

40 B. Testosterone Elevates Intracellular Ca

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As discussed earlier, androgen elevates [Ca2þ]i in Sertoli cells (Gorczynska and Handelsman, 1995; Lyng et al., 2000). In contrast, aromatization of testosterone to 17 -estradiol reduced Ca2þ entry into Sertoli cells. Furthermore, other steroids including progesterone were ineVective in altering [Ca2þ]i levels, implying that androgen alone activates Ca2þ-dependent nongenomic signaling in Sertoli cells (Gorczynska and Handelsman, 1995). In Sertoli cells, testosterone-mediated elevation of [Ca2þ]i required the influx of extracellular Ca2þ, suggesting that calcium channels in the plasma membrane play a role in testosterone–calcium signaling (Lyng et al., 2000). Sertoli cells express L, N, P/Q, and T type voltage-sensitive calcium channels (VSCC) (D’Agostino et al., 1992; Fragale et al., 2000). However, testosterone-induced increases in [Ca2þ]i were reduced more than 60% by pretreatment with verapamil, a VSCC blocker that acts principally on L-type channels, suggesting that L-type channels are the major conveyers of testosterone-induced Ca2þ into Sertoli cells (Lyng et al., 2000). It has been proposed that Ca2þ-dependent nongenomic actions of androgen in Sertoli cells involve the closing of an ATP-dependent Kþ channel because increases in [Ca2þ]i after stimulation with 200–300 nM testosterone were nullified by the Kþ channel agonist diazoxide (Silva et al., 2002). This finding raises the possibility that depolarization of Sertoli cells due to testosterone closing of Kþ-ATP channels is the mechanism that causes Ca2þ uptake through L-type voltage-dependent Ca2þ channels. While the previous study awaits confirmation, the possibility remains that androgens may alternatively open Kþ channels in Sertoli cells because testosterone and DHT reportedly contribute to vasodilation by opening a large conductance, calcium- and voltage-activated Kþ channel in coronary myocytes (Deenadayalu et al., 2001).

C. Testosterone Activates Mitogen-Activated Protein Kinase and the CREB Transcription Factor in Sertoli Cells Signaling pathways activated by testosterone in Sertoli cells have been further characterized in my laboratory. These studies suggest that androgen stimulation of Sertoli cells results in the activation of the MAP kinase pathway in Sertoli cells that in turn causes the phosphorylation of the CREB transcription factor on serine 133, a mechanism known to trigger CREB transactivation properties and activate CREB-mediated transcription (Gonzalez and Montminy, 1989). The potential impact of CREB being phosphorylated in response to androgen stimulation is amplified by previous


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studies indicating that phosphorylated CREB in Sertoli cells is essential to support germ cell development and survival (Scobey et al., 2001). CREB is a focal point for numerous signaling pathways and regulatory mechanisms (reviewed in Shaywitz and Greenberg, 1999). Serine 133 in CREB is the target for many activating kinases including cAMP-dependent PKA, Ca2þ/calmodulin-dependent protein kinases (CaM kinases) types I and IV, and the PI3-kinase–activated AKT/PKB kinase, as well as the MSK, p90rsk, and MAPKAP-K2 kinases that are stimulated by MAP kinase pathways (Dash et al., 1991; Deak et al., 1998; Gonzalez and Montminy, 1989; Sheng et al., 1991; Tan et al., 1996; Xing et al., 1996). Once phosphorylated on serine 133, CREB bound to cAMP response element (CRE) motifs (TGACGTCA) in gene promoters is able to associate with the CREB binding protein (CBP) coactivator (Kwok et al., 1994), which facilitates the recruitment of RNA polymerase to the transcription initiation site (Chrivia et al., 1993; Kwok et al., 1994; Meyer and Habener, 1993). Our initial studies indicated that physiological levels of testosterone (10– 250 nM), but not estradiol, rapidly (within 15 minutes) induced the phosphorylation and activation of the Erk MAPK and CREB in Sertoli cells from 15-day-old rats. The kinetics of androgen-induced CREB phosphorylation were assessed using the nonhydrolysable androgen agonist R1881 (100 nM). CREB phosphorylation increased 2.7- to 3.0-fold within 1–15 minutes after R1881 stimulation, and 4.8- to 5.0-fold 1–2 hours after the initial R1881 stimulation (Fig. 4a,b). Erk phosphorylation followed similar kinetics and the MAP kinase pathway inhibitor PD98059 blocked testosterone-mediated induction of CREB phosphorylation, thus supporting the hypothesis that androgen-induced CREB phosphorylation is mediated via the MAP kinase pathway (Fig. 4c). In contrast, the p38 kinase inhibitor (SB 203580) and a PI3-kinase inhibitor (wortmanin) resulted in intermediate or no inhibition, respectively. H-89, an inhibitor of PKA, also reduced P-CREB levels to basal levels, and the blocking of Ca2þ influx into Sertoli cells using the Ca2þ chelator EGTA prevented increased phosphorylation of CREB by testosterone. The inhibition of CREB phosphorylation by EGTA and H-89 is consistent with previous studies showing that elevated [Ca2þ]i levels can stimulate MAP kinase activity through PKA activation of the Ras or Ras-like GTP binding proteins (Enslen et al., 1996; Grewal et al., 2000; Impey et al., 1998; Yao et al., 1998). Erk is known to activate the p90rsk and MSK kinases that can phosphorylate CREB on serine 133 (Deak et al., 1998; Xing et al., 1996). The dynamics of testosterone-induced CREB phosphorylation in Sertoli cells is similar to that observed in depolarized hippocampal pyramidal neurons in response to Ca2þ influx (Dolmetsch et al., 2001; Wu et al., 2001). In these reports, CREB was found to be rapidly and transiently phosphorylated (1–5 minutes) by CaM kinase

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Figure 4 Androgen induces rapid, transient as well as delayed, long-term phosphorylation of CREB and Erk. (a) The kinetics of testosterone-induced phosphorylation of CREB and Erk. Primary Sertoli cells were stimulated with ethanol for 15 minutes (0) or 100 nM R1881 for the times shown. Western analyses were first performed with an antiserum against P-CREB. Fractionation and probing/reprobing of the samples were repeated using antisera, recognizing


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IV, but further phosphorylation of CREB by MAP kinase was delayed until 30–60 minutes after stimulation. Together, these observations raise the possibility that in Sertoli cells CREB is phosphorylated via CaM kinase IV 1–15 minutes after testosterone stimulation but that after 1–2 hours CREB is phosphorylated predominantly due to higher levels of activated MAP kinase. Activation of CREB may result in part from the testosterone-induced increases in [Ca2þ]i levels observed previously (Gorczynska and Handelsman, 1995; Lyng et al., 2000) as blockers of Ca2þ influx into Sertoli cells (2 mM EGTA and 10 mM lanthanum chloride) inhibited phosphorylation of CREB in our studies. Src kinase, which has been shown to be activated after direct interactions with AR (Migliaccio et al., 2000), also contributed to the phosphorylation of CREB as an inhibitor of Src kinase (1 mM PP2) reduced testosterone-induced phosphorylation of CREB to basal levels. Furthermore, Src was found to be phosphorylated (activated) with kinetics similar to that observed for CREB. Notably, Ca2þ influx and Src both can activate the MAPK signaling cascade. Further studies will be required to determine whether Src kinase and Ca2þ-mediated events act in concert or independently to activate MAPK and CREB. Although other receptors may be involved, additional studies indicated that the initial signals responsible for testosterone-induced Erk and CREB phosphorylation likely require classical AR. This hypothesis was supported by our finding that the AR antagonist flutamide inhibited testosterone-mediated phosphorylation of CREB. Furthermore, CREB was not phosphorylated in Sertoli cells lacking AR activity either due to a testicular feminization (tfm) mutation or to RNA interference knockdown of AR expression. Presently, there is no evidence that RSHBG or a GPR30-like receptor are required for the observed nongenomic androgen actions in Sertoli cells because in these cells testosterone does not stimulate the production of cAMP, a hallmark of these receptors. CREB has been linked to the activation of Sertoli cell genes that contribute to germ cell development and survival. The genes potentially regulated by CREB include those encoding the transcription factors c-fos P-Erk or all forms of CREB or Erk. (b) Quantification of P-CREB and P-Erk levels. The relative levels of P-CREB and P-Erk normalized for CREB and Erk expression are provided. No changes in protein expression were observed over 24 hours with vehicle (EtOH) controls (data not shown). (c) MAP kinase, PKA, and Ca2+ influx inhibitors prevent testosteroneinduced CREB phosphorylation. Inhibitors of MEK kinase (PD98059, 50 mM), p38 kinase (SB203580, 10 mM), PI3 kinase (wortmanin, 100 nM), PKA (H-89, 10 mM), Ca2þ influx (EGTA, 2 mM), or vehicle (DMSO) were added to primary rat Sertoli cells before stimulation with EtOH (vehicle), testosterone (T) (100 nM), or forskolin (Forsk) (10 mM) for 15 minutes. Western immunoblot results using an antisera specifically recognizing CREB phosphorylated on serine 133 (P-CREB) are shown. Fractionation and probing of the samples were repeated using an antiserum recognizing all forms of CREB (CREB).

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and C/EBP (Niehof et al., 1997) that may in turn regulate the production of other Sertoli cell factors required by germ cells. The AR is also inducible by CREB (Blok et al., 1992; Lidzey et al., 1993; Mizokami et al., 1994; Sanborn et al., 1991; Verhoven and Cailleau, 1988). Transferrin (Chaudhary and Skinner, 1999; Suire et al., 1995), which is required to transport iron to germ cells, and insulin-like growth factor (IGF-1) (Suwanichkul et al., 1993) are also induced by CREB. Furthermore, CREB stimulates lactate dehydrogenase (LDH-A) (the gene that controls the synthesis of the major fuel source for germ cells) through a CRE within the gene promoter (Short et al., 1994). Finally, the CREB gene promoter is also stimulated by CREB (Walker et al., 1995). The stimulation of CREB-mediated gene expression is thought to require long-term (>20 minutes) elevations in P-CREB (Bito et al., 1996; Liu and Graybiel, 1996; Wu et al., 2001). Therefore, the prolonged stimulation of CREB phosphorylation (up to 2 hours or more) by androgen in Sertoli cells is a significant discovery because of the potential for induction of CREBmediated genes that are required to support spermatogenesis. We have obtained the first evidence that nonclassical androgen actions can result in altered gene expression in Sertoli cells. Specifically, our initial studies suggest that androgen stimulation of primary rat Sertoli cells results in the activation of the CREB-regulated LDH-A and CREB genes. Based on these results and reports of testosterone actions in other cell types, a working model was devised that includes two potential pathways by which testosterone may activate MAP kinase, CREB phosphorylation, and CREB-mediated transcription in Sertoli cells (Fig. 5). In the first pathway, testosterone binds to AR, causing the recruitment and activation of Src, thereby initiating a series of events leading to the activation of the MAP kinase cascade by the Ras G-protein. In the second pathway, testosterone binding to AR causes an increase in [Ca2þ]i that activates numerous potential intermediates that are capable of stimulating Ras or a Ras-like G-protein. Determining the factors that are required for testosterone induction of MAP kinase and CREB activity in Sertoli cells is essential to better understanding testosterone regulation of spermatogenesis and androgen regulation of other target tissues. Previously, CREB was thought to be phosphorylated in Sertoli cells only via cAMP and Ca2þ-mediated signaling pathways initiated by the binding of follicle-stimulating hormone (FSH) to GPCRs on the Sertoli cell membrane (reviewed in Simoni et al., 1997). FSH, one of the few known regulators of the MAP kinase pathway in Sertoli cells, has been shown to regulate Sertoli cell proliferation through the temporal control of MAP kinase (Crepieux et al., 2001). Together, FSH and testosterone hormonal signals provide for maximal spermatozoa production; however, in the absence of FSH, androgen is capable of supporting spermatogenesis at a reduced eYciency

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2. Nongenomic Actions of Androgen in Sertoli Cells T

1 Sertoli cell

T AR SRC

Ca2+

RAS like G-protein

Shc

T

2

AR MAPKKK RAF-1/

T

Ca2+

B-RAF

ADENYLATE CYCLASE

PKC MAP kinase MAPKK pathway

MEK

MAPK

ERK

CaM p38K Pathway cAMP GEFs

cAMP

PKA

p90rsk

MSK

MAPKAP

Nucleus P CREB

CaM Kinase

CREB-Regulated gene

Figure 5 Potential testosterone signaling pathways in Sertoli cells: two potential pathways are proposed for testosterone-induced CREB phosphorylation. In one pathway (left side, 1), testosterone (T) binding to AR allows AR to bind with and activate Src tyrosine kinase, resulting in the stimulation Ras and Raf-1 kinase and the activation of the MAP kinase pathway. In the second pathway (right side, 2), testosterone binding to AR induces Ca2þ influx into Sertoli cells, causing calmodulin (CaM) to stimulate CaM kinase to translocate to the nucleus and transiently phosphorylate CREB within 1 minute. Ca2þ also induces a slower, more persistent pathway in which protein kinase C (PKC), guanine nucleotide exchange factors (GEFs), or PKA stimulates Ras or a Ras-like GTP binding protein, resulting in the activation of the MAP kinase pathway. Both pathways are capable of inducing CREB phosphorylation and CREB-mediated gene expression.

(Sharpe, 1994). The finding that both FSH and testosterone stimulate the MAP kinase pathway suggests that there may be common mechanisms by which the hormones support spermatogenesis. Furthermore, the activation of CREB is a common mechanism for transcriptional control of genes that are essential for spermatogenesis. It is likely that future studies will identify additional testosterone-responsive transcription factors such as Elk-1, myc, fos, and jun in Sertoli cells that together establish the gene expression set points required to maintain spermatogenesis.

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V. Summary Nongenomic actions mediated by androgens have now been described in more than 10 cell types. Some of these cells transduce androgen signals using surface receptors that await final characterization, whereas other cells employ the classical AR. Various second messengers can be activated by androgens, including cAMP, IP3, phospholipase C, DAG, and Ca2þ. Each of these second messengers is capable of activating multiple kinases. One of the most important kinase networks to be regulated by androgens is the MAP kinase cascade. This series of kinase reactions is capable of altering the activity of many transcription factors with important implications for the regulation of gene expression. Because there is evidence that androgen is capable of regulating CREB-mediated gene expression via the MAP kinase pathway, it is now somewhat misleading to characterize androgen actions in Sertoli cells as nongenomic. Instead, it may be more appropriate to label these activities as independent of AR-DNA interactions, or more simply as nonclassical. The nonclassical regulation of gene expression in Sertoli cells is particularly relevant for providing an answer to the paradox of how testosterone can support spermatogenesis yet regulate few genes via AR-promoter interactions. It is expected that with the increasing use of microarray and related technologies, additional AR-regulated genes will be identified. However, the androgen-induced increases in [Ca2þ]i, the activation of Src kinase, and the MAP kinase cascade that have been characterized thus far have the potential to regulate the expression of many more genes than is possible by direct AR-promoter interactions. Thus, it is likely that nonclassical actions of testosterone in Sertoli cells will be found to be a necessary complement to the classical actions that are required to maintain spermatogenesis.

Acknowledgments Work from the author’s laboratory that is cited herein was supported by the NIH grant HD01206. I would like to thank Drs. Steve Hammes, Sergio Onate, and Tony Plant for stimulating discussions and critical reading of this chapter and Andrea Soltes for assistance in preparation of the manuscript.

References Ahola, T. M., Alkio, N., Manninen, T., and Ylikomi, T. (2002). Progestin and G proteincoupled receptor 30 inhibit mitogen-activated protein kinase activity in MCF-7 breast cancer cells. Endocrinology 143, 4620–4626.


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Bagchi, M. K., Tsai, M. J., O’Malley, B. W., and Tsai, S. Y. (1992). Analysis of the mechanism of steroid hormone receptor-dependent gene activation in cell-free systems. Endocr. Rev. 13, 525–535. Bagowski, C. P., Myers, J. W., and Ferrell, J. E., Jr. (2001). The classical progesterone receptor associates with p42 MAPK and is involved in phosphatidylinositol 3-kinase signaling in Xenopus oocytes. J. Biol. Chem. 276, 37708–37714. Benten, W. P. M., Benten, M., Lieberherr, M., Stamm, O., Wrehlke, C., Guo, Z., and Wunderlich, F. (1999a). Testosterone signaling through internalizable surface receptors in androgen receptor-free macrophages. Mol. Biol. Cell 10, 3113–3123. Benten, W. P. M., Liberherr, M., Giese, G., Wrehlke, C., Stamm, O., Sekeris, C. E., Mossman, H., and Wunderlich, F. (1999b). Functional testosterone receptors in plasma membranes of T cells. FASEB J. 13, 123–133. Bito, H., Deisseroth, K., and Tsien, R. W. (1996). CREB phosphorylation and dephosphorylation: A Ca(2þ)- and stimulus duration-dependent switch for hippocampal gene expression. Cell 87, 1203–1214. Blok, L. J., Hoogenbrugge, J. W., Themmen, A. P. N., Baarends, W. M., Post, M., and Grootegoed, A. (1992). Transient down-regulation of androgen receptor messenger ribonucleic acid (mRNA) expression in Sertoli cells by follicle-stimulating hormone is followed by up-regulation of androgen receptor mRNA and protein. Endocrinology 131, 1343–1349. Campbell, C. H., Bulayeva, N., Brown, D. B., Gametchu, B., and Watson, C. S. (2002). Regulation of the membrane estrogen receptor-alpha: Role of cell density, serum, cell passage number, and estradiol. FASEB J. 16, 1917–1927. Cato, A. C., Nestl, A., and Mink, S. (2002). Rapid actions of steroid receptors in cellular signaling pathways. Sci. STKE 2002, (138):RE9. Chambliss, K. L., Yuhanna, I. S., Anderson, R. G., Mendelsohn, M. E., and Shaul, P. W. (2002). ER beta has nongenomic action in caveolae. Mol. Endocrinol. 16, 938–946. Chaudhary, J., and Skinner, M. K. (1999). E-box and cyclic adenosine monophosphate response elements are both required for follicle-stimulating hormone-induced transferrin promoter activation in Sertoli cells. Endocrinology 140, 1262–1271. Cheng, C. Y., Mather, J. P., Byer, A. L., and Bardin, C. W. (1986). Identification of hormonally responsive proteins in primary Sertoli cell culture medium by anion-exchange high performance liquid chromatography. Endocrinology 118, 480–488. Chrivia, J. C., Kwok, R. P., Lamb, N., Hagiwara, M., Montminy, M. R., and Goodman, R. H. (1993). Phosphorylated CREB binds specifically to the nuclear protein CBP. Nature 365, 855–859. Crepieux, P., Marion, S., Martinat, N., Fafeur, V., Vern, Y. L., Kerboeuf, D., Guillou, F., and Reiter, E. (2001). The ERK-dependent signalling is stage-specifically modulated by FSH, during primary Sertoli cell maturation. Oncogene 20, 4696–4709. D’Agostino, A., Mene, P., and Stefanini, M. (1992). Voltage-gated calcium channels in rat Sertoli cells. Biol. Reprod. 46, 414–418. Dash, P. K., Karl, K. A., Colicos, M. A., Prywes, R., and Kandel, E. R. (1991). cAMP response element-binding protein is activated by Ca2þ/calmodulin—as well as cAMP-dependent protein kinase. Proc. Natl. Acad. Sci. USA 88, 5061–5065. Deak, M., Clifton, A. D., Lucocq, L. M., and Alessi, D. R. (1998). Mitogen- and stressactivated protein kinase-1 (MSK1) is directly activated by MAPK and SAPK2/p38, and may mediate activation of CREB. EMBO J. 17, 4426–4441. Deenadayalu, V. P., White, R. E., Stallone, J. N., Gao, X., and Garcia, A. J. (2001). Testosterone relaxes coronary arteries by opening the large-conductance, calcium-activated potassium channel. Am. J. Physiol. Heart Circ. Physiol. 281, H1720–H1727.

48

Walker

Dolmetsch, R. E., Pajvani, U., Fife, K., Spotts, J. M., and Greenberg, M. E. (2001). Signaling to the nucleus by an L-type calcium channel-calmodulin complex through the MAP kinase pathway. Science 294, 333–339. Dunn, J. F., Nisula, B. C., and Rodbard, D. (1981). Transport of steroid hormones: Binding of 21 endogenous steroids to both testosterone-binding globulin and corticosteroid-binding globulin in human plasma. J. Clin. Endocrinol. Metab. 53, 58–68. Enslen, H., Tokumitsu, H., Stork, P. J. S., Davis, R. J., and Soderling, T. R. (1996). Regulation of mitogen-activated protein kinases by a calcium/calmodulin-dependent protein kinase cascade. Proc. Natl. Acad. Sci. USA 93, 10803–10808. Falkenstein, E., Tillmann, H. C., Christ, M., Feuring, M., and Wehling, M. (2000). Multiple actions of steroid hormones—a focus on rapid, nongenomic effects. Pharmacol. Rev. 52, 513–555. Ferrell, J. E., Jr. (1999). Xenopus oocyte maturation: New lessons from a good egg. Bioessays 21, 833–842. Filardo, E. J. (2002). Epidermal growth factor receptor (EGFR) transactivation by estrogen via the G-protein-coupled receptor, GPR30: A novel signaling pathway with potential significance for breast cancer. J. Steroid Biochem. Mol. Biol. 80, 231–238. Filardo, E. J., Quinn, J. A., Bland, K. I., and Frackelton, R. A. (2000). Estrogen-induced activation of Erk-1 and Erk-2 requires the G protein coupled receptor homolog, GPR30, and occurs via transactivation of the epidermal growth factor receptor through release of HB-EGF. Mol. Endocrinol. 14, 1649–1660. Filardo, E. J., Quinn, J. A., Frackelton, A. R., Jr., and Bland, K. I. (2002). Estrogen action via the G protein-coupled receptor, GPR30: Stimulation of adenylyl cyclase and cAMPmediated attenuation of the epidermal growth factor receptor-to-MAPK signaling axis. Mol. Endocrinol. 16, 70–84. Finkbeiner, S., and Greenberg, M. E. (1996). Ca(2þ)-dependent routes to Ras: Mechanisms for neuronal survival, differentiation, and plasticity? Neuron 16, 233–236. Fragale, A., Aguanno, S., Kemp, M., Reeves, M., Price, K., Beattie, R., Craig, P., Volsen, S., Sher, E., and D’Agostino, A. (2000). Identification and cellular localisation of voltageoperated calcium channels in immature rat testis. Mol. Cell. Endocrinol. 162, 25–33. Gonzalez, G., and Montminy, M. R. (1989). Cyclic AMP stimulates somatostatin gene transcription by phosphorylation of CREB at Serine 133. Cell 59, 675–680. Gorczynska, E., and Handelsman, D. J. (1995). Androgens rapidly increase the cytosolic calcium concentration in Sertoli cells. Endocrinology 136, 2052–2059. Grewal, S. S., Fass, D. M., Yao, H., Ellig, C. L., Goodman, R. H., and Stork, P. J. (2000). Calcium and cAMP signals differentially regulate cAMP-responsive element-binding protein function via a Rap1-extracellular signal-regulated kinase pathway. J. Biol. Chem. 275, 34433–34441. Huchon, D., Ozon, R., Fischer, E. H., and Demaille, J. G. (1981). The pure inhibitor of cAMPdependent protein kinase initiates Xenopus laevis meiotic maturation: A 4-step scheme for meiotic maturation. Mol. Cell. Endocrinol. 22, 211–222. Impey, S., Obrietan, K., Wong, S. T., Poser, S., Yano, S., Wayman, G., Deloulme, J. C., Chan, G., and Storm, D. R. (1998). Crosstalk between ERK and PKA is required for Ca2þ stimulation of CREB-dependent transcription and ERK nuclear translocation. Neuron 21, 869–883. Kim, H. P., Lee, J. Y., Jeong, J. K., Bae, S. W., Lee, H. K., and Jo, I. (1999). Nongenomic stimulation of nitric oxide release by estrogen is mediated by estrogen receptor alpha localized in caveolae. Biochem. Biophys. Res. Commun. 263, 257–262. Kokontis, J., and Liao, S. (1999). Molecular action of androgen in the normal and neoplastic prostate. Vitam. Horm. 55, 219–307.


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Kousteni, S., Bellido, T., Plotkin, L. I., O’Brien, C. A., Bodenner, D. L., Han, L., Han, K., DiGregio, G. B., Katzenellenbogen, J. A., Katzenellenbogen, B. S., Roberson, P. K., Weinstein, R. S., Jilka, R. L., and Manolagas, S. C. (2001). Nongenotropic, sex-nonspecific signaling through the estrogen or androgen receptors: Dissociation from transcriptional activity. Cell 104, 719–730. Kousteni, S., Chen, J. R., Bellido, T., Han, L., Ali, A. A., O’Brien, C. A., Plotkin, L., Fu, Q., Mancino, A. T., Wen, Y., Vertino, A. M., Powers, C. C., Stewart, S. A., Ebert, R., Parfitt, A. M., Weinstein, R. S., Jilka, R. L., and Manolagas, S. C. (2002). Reversal of bone loss in mice by nongenotropic signaling of sex steroids. Science 298, 843–846. Kwok, R. P. S., Lundbland, J. R., Chrivia, J. C., Richards, J. P., Bachinger, H. P., Brennan, R. G., Roberts, S. G. E., Green, M. R., and Goodman, R. H. (1994). Nuclear protein CBP is a coactivator for the transcription factor CREB. Nature 370, 223–226. Leung, G. P., Cheng-Chew, S. B., and Wong, P. Y. (2001). Nongenomic effect of testosterone on chloride secretion in cultured rat efferent duct epithelia. Am. J. Physiol. Cell. Physiol. 280, C1160–C1167. Lidzey, J., Grossmann, M., Kumar, M. V., and Tindall, D. J. (1993). Regulation of the 50 -flanking region of the mouse androgen gene by cAMP and androgen. Mol. Endocrinol. 7, 1530–1540. Lieberherr, M., and Grosse, B. (1994). Androgens increase intracellular calcium concentration and inositol 1,4,5-triphosphate and diacylglycerol formation via a pertussis toxin-sensitive G-protein. J. Biol. Chem. 269, 7217–7223. Lieberherr, M., Grosse, B., Kachkache, M., and Balsan, S. (1993). Cell signaling and estrogens in female rat osteoblasts: A possible involvement of unconventional nonnuclear receptors. J. Bone Miner. Res. 8, 1365–1376. Lim, K., Yoo, J. H., Kim, K. Y., Kweon, G. R., Kwak, S. T., and Hwang, B. D. (1994). Testosterone regulation of proto-oncogene c-myc expression in primary Sertoli cell cultures from prepubertal rats. J. Androl. 15, 543–550. Lindsey, J. S., and Wilkinson, M. F. (1996). Pem: A testosterone- and LH-regulated homeobox gene expressed in mouse Sertoli cells and epididymus. Dev. Biol. 179, 471–484. Liu, F. C., and Graybiel, A. M. (1996). Spatiotemporal dynamics of CREB phosphorylation: Transient versus sustained phosphorylation in the developing striatum. Neuron 17, 1133–1144. Losel, R., and Wehling, M. (2003). Nongenomic actions of steroid hormones. Nat. Rev. Mol. Cell. Biol. 4, 46–55. Lu, M. L., Schneider, M. C., Zheng, Y., Zhang, X., and Richie, J. P. (2001). Caveolin-1 interacts with androgen receptor: A positive modulator of androgen receptor mediated transactivation. J. Biol. Chem. 276, 13442–13451. Lutz, L. B., Cole, L. M., Gupta, M. K., Kwist, K. W., Auchus, R. J., and Hammes, S. R. (2001). Evidence that androgens are the primary steroids produced by Xenopus laevis ovaries and may signal through the classical androgen receptor to promote oocyte maturation. Proc. Natl. Acad. Sci. USA 98, 13728–13733. Lutz, L. B., Kim, B., Jahani, D., and Hammes, S. R. (2000). G protein beta gamma subunits inhibit nongenomic progesterone-induced signaling and maturation in Xenopus laevis oocytes: Evidence for a release of inhibition mechanism for cell cycle progression. J. Biol. Chem. 275, 41512–41520. Lyng, F. M., Jones, G. R., and Rommerts, F. F. G. (2000). Rapid androgen actions on calcium signaling in rat Sertoli cells and two human prostatic cell lines: Similar biphasic responses between 1 picomolar and 100 nanomolar concentrations. Biol. Reprod. 63, 736–747. Machelon, V., Nome, F., Grosse, B., and Lieberherr, M. (1996). Progesterone triggers rapid transmembrane calcium influx and/or calcium mobilization from endoplasmic reticulum, via

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a pertussis-insensitive G-protein in granulosa cells in relation to luteinization process. J. Cell. Biochem. 61, 619–628. Machelon, V., Nome, F., and Tesarik, J. (1998). Nongenomic effects of androstenedione on human granulosa luteinizing cells. J. Clin. Endocrinol. Metab. 83, 263–269. Maller, J. L. (2001). The elusive progesterone receptor in Xenopus oocytes. Proc. Natl. Acad. Sci. USA 98, 8–10. Maller, J. L., Butcher, F. R., and Krebs, E. G. (1979). Early effect of progesterone on levels of cyclic adenosine 30 :50 -monophosphate in Xenopus oocytes. J. Biol. Chem. 254, 579–582. Maller, J. L., and Krebs, E. G. (1977). Progesterone-stimulated meiotic cell division in Xenopus oocytes: Induction by regulatory subunit and inhibition by catalytic subunit of adenosine 30 :50 -monophosphate-dependent protein kinase. J. Biol. Chem. 252, 1712–1718. Maller, J. L., and Krebs, E. G. (1980). Regulation of oocyte maturation. Curr. Top. Cell. Regul. 16, 271–311. Masui, Y., and Markert, C. L. (1971). Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes. J. Exp. Zool. 177, 129–145. Mendel, C. M. (1989). The free hormone hypothesis: A physiologically based mathematical model. Endocr. Rev. 10, 232–274. Meyer, T. E., and Habener, J. F. (1993). Cyclic AMP response element binding protein CREB and related transcription-activating DNA binding proteins. Endocr. Rev. 14, 269–290. Migliaccio, A., Castoria, G., Di Domenico, M., de Falco, A., Barone, M. V., Ametrano, D., Zannini, M. S., Abbondanza, C., and Auricchio, F. (2000). Steroid-induced androgen receptor-oestradial receptor -Src complex triggers prostate cancer cell proliferation. EMBO J. 20, 5406–5417. Mizokami, A., Yeh, S. Y., and Chang, C. (1994). Identification of 30 -50 -cyclic adenosine monophosphate response element and other cis-acting elements in the human androgen receptor gene promoter. Mol. Endocrinol. 8, 77–88. Nakhla, A. M., Khan, M. S., and Rosner, W. (1990). Biologically active steroids activate receptor-bound human sex hormone-binding globulin to cause LNCaP cells to accumulate adenosine 30 ,50 -monophosphate. J. Clin. Endocrinol. Metab. 71, 398–404. Nakhla, A. M., Leonard, J., Hryb, D. J., and Rosner, W. (1999). Sex hormone-binding globulin receptor signal transduction proceeds via a G protein. Steroids 64, 213–216. Niehof, M., Manns, M. P., and Trautwein, C. (1997). CREB controls LAP/C/EBP transcription. Mol. Cell. Biol. 17, 3600–3613. Norfleet, A. M., Thomas, M. L., Gametchu, B., and Watson, C. S. (1999). Estrogen receptoralpha detected on the plasma membrane of aldehyde-fixed GH3/B6/F10 rat pituitary tumor cells by enzyme-linked immunocytochemistry. Endocrinology 140, 3805–3814. Okamoto, T., Schlegel, A., Scherer, P. E., and Lisanti, M. P. (1998). Caveolins, a family of scaffolding proteins for organizing ‘‘preassembled signaling complexes’’ at the plasma membrane. J. Biol. Chem. 273, 5419–5422. Onate, S. A. (2001). Steroid receptor and ligand-dependent interaction with coactivator proteins. In ‘‘Methods Mol Biol’’ (P. N. MacDonald, Ed.), pp. 199–209. Humana Press, Inc., Towota, NJ. Pappas, T. C., Gametchu, B., and Watson, C. S. (1995). Membrane estrogen receptors identified by multiple antibody labeling and impeded-ligand binding. FASEB J. 9, 404–410. Pearson, G., Robinson, F., Gibson, T. B., Xu, B. E., Karandikar, M., Berman, K., and Cobb, M. H. (2001). Mitogen-activated protein (MAP) kinase pathways: Regulation and physiological functions. Endocr. Rev. 22, 153–183. Peterziel, H., Mink, S., Schonert, A., Becker, M. K. H., and Cato, A. C. B. (1999). Rapid signaling by androgen receptor in prostate cancer cells. Oncogene 18, 6322–6329. Plant, T. M., and Marshall, G. R. (2001). The functional significance of FSH in spermatogenesis and the control of its secretion in male primates. Endocr. Rev. 22, 764–786.


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Pratt, W. B., Czar, M. J., Stancato, L. F., and Owens, J. K. (1993). The hsp56 immunophilin component of steroid receptor heterocomplexes: Could this be the elusive nuclear localization signal-binding protein? J. Steroid Biochem. Mol. Biol. 46, 269–279. Pratt, W. B., and Toft, D. O. (1997). Steroid receptor interactions with heat shock proteins and immunophilin chaperones. Endocr. Rev. 18, 306–360. Quigley, C. A., De Bellis, A., Marschke, K. B., El-awady, M. K., Wilson, E. M., and French, F. S. (1995). Androgen receptor defects: Historical, clinical, and molecular perspectives. Endocr. Rev. 16, 271–321. Ramirez, V. D., Zheng, J., and Siddique, K. M. (1996). Membrane receptors for estrogen, progesterone, and testosterone in the rat brain: Fantasy or reality. Cell. Mol. Neurobiol. 16, 175–198. Razandi, M., Oh, P., Pedram, A., Schnitzer, J., and Levin, E. R. (2002). ERs associate with and regulate the production of caveolin: Implications for signaling and cellular actions. Mol. Endocrinol. 16, 100–115. Razandi, M., Pedram, A., Greene, G. L., and Levin, E. R. (1999). Cell membrane and nuclear estrogen receptors (ERs) originate from a single transcript: Studies of ER and ER expressed in Chinese hamster ovary cells. Mol. Endocrinol. 13, 307–319. Razandi, M., Pedram, A., Park, S. T., and Levin, E. R. (2003). Proximal events in signaling by plasma membrane estrogen receptors. J. Biol. Chem. 278, 2701–2712. Revelli, A., Massobrio, M., and Tesarik, J. (1998). Nongenomic actions of steroid hormones in reproductive tissues. Endocr. Rev. 19, 3–17. Roberts, K., and Griswold, M. D. (1989). Testosterone induction of cellular proteins in cultured Sertoli cells from hypophysectomized rats and rats of different ages. Endocrinology 125, 1174–1179. Rommerts, F. F. G. (1988). How much androgen is required for maintenance of spermatogenesis? J. Endocrinol. 116, 7–9. Rosner, W., Hryb, D. J., Khan, M. S., Nakhla, A. M., and Romas, N. A. (1999). Sex hormonebinding globulin mediates steroid hormone signal transduction at the plasma membrane. J. Steroid Biochem. Mol. Biol. 69, 481–485. Rusanescu, G., Qi, H., Thomas, S. M., Brugge, J. S., and Halegoua, S. (1995). Calcium influx induces neurite growth through a src-ras signaling cassette. Neuron 15, 1415–1425. Russell, K. S., Haynes, M. P., Sinha, D., Clerisme, E., and Bender, J. R. (2000). Human vascular endothelial cells contain membrane binding sites for estradiol, which mediate rapid intracellular signaling. Proc. Natl. Acad. Sci. USA 97, 5930–5935. Sadler, S. E., Bower, M. A., and Maller, J. L. (1985). Studies of a plasma membrane steroid receptor in Xenopus oocytes using the synthetic progestin RU 486. J. Steroid Biochem. 22, 419–426. Sadler, S. E., and Maller, J. L. (1985). Inhibition of Xenopus oocyte adenylate cyclase by progesterone: A novel mechanism of action. Adv. Cyclic Nucleotide Protein Phosphorylation Res. 19, 179–194. Sanborn, B. M., Caston, L. A., Chang, C., Liao, S., Speller, R., Porter, L. D., and Ku, C. Y. (1991). Regulation of androgen receptor mRNA in rat Sertoli and peritubular cells. Biol. Reprod. 45, 634–641. Schlegel, A., Wang, C., Katzenellenbogen, B. S., Pestell, R. G., and Lisanti, M. P. (1999). Caveolin-1 potentiates estrogen receptor alpha (ERalpha) signaling. Caveolin-1 drives ligand-independent nuclear translocation and activation of ERalpha. J. Biol. Chem. 274, 33551–33556. Scobey, M. J., Bertera, S., Somers, J. P., Watkins, S. C., Zeleznik, A. J., and Walker, W. H. (2001). Delivery of a cyclic adenosine 30 ,50 -monophosphate response element binding protein (CREB) to seminiferous tubules results in impaired spermatogenesis. Endocrinology 142, 948–954.

52

Walker

Shang, Y., Hu, X., DiRenzo, J., Lazar, M. A., and Brown, M. (2000). Cofactor dynamics and sufficiency in estrogen receptor-regulated transcription. Cell 103, 843–852. Shang, Y., Myers, M., and Brown, M. (2002). Formation of the androgen receptor transcription complex. Mol. Cell 9, 601–610. Sharpe, R. M. (1994). Regulation of spermatogenesis. In ‘‘The Physiology of Reproduction’’ (E. Knobil and J. D. Neil, Eds.), pp. 1363–1434. Raven Press, New York. Shaywitz, A. J., and Greenberg, M. E. (1999). CREB: A stimulus-induced transcription factor activated by a diverse array of extracellular signals. Annu. Rev. Biochem. 68, 821–861. Sheng, M., Thompson, M. A., and Greenberg, M. E. (1991). CREB: A Ca2þ-regulated transcription factor phosphorylated by calmodulin-dependent kinases. Science 252, 1427–1430. Sheng, Y., Tiberi, M., Booth, R. A., Ma, C., and Liu, X. J. (2001). Regulation of Xenopus oocyte meiosis arrest by G protein beta gamma subunits. Curr. Biol. 11, 405–416. Short, M. L., Huang, D., Milkowski, D. M., Short, S., Kunstman, K., Soong, C. J., Chung, K. C., and Jungmann, R. A. (1994). Analysis of the rat lactate dehydrogenase A subunit gene promoter/regulatory region. Biochem. J. 304, 391–398. Silva, F. R. M. B., Leite, L. D., and Wassermann, G. F. (2002). Rapid signal transduction in Sertoli cells. Eur. J. Endocrinol. 147, 425–433. Simoni, M., Gromoli, J., and Niesclag, E. (1997). The follicle-stimulating hormone receptor: Biochemistry, molecular biology, physiology, and pathophysiology. Endocr. Rev. 18, 739–773. Skinner, M. K. (1991). Cell-cell interactions in the testis. Endocr. Rev. 12, 45–77. Smith, L. D., and Ecker, R. E. (1971). The interaction of steroids with Rana pipiens oocytes in the induction of maturation. Dev. Biol. 25, 232–247. Song, R. X. D., McPherson, R. A., Adam, L., Bao, Y., Shupnik, M., Kumar, R., and Saten, R. J. (2002). Linkage of rapid estrogen action to MAPK activation by ER-Shc association and Shc pathway activation. Mol. Endocrinol. 16, 116–127. Steinsapir, J., Socci, R., and Reinach, P. (1991). Effects of androgen on intracellular calcium of LNCaP cells. Biochem. Biophys. Res. Comm. 179, 90–96. Stevis, P. E., Deecher, D. C., Suhadolnik, L., Mallis, L. M., and Frail, D. E. (1999). Differential effects of estradiol and estradiol-BSA conjugates. Endocrinology 140, 5455–5458. Suire, S., Fontaine, I., and Guillou, F. (1995). Follicle stimulating hormone (FSH) stimulates transferrin gene transcription in rat Sertoli cells: cis and trans-acting elements involved in FSH action via cyclic adenosine 30 ,50 -monophosphate on the transferrin gene. Mol. Endocrinol. 9, 756–766. Suwanichkul, A., DePaolis, L. A., Lee, P. D., and Powell, D. R. (1993). Identification of a promoter element which participates in cAMP-stimulated expression of human insulin-like growth factor-binding protein-1. J. Biol. Chem. 268, 9730–9736. Tan, Y., Rouse, J., Zhang, A., Cariati, S., Cohen, P., and Comb, M. J. (1996). FGF and stress regulate CREB and ATF-1 via a pathway involving p38 MAP kinase and MAPKAP kinase-2. EMBO J. 15, 4629–4642. Towle, A. C., and Sze, P. Y. (1983). Steroid binding to synaptic plasma membrane: Differential binding of glucocorticoids and gonadal steroids. J. Steroid Biochem. 18, 135–143. Tsai, M. J., and O’Malley, B. W. (1994). Molecular mechanisms of action of steroid/thyroid receptor superfamily members. Annu. Rev. Biochem. 63, 451–486. Uittenbogaard, A., Ying, Y., and Smart, E. J. (1998). Characterization of a cytosolic heat-shock protein-caveolin chaperone complex: Involvement in cholesterol trafficking. J. Biol. Chem. 273, 6525–6532. Veldscholte, J., Berrevoets, C. A., Ris-Stalpers, C., Kuiper, G. G., Jenster, G., Trapman, J., Brinkmann, A. O., and Mulder, E. (1992). The androgen receptor in LNCaP cells contain a


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mutation in the ligand binding domain which affects steroid binding characteristics and response to antiandrogens. J. Steroid Biochem. Mol. Biol. 41, 665–669. Vendola, K. A., Zhou, J., Adesanya, O. O., Weil, S. J., and Bondy, C. A. (1998). Androgens stimulate early stages of follicular growth in the primate ovary. J. Clin. Invest. 101, 2622–2629. Vendola, K., Zhou, J., Wang, J., and Bondy, C. A. (1999a). Androgens promote insulin-like growth factor-I and insulin-like growth factor-I receptor gene expression in the primate ovary. Hum. Reprod. 14, 2328–2332. Vendola, K., Zhou, J., Wang, J., Famuyiwa, O. A., Bievre, M., and Bondy, C. A. (1999b). Androgens promote oocyte insulin-like growth factor I expression and initiation of follicle development in the primate ovary. Biol. Reprod. 61, 353–357. Verhoven, G., and Cailleau, J. (1988). Follicle-stimulating hormone and androgens increase the concentration of the androgen receptor in Sertoli cells. Endocrinology 122, 1541–1550. Walker, W. H., Fucci, L., and Habener, J. F. (1995). Expression of the gene encoding transcription factor adenosine 30 ,50 -monophosphate (cAMP) response element-binding protein: Regulation by follicle-stimulating hormone-induced cAMP signaling in primary rat Sertoli cells. Endocrinology 136, 3534–3545. Watson, C. S., Campbell, C. H., and Gametchu, B. (2002). The dynamic and elusive membrane estrogen receptor-alpha. Steroids 67, 429–437. Wehling, M. (1997). Specific, nongenomic actions of steroid hormones. Annu. Rev. Physiol. 59, 365–393. Weil, S., Vendola, K., Zhou, J., and Bondy, C. A. (1999). Androgen and follicle-stimulating hormone interactions in primate ovarian follicle development. J. Clin. Endocrinol. Metab. 84, 2951–2956. Wong, C. W., McNally, C., Nickbarg, E., Komm, B. S., and Cheskis, B. J. (2002). Estrogen receptor-interacting protein that modulates its nongenomic activity-crosstalk with Src/Erk phosphorylation cascade. Proc. Natl. Acad. Sci. USA 99, 14783–14788. Wu, G. Y., Deisseroth, K., and Tsien, R. W. (2001). Activity-dependent CREB phosphorylation: Convergence of a fast, sensitive calmodulin kinase pathway and a slow, less sensitive mitogen-activated protein kinase pathway. Proc. Natl. Acad. Sci. USA 98, 2808–2813. Wunderlich, F., Benten, W. P., Lieberherr, M., Guo, Z., Stamm, O., Wrehlke, C., Sekeris, C. E., and Mossmann, H. (2002). Testosterone signaling in T cells and macrophages. Steroids 67, 535–538. Xing, J., Ginty, D. D., and Greenberg, M. (1996). Coupling of the RAS-MAPK pathway to gene activation by RSK2, a growth factor-regulated CREB kinase. Science 273, 959–963. Yang, W. H., Lutz, L. B., and Hammes, S. R. (2003). Xenopus laevis ovarian CYP17 is a highly potent enzyme expressed exclusively in oocytes: Evidence that oocytes play a critical role in xenopus ovarian androgen production. J. Biol. Chem. 278, 9552–9559. Yao, H., York, R. D., Misra-Press, A., Carr, D. W., and Stork, P. J. S. (1998). The cyclic adenosine monophosphate-dependent protein kinase (PKA) is required for the sustained activation of mitogen-activated kinases and gene expression by nerve growth factor. J. Biol. Chem. 273, 8240–8247. Zhu, X., Li, H., Liu, J. P., and Funder, J. W. (1999). Androgen stimulates mitogen-activated protein kinase in human breast cells. Mol. Cell. Endocrinol. 152, 199–206.

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Regulation of Chromatin Structure and Gene Activity by Poly(ADP-Ribose) Polymerases Alexei Tulin, Yurii Chinenov*, and Allan Spradling HHMI Laboratories, Carnegie Institution of Washington, Baltimore, Maryland 21210 *HHMI Laboratories, University of Michigan Medical Center, Ann Arbor, Michigan 48109 I. Introduction II. Background A. Poly(ADP-Ribose) Polymer Metabolism B. The Poly(ADP-Ribose) Polymerases Protein Family C. Evolutionary Relationships III. Mechanism of Action A. Poly(ADP-Ribose) Polymerase is Widely Distributed along Chromosomes IV. Poly(ADP-Ribose) Polymerase and Transcription A. Immunity Genes B. p53 C. Heat Shock Genes D. Steroid Hormone-Dependent Genes E. rDNA Genes and Nucleoli V. Multiple Routes to Poly(ADP-Ribose) Polymerase Activation VI. Specificity of Transcriptional Activation VII. Poly(ADP-Ribose) Polymerase and Other Chromatin Processes A. Heterochromatin Condensation B. Centromere and Centrosome Activity C. Episomal Chromosomes VIII. Concluding Thoughts Acknowledgments References

Poly(ADP-ribose) polymerase 1 (PARP1) is an abundant nuclear protein that plays an important role in repairing DNA and responding to infection. Here we review recent evidence that PARP1 and related proteins also carry out crucial functions regulating genes during normal development. Genetic studies in mammals and Drosophila have implied that PARPs mediate rapid responses to environmental stimuli, including infection, stress, hormones, and growth signals. In addition, these polymerases may control fundamental processes that diVerentially mold and remodel chromatin within the many cell types of a developing embryo. We discuss a unified mechanism of PARP action during DNA repair, gene transcription, and chromatin modulation. ß 2003, Elsevier Inc. Current Topics in Developmental Biology, Vol. 56 Copyright 2003, Elsevier Inc. All rights reserved. 0070-2153/03 $35.00

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I. Introduction The first evidence that cells contain a polymerase that synthesizes long polymers of poly(ADP-ribose) (pADPr) in vivo from NAD was reported by Chambon et al. (1963). A variety of nuclear proteins including histones, RNA polymerase, transcription factors, and poly(ADP-ribose) polymerase (PARP) itself are covalently modified by this activity, which was found to increase more than 100-fold in response to genotoxic stress. During most of the ensuing decades, research focused largely on the roles played by PARP (and the enzymes pADPr glycohydrolase [PARG] and pADPr lyase that degrade ADP-ribose polymers) in response to DNA damage and related phenomena (reviewed in D’Amours et al., 1999; de Murcia and Shall, 2000; Zeigler and Oei, 2001). Genetic studies (Shieh et al., 1998) and whole genome sequencing revealed that virtually all metazoan organisms contain multiple genes encoding PARP-related proteins. These include proteins such as PARP1, PARP2, PARP3, tankyrase, vault PARP (vPARP) (mammals), and PARP-e (Drosophila). With the discovery of multiple PARPs, awareness has grown that the residual PARP activity in undamaged cells does not simply represent background repair processes but rather that poly(ADP-ribose) modifications play important roles in telomere maintenance (Smith and de Lange, 2000), cell cycle checkpoints (see Pleschke et al., 2000), apoptosis (reviewed in Chiarugi and Moskowitz, 2002), transcription (see de Murcia and Shall, 2000), and even in certain cytoplasmic events (Arnold and Grune, 2002; Kickhoefer et al., 1999; Mossink et al., 2002). Genetic studies of PARP-deficient mice (Wang et al., 1997) and Drosophila (Tulin and Spradling, 2003; Tulin et al., 2002; see Pirrotta, 2003) have greatly increased our understanding of these non-canonical PARP functions. Today, our view of the PARP family has reached a turning point; it is becoming clear that these abundant and ubiquitous proteins regulate fundamental aspects of chromosome and gene activity. In this review, we focus on how PARP functions to mediate chromatin structure and transcription. We discuss a unified mechanism for these roles and for the proposed action of PARP during DNA repair. First, however, we briefly review some basic information about PARP genes and proteins.

II. Background A. Poly(ADP-Ribose) Polymer Metabolism The basic enzymatic reactions catalyzed by PARP involve transferring ADP-ribose from NAD to either a protein acceptor or to an existing ADPribose chain (Fig. 1). One of the most remarkable aspects of PARP from a

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Figure 1 ADP-ribose metabolism. The three major types of enzymatic activity of PARP proteins (green) are shown. (a) Initiation includes recognizing a protein acceptor (grey) containing a glutamic acid (Glu) and transferring an ADP-ribose moiety from NAD (A ¼ adenine, N ¼ nicotinamide). Phosphate groups are dark circles. (b) Elongation involves PARP binding the pADPr chain and using the 20 OH of the terminal ribose to accept the next ADP-ribose moiety from NAD. (c) Branching involves PARP binding the pADPr chain and using the 20 OH group of a nonterminal ribose to accept the next ADP-ribose moiety from NAD. (d) pADPr catabolism PARG (poly[ADP-ribose] glycohydrolase) cleaves internal 100 –20 glycosyl bonds in pADPr. The enzymes pADPr lyase (Oka et al., 1984) and pADPr phosphodiesterase (Futai et al., 1968) target diVerent bonds in pADPr as indicated.

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biochemical perspective is that it is capable of three distinct activities, an unprecedented flexibility for a polymerase. The target is most commonly the COOH residue of a glutamic acid (Glu), but modification of other amino acids may occasionally occur. The first reaction is chain initiation (Fig. 1). Mono (ADP-ribosyl) transferases (e.g., cholera toxin) are limited to this activity. The second reaction, linear chain elongation (Fig. 2), requires that PARP recognize a diVerent target, the terminal ribose 20 hydroxyl group of a preexisting pADPr polymer. Glu 988, a completely conserved amino acid within the catalytic domain, appears to be critical to the elongation reaction (Ruf et al., 1998). The average chain length synthesized varies between PARP family members and under diVerent conditions. PARP1-type proteins synthesize the longest chains (averaging 80 or more residues), PARP2s are intermediate, and tankyrases exhibit the lowest processivity, generating chains containing only about 20 ADPr moieties (Rippmann et al., 2002).

Figure 2 Structure of poly(ADP-ribose) polymerases. (a) The domain structure of PARP protein family members. Letters A–G correspond to recognized subdomains of PARP1. A–C ¼ DNA binding domain; D ¼ automodification domain; E–G ¼ catalytic domains. Homologous regions in other PARP family proteins are indicated by the same color. fI and fII ¼ PARP1/ LigIII type Zn fingers; f ¼ DNA binding Zn finger in tiPARP; NLS ¼ nuclear location signal; BRCT ¼ BRCT domain; PS ¼ PARP signature (NAD binding site). The putative DNAbinding domain of PARP2 is indicated by the violet box. The domains labeled: WWE (brown), SAM (blue), Ankyrin repeats (black dots), and von Willebrand A (red) are putative protein– protein interaction domains (see text). Arrows above indicate sites of PARP protein cleavage during apoptosis in mammals (M) and in Drosophila (Dr) (see panel B). (b) Apoptotic cleavage of Drosophila PARP1 and PARP-e at a noncanonical site. A Western blot of proteins extracted from second instar larvae expressing PARP-e–GFP (left) or PARPI–DsRed (right) that had not ( ) or had (þ) been pretreated for 20 minutes with 10 mM methyl methane sulfonate (MMS) to elevate levels of cellular apoptosis is shown. Probing the blot with antibodies against GFP or DsRed detected apoptosis-elevated cleavage of a specific C-terminal fragment at the junction of domains D and E (arrow in panel A). (See Color Insert.)


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The third reaction is chain branching (Fig. 1), an activity that is confined to PARP1-like family members (Rippmann et al., 2003). The next monomer is added to a nonterminal rather than a terminal ribose 20 site, creating a bifurcation in the growing chain that can be elongated further. The binding properties of the acceptor site suggest a possible mechanism for branch induction (Ruf et al., 1998). The fact that PARP1-generated chains are branched (on the order of one branch per 20 residues) may give these polymers novel protein binding characteristics that contribute to PARP function. pADPr levels in vivo are determined by the relative rates of its degradation and synthesis (reviewed by Davidovic et al., 2001). A substantial amount of pADPr cleaving activity is found in cell extracts (Wielckens et al., 1982), and three enzymes, pADPr glycohydrolase (PARG), pADPr lyase, and pADPr phosphodiesterase, have been characterized. The relative accessibility and activity of pADPr polymer degradative enzymes may itself be subject to regulation. However, at present, much more is known about the synthesis of poly(ADP-ribose) adducts than about their turnover. PARG makes the largest contribution to pADPr catabolic activity (see Fig. 1). This evolutionarily conserved protein hydrolyzes the 100 -20 bond in pADPr (Miwa and Sigimura, 1971; Ueda et al., 1972). The Drosophila genome has a single gene encoding a PARG homologue (Adams et al., 2000). As shown in Fig. 2, PARG is capable of removing a protein-linked pADPr chain down to the initial residue. pADPr phosphodiesterase (Futai et al., 1968) and lyase (Oka et al., 1984) cleave diVerent bonds (Fig. 2). Presumably lyase is responsible for removing the initial residue from modified proteins. At the present time, little is known about the gene or protein encoding this activity, however.

B. The Poly(ADP-Ribose) Polymerases Protein Family The most abundant form of PARP in metazoan cells is typified by the mammalian PARP1 protein (see Fig. 2). Three major domains revealed by partial protease cleavage mediate DNA binding, automodification, or catalytic activity (Kameshita et al., 1984). Further study separates these regions into subdomains (labeled A–G in Fig. 2) that appear to reflect real functional boundaries. During apoptosis, caspase 3 cleaves PARP1 between domains B and C (D’Amours et al., 1998; Lazebnik et al., 1994), whereas under similar conditions we find that Drosophila PARP-I is digested between domains D and E (Fig. 2b). The DNA-binding domain (A) plays a critical role during DNA repair. Its two Zn fingers (fI and fII) have been evolutionarily conserved in PARP1 proteins and in DNA ligase III enzymes. The second finger specifically

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recognizes and binds DNA nicks, whereas the first finger stabilizes this interaction. Domain C in mammalian PARP1 has a predicted helix-turn-helix motif, but it is not known if it binds DNA. Between domains B and C lies a bipartite nuclear location signal (NLS). The DNA or chromatin aYnity of some PARP family members does not depend on the Zn finger domain. For example, plant PARP2 proteins (Babiychuk et al., 1998) contain SAP domains (Fig. 2, dark blue box), a DNA-binding domain shared with many other proteins involved in chromosome organization (Aravind and Koonin, 2000). Mammalian PARP2 lacks a conserved DNA binding domain but like PARP1 is activated by DNA damage (Ame´ et al., 1999). In contrast, tankyrase has a SAM domain linked to ankyrin-like repeats that mediates its binding to the telomeric DNA-binding protein TRF-1 (Smith et al., 1998). The automodification domain (D) controls PARP dimerization (Buki et al., 1995) and serves as a major means of regulation. PARP is active as a dimer (Mendoza-Alvarez and Alvarez-Gonzalez, 1993), but ADP-ribose modification of region D causes dimers to dissociate and lose activity. Within domain D lies a BRCT domain, a protein interaction motif found in a variety of DNA damage-responsive proteins (Bork et al., 1997; see Fig. 2), which in Drosophila overlaps with a putative leucine zipper (Uchida et al., 1993). A cluster of 14–28 Glu residues located near the center of the automodification domain serves as a major acceptor of ADP-ribose. The fact that this Glu track is the main target of PARP catalytic activity within the cell is probably due to its location near the catalytic site (Kameshita et al., 1986) in PARP dimers (Buki et al., 1995). The negative feedback loop mediated by the automodification domain limits the time during which PARP molecules can remain active. BRCT domains are also present in vault PARP (V-PARP) and in the PARP2/3-like proteins of Neurospora crassa and Dictyostelium discoideum, but it is not known if these proteins undergo a similar autoregulatory mechanism. Isoforms of PARP that lack the automodification domain are produced in some cells by diVerential splicing (Kawamura et al., 1998), but their function is currently unknown. The catalytic domain of PARP lies at the C-terminus and is the most evolutionally conservative part of protein (Fig. 2, domains E, F, and G). It includes a characteristic 50 amino acid segment within domain G (positions 859–908 in human PARP1) known as the ‘‘PARP signature (PS)’’ which corresponds to the NAD-binding site. The PARP catalytic region is traditionally subdivided into regulatory (F) and catalytic (G) domains. The G domain alone is capable of weak PARP enzymatic function. However, the activity of the regulatory and catalytic domains together is 500-fold greater (Kameshita et al., 1984; Kameshita et al., 1986). Whether the F domain actually regulates PARP activity in vivo is unclear, however. Tankyrases combine ankyin repeats, a SAM-domain responsible for protein–protein interactions and the PARP-catalytic domain without its associated regulatory domain.


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Drosophila produces by diVerential splicing a catalytically inactive regulatory PARP isoform, PARP-e, that retains subdomain E but lacks both subdomains F and G (Tulin et al., 2002). Almost nothing is known about the protein domains that mediate the cytoplasmic activities of PARP proteins. (v-PARP is found primarily within cytoplasmic ‘‘vault’’ particles, which are poorly understood RNP complexes of unknown function (Kickhoefer et al., 1999). vPARP is one of three abundant proteins within vault particles, but the basis of its association is not known. Although best known for its action at telomeres, some tankyrase is found in the cytoplasm in association with the Golgi apparatus and nuclear pore complexes (Kaminker et al., 2001). However, protein domains important for these aYnities have not been reported.

C. Evolutionary Relationships An evolutionary tree of the PARP protein family is shown in Fig. 3, where related subfamilies share colored shading. Sequences of PARP-related proteins were extracted from the nonredundant protein database using an iterative search algorithm implemented in PROBE (Neuwald et al., 1997). The catalytic domains of PARP-related proteins were realigned with ClustalW with minimal manual editing (Thompson et al., 1994), and then the neighbor-joining tree was built (Kumar, 2001; Yang, 1997). The domain structure of each protein group is summarized at the side. The ancestors of the PARP family appear within the Archaea, like many other proteins involved in DNA and chromosome metabolism. For example, a protein with PARP activity has been purified and characterized from the thermophilic species Sulfolobus solfataricus (Faraone-Mennella et al., 1998). Many common bacterial toxins are mono (ADP-ribosyl) transferases that are closely related to eukaryotic NAD arginine ADP-ribosyl transferases (ARTs) (Okazaki et al., 1997). Although the catalytic centers of ARTs and the bacterial toxins are weakly similar to the PARP catalytic domain, the evolutionary relationship between these two groups of ADPribosyl transferases and PARP remains unclear. A broad radiation within the Eukaryota has led to six recognizable subgroups of proteins with PARP activity (see Fig. 3). PARP1-related proteins are found in both plants and metazoan animals and hence appeared very early in evolution. PARP2 proteins are even more widely distributed because they exist in organisms as diverse as slime molds, fungi, plants, and mammals. Tankyrases constitute a distinctive PARP subgroup that is limited to metazoan animals (Kaminker et al., 2001; Smith et al., 1998). Vertebrates generally have two tankyrase genes (Cook et al., 2002; Sbodio et al., 2002).

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Figure 3 Phylogenetic relationship between PARP-related proteins. A family tree of PARPrelated proteins was constructed as described in the text. A double arrowheaded line indicates a ‘‘branch’’ of two or more genes sharing the domain structure shown at the right. The following subgroups are indicated: PARP1 (pink), PARP2 (dotted orange box; shaded members share a putative N-terminal DNA-binding domain), PARP3 (green), vault PARP (light blue), tankyrase (dark blue), tiPARP (yellow). A key at the bottom identifies the domains indicated at the right of each branch. (See Color Insert.)

vPARP constitutes a vertebrate-specific subclass (Mossink et al., 2002). vPARP has a characteristic von Willebrand factor (vWF) A domain, a motif found in both extracellular and nuclear proteins that has been proposed to form a metal ion-dependent protein–protein adhesion site (Lee et al., 1995). vPARP makes only a short polymer. It modifies itself and the major vault protein. Mice lacking vPARP are viable and fertile (Kickhoefer et al., 1999). Vertebrates contain another PARP variant with a tankyrase-like catalytic domain known as tetrachlorodibenzo-p-dioxin (TCDD)-inducible PARP (tiPARP) (Ma, 2002; Ma et al., 2001). In this variant, a PARP catalytic domain is present along with a novel Zn finger that binds DNA. tiPARP


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is expressed in response to TCDD and exhibits high activity toward histones. The presence of a specific DNA-binding domain in tiPARP suggests that it plays a role in inducing a response gene following dioxin exposure. Finally, there are a number of PARP-related genes with more novel domain architectures. For example, the single Neurospora PARP is neither a classical PARP1 nor PARP2 because it encodes a BRCT domain but lacks Zn fingers. Dictyostelium has a gene with the same domain structure, as well as a related gene in which a single PARP1-like Zn finger is found in place of the BRCT domain. All four PARP genes in Caenorhabditis elegans lack the BRCT domain (Gagnon et al., 2002). Functional studies of these atypical PARPs are needed to understand the roles they play in these organisms.

III. Mechanism of Action Previous studies have documented that PARP modulates multiple aspects of chromatin structure, including DNA repair (see de Murcia and Shall, 2000), telomere elongation (Smith and de Lange, 2000), centromere activity (Saxena et al., 2002a), and the availability of nuclear matrix attachment sites (Galande, 2002). In two of these cases, mechanisms were proposed in which PARP inactivated DNA-bound inhibitors by poly(ADP-ribose) modification (Fig. 4). Thus, following DNA damage, PARP1 becomes activated when nicks are recognized by the Zn fingers in the DNA-binding domain. The active enzyme modifies local chromatin targets so that they dissociate from the DNA, opening access to repair enzymes (Althaus et al., 1994; Althaus et al., 1995; Panzeter et al., 1992; Panzeter et al., 1993) (Fig. 4a). Histone H1 and many other chromatin proteins exhibit a high aYnity for pADPr (Gagne et al., 2003). Hence the proteins released as a result of PARP activity may bind to the long pADPr chains present on PARP itself and remain locally available for reassembly when pADPr is degraded (see Fig 4a) (Althaus, 1992; Panzeter et al., 1993). Likewise, tankyrase becomes activated by unknown signals and modifies the telomere binding protein TRF-1, causing it to dissociate. TRF-1 normally blocks telomerase from accessing the chromosome end, hence its removal leads to telomere elongation (Fig. 4b). Drosophila PARP has been shown to be necessary to maintain nucleoli and to activate genes within polytene chromosome puVs (Tulin and Spradling, 2003; Tulin et al., 2002). Shortly after hormone treatment, infection, or stress, pADPr polymers accumulate at the target loci. As a result of parp action, the chromatin structure loosens, leading to the appearance of a visible ‘‘puV’’ as gene transcription commences. Parp mutant Drosophila lack nucleoli, fail to form puVs, and produce greatly reduced amounts of target

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Figure 4 Unified models of PARP action. (a) Model of PARP action during DNA repair (Althaus 1992; Panzeter et al., 1993). An organized chromatin domain is shown at the top containing nucleosomes (blue), inactive PARP molecules (closed green ring), and other chromatin proteins (see key below figure). Binding to damaged DNA activates PARP (open green rings), generates a poly(ADP-ribose) (red) network, and dissociates chromatin proteins, providing DNA access to repair proteins (middle). Degradation of poly(ADP-ribose) releases bound or modified proteins, which reassemble chromatin (bottom). (b) Model of tankyrase function at telomeres (Smith and de Lange, 2000). Unknown signals activate tankyrase proteins on telomeres, which add poly(ADP-ribose) (red) to TRF-1 (yellow), causing them to dissociate from telomeric DNA and admit a telomerase complex. Degradation of poly(ADP-ribose) induces the reassembly of TRF-1 and TRF-2, blocking further elongation. (c) Model of PARP action on genes during development (Tulin and Spradling, 2003). PARP molecules (green) near an inactive target gene (top) receive an inducing signal and become activated. Target chromatin proteins including histone H1 and PARP itself become modified with pADPr (red), loosening local chromatin structure and facilitating target gene transcription (red arrow). Not all loosened genes become active (black arrow). Released proteins remain nearby bound to pADPr, where they may interact with chromatin modifying enzymes (thick arrows). Following autoinactivation of PARP, removal of the inducing signal, and cleavage of pADPr by PARG, the chromatin reassembles in an unchanged or reprogrammed state. (See Color Insert.)


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gene products. These observations, along with the previously studied roles of PARP in repair and telomere maintenance, led to the suggestion that PARP acts in a wide variety of situations by locally derepressing gene transcription or other chromosomal processes that are blocked by the tight binding of histones and other inhibitory chromatin proteins (Fig. 4c).

A. Poly(ADP-Ribose) Polymerase is Widely Distributed along Chromosomes If PARP is to act in this manner, then it must either be widely distributed along chromosomes or have the ability to translocate to domains requiring modification. Considerable evidence suggests that PARP is a regular constituent of chromatin (Desnoyers et al., 1996; Yamanaka et al., 1988). The distribution of epitope-tagged PARP molecules along Drosophila polytene chromosome is highly uniform and parallels the distribution of chromatin (Fig. 5). Immunofluorescence studies of mammalian nuclei using anti-PARP antibodies also indicate that PARP is widespread (Dantzer et al., 1998; Molinete et al., 1993). The molecular basis of PARP1’s general association with chromatin remains poorly known. Because the Zn fingers specifically recognize damaged DNA, it seems more likely that PARP interacts with a widespread chromatin protein. Considerable evidence suggests that PARP1 interacts

Figure 5 Wide distribution of PARP along chromosomes and within nucleoli. The distribution of PARP–GFP protein in Drosophila diploid (a) and polytene (b) cell nuclei are shown. Red: DNA; green: PARP-GFP. The nucleoli are strongly labeled (arrows). Arrowhead in (b) indicates a polytene chromosome puV, where transcription is highly active. The inset shows PARP protein (green) in the ecdysone-induced puVs at 75A, 75B. (c) Immunofluorescent detection of the nucleolar protein fibrillarin (red) and DNA (green) is shown in second instar salivary glands from wild-type second (top) and Parp mutant (bottom). In the mutant, fibrillarin is absent from nucleoli and resides in cytoplasmic particles. (See Color Insert.)

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directly with histones (Poirier et al., 1982; see D’Amours et al., 1999). Histone H1 and H2B are preferential targets for PARP binding (Buki et al., 1995) and enzymatic modification (Aubin et al., 1983; Krupitza and Cerruti, 1988). Two ribosomal proteins with H1-like N-terminal tails, Drosophila L22 and L23a, specifically bind PARP (Koyama et al., 1999) and may play a role in targeting PARP to nucleoli.

IV. Poly(ADP-Ribose) Polymerase and Transcription It has been well established that PARP can bind to a variety of transcription factors, including NF-B (Hassa and Hottiger, 2002), YY1 (Oei and Shi, 2001; Oei et al., 1997), Oct-1 (Oei et al., 1998), and AP-2 (Kannan et al., 1999), whereas other factors are targets of its enzymatic activity, including p53 (Wesierska-Gadek and Schmid, 2001) and fos (Amstad et al., 1992). Furthermore, evidence has been presented, some contradictory, that PARP plays a role in the induction or repression of target genes as a result of such interactions, or in some cases due to direct promoter binding (Soldatenkov et al., 2002). The strongest support for a transcriptional role for PARP in vivo is in the induction of innate immune response genes under the control of NF-B transcription factors. However, despite clear evidence that NF-B activity is greatly reduced in PARP1 mice or in cells treated with PARP inhibitors (Hassa and Hottiger, 1999; Oliver et al., 1999; Soriano et al., 2001), the mechanism by which PARP activates immune response genes has remained controversial (reviewed by Hassa and Hottiger, 2002). Recent genetic studies in Drosophila indicate that PARP is required to normally activate a variety of genes involved in rapidly responding to immune challenge, stress, and steroid hormones (Tulin and Spradling, 2003). The induction of most of these genes is accompanied by a dramatic loosening of the local chromatin structure that was described many years ago in polytene chromosomes as ‘‘puYng’’ (see Fig. 5b). These studies suggest that PARP does not act simply by altering transcription complexes but rather by modifying the chromatin structure of target gene loci. Many of the interactions previously noted between PARP and specific transcription factors may reflect mechanisms used to bring PARP to target loci and/or to activate preexisting PARP molecules at the site of the target genes. The activated PARP molecules would modify local chromatin proteins, thereby loosening repressive chromatin structures and promoting high rates of transcription and mRNA production. Such a mechanism is very similar to the proposed role for PARP in DNA repair and for tankyrase at telomeres (Fig. 4c vs Figs. 4a and 4b). According to this model, PARP molecules must become enzymatically active in order to stimulate chromatin loosening. In the case of DNA repair, or telomere elongation, PARP1 is known to be activated by DNA breaks, and


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tankyrase displays DNA-independent enzymatic activity. How would PARP molecules be activated at the sites of genes targeted for induction? Primarily inducible genes, rather than those modulated according to the normal developmental program, seem to require PARP action. To consider what aspects of these systems might promote activation, we now review the classes of inducible genes in which PARP appears to play an important role (Fig. 6).

A. Immunity Genes Mice and flies mutant for PARP are immunocompromised and fail to mount a normal genetic response to infection and to inflammatory stimuli. NF-B is normally inactive because it is held in a cytoplasmic complex. In PARP1

Figure 6 PARP and the transcription of environmentally responsive genes. Models are shown for the induction by specific factors of several classes of environmentally responsive genes in which PARP is likely to play a role. These include Hsp genes by heat shock factor (Hsf, green), hormone-dependent response genes by steroid hormone receptors (SHR, red), immune response genes by NF-kappa-B (NF-B, blue) family members, p21WAF and MDM2 by p53 (yellow), and rDNA genes by a postulated nucleolar factor (NuF, orange). Upon activation (as illustrated for rDNA and NF-B pathways), the factors interact with resident PARP molecules (green), upregulate PARP activity, and loosen the chromatin structure of the target gene to facilitate target gene activity. (See Color Insert.)

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mutant mice, NF-B still translocates into the nucleus but does not bind and activate target genes (Hassa and Hottiger, 1999; Oliver et al., 1999). PARP is required for a subset of NF-B–dependent genes, including iNOS, IL-1, -2, -6, -8; and TNFa (Oliver et al., 1999). However, the mechanism leading to this nuclear block remains unclear. PARP1 can bind both the p50 and p65 subunits of NF-B. One model is that unmodified PARP sequesters any nuclear NF-B protein, preventing it from acting on target genes (Chang and Alvarez-Gonzalez, 2001). Following activation, PARP automodification releases NF-B. Consistent with such a requirement for enzymatic activity, treatment of macrophages with the PARP inhibitor 3-AB blocks the induction of iNOS, IL-6, and TNFa genes (Le Page et al., 1998; PellatDeceunynck et al., 1994). However, sequestration by PARP1 is not consistent with the observation that PARP1 mutants are immunocompromised rather than constitutively activated. Alternative models have been put forward in which PARP simply acts as a coactivator, independent of its DNA-binding domain or enzymatic activity (Ha et al., 2002; Hassa et al., 2001).

B. p53 p53 plays a critically important role as a checkpoint protein that translocates from the cytoplasm to the nucleus of cells following DNA damage, where it coordinates homeostatic cellular responses including the induction of cell cycle arrest (Fig. 6, see Nikolaev et al., 2003). p53 does not sense DNA damage directly but is thought instead to detect PARP activation (Wang et al., 1998). p53 has a high aYnity for pADPr (Pleschke et al., 2000), so one possibility is that it detects fragments of pADPr generated by activated PARP1 that are exported from nucleus. Following receipt of some such signal and nuclear entry, p53 forms a homotetramer and transcriptionally activates target genes such as p21WAF1 and MDM2 (Vaziri et al., 1997; Wang et al., 1998). Elimination of PARP enzymatic function impairs p53-dependent transcription (Valenzuela et al., 2002; Wang et al., 1998). Moreover, overexpression of PARP causes the cell cycle to arrest (Bhatia et al., 1996; Kaiser et al., 1992; Wieler et al., 2003), and this could be explained by the DNA-damage independent activation of p53 (WesierskaGadek and Schmid, 2001). p53 is itself subject to ADP-ribosylation by PARP1, and the modified protein loses its DNA-binding capacity (Mendoza-Alvarez and Alvarez-Gonzalez, 2001; Simbulan-Rosenthal et al., 2001). The ATM kinase, another important mediator of DNA damage responses, is also activated by the same types of DNA damage sensed by PARP (Bakkenist and Kastan, 2003) and may participate in some of these same pathways (Menisser-de Murcia et al., 2001).


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C. Heat Shock Genes Heat shock response genes encode a diverse set of chaperone proteins that are strongly induced following heat shocks or other stressful conditions that increase the amount of incorrectly folded proteins within the cell (see Wang and Lindquist, 1998). The response is controlled by a conserved transcription factor, ‘‘heat shock factor’’ (Hsf ), that forms an active trimeric complex, moves to the site of the target genes, and rapidly initiates transcription. This ancient stress response system is widely conserved among both plant and animal kingdoms. Heat shock response genes in Drosophila require PARP to be induced (Tulin and Spradling, 2003). Large puVs containing pADPr rapidly form at the sites of target gene transcription. Hsf may directly or indirectly activate chromosomal PARP molecules at target loci. Heat shock puVs are visible at target genes within 1–2 minutes of induction, so if Hsf activates PARP indirectly it must be by a fast-acting mechanism.

D. Steroid Hormone-Dependent Genes Not only stress-activated genes but also some developmentally regulated genes may require PARP (see Fig. 6). In particular, a cascade of genes that are induced by the steroid-molting hormone ecdysone form puVs and accumulate pADPr at the time of ecdysone release (Tulin and Spradling, 2003). The ecdysone response is mediated by a conserved family of steroid hormone receptors that, upon hormone binding, activate target response genes. PARP is known to bind specifically to mammalian retinoid X receptors (Miyamoto et al., 1999); however, it acted as an inhibitor in the transient transfection assay used. The heat shock protein chaperones Hsp70 and Hsp90 are involved in protein complexes needed to activate steroid hormone response genes (reviewed in Morimoto, 2002). Nonetheless, it remains clear how the PARP1 molecules at the site of steroid response genes become activated.

E. rDNA Genes and Nucleoli rDNA transcription and ribosome production in the nucleolus must be closely regulated with a cell’s nutritional status and growth rate (see Frank et al., 2002). Unlike most other loci, rDNA genes are not controlled by the rate of transcription initiation. Instead, rRNA synthesis is controlled digitally by specifying how many genes, which all reside in tandem clusters within special nucleolus organizer regions, are actually active at any given moment.

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How individual genes are selected for activity is not known but likely involves chromatin organization. The activated genes unfold and give rise to one or more nucleoli, like giant chromosome puVs. Nucleoli contain large amounts of PARP (Fig 5a, 5b), PARP activity, and pADPr (Desnoyers et al., 1996; Tulin et al., 2002; Yamanaka et al., 1988). PARP is required for nucleolar function (Tulin and Spradling, 2003; Tulin et al., 2002). Indeed, nucleoli disintegrate completely in PARP mutants, leaving normal components such as NOP140 and fibrillarin in the cytoplasm (Fig. 5c). The strong requirement of rDNA genes for PARP can be rationalized in the following manner. PARP may be required to unfold and activate individual rDNA genes and to prevent active genes from repacking into an inactive state. This might be accomplished by modifying target nucleolar proteins that would otherwise inhibit transcription and promote condensation of rDNA chromatin. Several abundant nucleolar proteins are candidates for the role of transcriptional repressor. The C. elegans gene ncl-1 and its Drosophila homology brat repress nucleolar size and cellular rDNA content in vivo (Frank et al., 2002). Nucleolin has been shown to negatively regulate rDNA transcription (Roger et al., 2002). Furthermore, nucleolin (as well as nucleoplasmin/B23 [Chan, 1992]) undergo modification by pADPr (Leitinger and WesierskaGadek, 1993). Fibrillarin and NOP140 may also be involved as they move to the cytoplasm in PARP mutant cells (Fig. 5c, Tulin et al., 2002). Unidentified signals that presumably reflect the cellular growth rate may modulate the localization and activity of PARP in nucleoli, thereby determining the extent to which it derepresses bound nucleolin and other potential target proteins. In addition, the extensive pADPr network generated in the nucleolus by PARP activity may serve as a structural scaVold for various nucleolar components supporting rRNA processing and ribosome assembly. Little is known about the mechanisms that regulate the amount and location of PARP in nucleoli. The process does appear to depend on nucleolar activity because a significant amount of PARP leaves the nucleoli when rDNA transcription is inhibited by DRB (Desnoyers et al., 1996).

V. Multiple Routes to Poly(ADP-Ribose) Polymerase Activation Based on the evidence, activated PARP1 plays important roles in gene transcription under a wide variety of circumstances. According to our current understanding, PARP1 becomes active following BRCT domain-mediated dimerization (Buki et al., 1995; Mendoza-Alvarez and Alvarez-Gonzalez, 1993) by recognizing and binding to DNA nicks or double-stranded breaks. The PARP/LigIII-type Zn fingers located in the DNA-binding


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domain (see Fig. 1) mediate damage recognition. Upon binding, a conformational change in the protein is proposed to promote assembly of an active catalytic center. These observations are supported by the observed requirement for randomly broken double-stranded DNA as a cofactor for PARP1 activity in vitro. The many PARP-requiring inducible gene systems unrelated to DNA repair reviewed previously suggest that activation pathways for PARP also exist that are independent of DNA damage (Fig. 7). Kun et al. (2002) showed that PARP1 molecules can recognize a wider range of DNA cofactors than just damaged DNA. They found that when assayed for trans poly(ADP-ribosyl)ation of histone H1, stem-loop–containing DNA lacking free ends or discontinuities activates PARP1 activity more eYciently than damaged DNA. Perhaps the PARP1 Zn fingers in vivo recognize promoter DNA that has been locally strand separated by specific transcription factors to facilitate polymerase entry (Fig. 7a). Another possibility is that PARPinteracting proteins exist that can induce the same conformational change as DNA binding (Fig. 7b). An Aly-like transcription factor in Arabidopsis is known to interact with the PARP Zn fingers (Storozhenko et al., 2001). The critical conformational change might equally well be mediated by protein binding to a diVerent portion of the protein, however.

Figure 7 Mechanisms of PARP protein activation. Three mechanisms proposed in the text that may lead to the activation of chromosomal PARP protein (green) at the site of target genes. (a) Activation by DNA hairpins or unfolding at promoters. (b) Activation by a protein cofactor (brown) that mediates a conformation change. (c) Activation by an enzyme (blue) that catalyzes a PARP protein modification (blue circle). See text for further details.

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A final possibility is that activation can be stimulated by secondary modification of the PARP1 protein itself (Fig. 7c). It is known, for example, that tankyrase can be activated by MAP kinase-mediated phosphorylation (Chi and Lodish, 2000). PARP has been shown to be subject to a variety of modifications. Interaction of PARP1 with histone H1 in vitro stimulates cdc2-dependent phosphorylation of PARP (Bauer et al., 2001). PARP1 phosphorylation correlates with increasing PARP activity during Xenopus oocyte maturation (Aoufouchi and Shall, 1997). Enzymes that could potentially modify and activate PARP1 are known to be involved in several of the systems discussed previously. For example, P-TEPb is recruited to newly induced heat shock genes, where its cyclin-dependent kinase activity might modify PARP (Egyhazi et al., 1998; Lis et al., 2000). The loss of PARP from the nucleolus following treatment with the casein kinase 2 (CKII) inhibitor DRB (Desnoyers et al., 1996) might indicate a role for CKII in activating PARP in nucleoli (Egyhazi et al., 1999). More attention should be paid to the state of PARP modification under conditions where PARP-dependent gene induction has occurred.

VI. Specificity of Transcriptional Activation It is now clear that PARP helps induce many genes during developmental and adult life. Interestingly, most PARP-dependent genes mediate rapid responses to environmental factors such as stress, infection, nutrition, or hormonal signals. PARP might be specifically required when chromatin needs to be changed rapidly or to be under precise control. However, it remains possible that it is a general transcription factor (Meisterernst et al., 1997) that plays a major role only when genes must be induced at the highest levels. PuVs expand along the chromosome far beyond the limits of the activated gene itself. Presumably, the size of the loosened region depends on how widely PARP1 molecules are activated on either side of the target gene. However, unrelated genes that happen to lie in the expanded region are not activated (Meyerowitz and Hogness, 1982). Thus, chromatin loosening may be necessary, but it does not appear to be suYcient for gene transcription, which remains dependent on gene-specific transcription factors. However, coordinately regulated genes are found in chromosomal clusters quite frequently in the Drosophila genome (Spellman and Rubin, 2002), and multiple related genes are sometimes turned on in a single puV. To maintain gene-specific control, some proteins must be able to resist PARP action when chromatin is loosened. RNA polymerase, for example, can no longer be modified by PARP once it has initiated transcription. Both TATA-binding protein and the transcription factor YY1 lose their aYnity for DNA following modification by pADPr in solution, but when bound


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in a transcription complex at a promoter, they resist PARP-induced dissociation (Oei et al., 1998). Transcription complexes at the major heat shock loci encoding Hsp70 are preassembled, and RNA polymerase has already initiated and then paused before heat shock (reviewed in Lis, 1998). Such a complex may be resistant to modification and allow these heat shock genes to undergo multiple rounds of transcription without being inactivated by PARP. Other target genes activated by PARP may utilize stable transcription complexes as well. PARP is likely to regulate some aspects of chromatin structure independent of its enzymatic activity. PARPe, an isoform in Drosophila that is found in oocytes and early embryos, lacks a catalytic domain due to alternative splicing. Yet the presence of PARPe is critical during early developmental stages and seems to control the activity of the alternative PARP promoter that gives rise to the PARP1-like isoform (PARPI). PARPe may act as a chromatin-binding protein or transcription factor. Remarkably, overexpression of PARPe, even later in development where it is not normally found, greatly elevates PARPI production (Tulin et al., 2002). Several other examples in which PARP proteins may act independently of their enzymatic activity have been identified. As mentioned previously, it is possible that PARP influences NF-B activation nonemzymatically. Furthermore, PARP1 binding to a specific promoter element can suppress transcription (Soldatenkov et al., 2002).

VII. Poly(ADP-Ribose) Polymerase and Other Chromatin Processes A. Heterochromatin Condensation The wide distribution of PARP protein along chromosomes and its overall abundance suggests that it may play a structural role even when inactive. The eVects of mutating parp-e in Drosophila strongly support this hypothesis (Tulin et al., 2002). DAPI-stained chromatin in parp-e mutant larval cells appears abnormally extended (Fig. 8a) and is more accessible to nuclease digestion. Heterochromatic regions rich in repetitive DNA sequences that normally condense early in development are preferentially aVected. Transcripts from the copia retrotransposon, whose multiple dispersed copies are normally silenced, accumulate to levels 50-fold higher than wild type. Inactivation of PARP mRNA after heterochromatin has already formed using RNAi causes very similar eVects (Tulin et al., 2002). Thus PARP is required to both initiate and maintain domains of compact, silent chromatin during development. There are several ways that PARP might compact chromatin. Higherorder chromatin packing into loops tethered on a central chromosomal scaVold is thought to be mediated by specific matrix attachment regions

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Figure 8 Proposed nonenzymatic role of PARP in chromatin structure. (a) DAPI staining of nuclei from second instar larval salivary glands of wild-type (top) and Parp mutant Drosophila (bottom). A single nucleus is presented at higher magnification in the insets. Nuclei in the mutant contain more diVuse chromatin and lack nucleoli (dark circular region). (From Tulin et al., 2002.) (b) Model for function of PARP protein in chromatin compaction of chromatin loops at MAR region–scaVold junctions and at the nuclear matrix/lamina (see Galantde, 2002). PARP is postulated to associate with scaVold-associated proteins and mediate junction formation. (See Color Insert.)

(MARs). Recently PARP1 was found in protein complexes that bind to ATrich regions within MARs (Galande and Kohwi-Shigematsu, 2000). Higher concentrations of PARP might facilitate loop formation, whereas reduced levels might prevent it (Fig. 8b). Another possibility is that PARP mediates the association of compacted chromatin regions with the nuclear lamina. Lamin, a major constituent of the lamina, is subject to modification by pADPr (Pedraza-Reyer and Alvarez-Gonzalez, 1990).

B. Centromere and Centrosome Activity Chromosome segregation during mitosis is a highly regulated process that is critical for cell survival. In preparation for M phase, chromosomes undergo condensation, a mitotic spindle is assembled under the control of the


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two centrosomes, and the spindle microtubules interact specifically with kinetochore proteins located at the site of the active chromosomal centromeres. PARP has been implicated in several of these events. Mammalian PARP1 (Earle et al., 2000; Saxena et al., 2002a) and PARP2 (Saxena et al., 2002b) proteins associate specifically with centromeric chromosomal regions and centromeric DNA sequences. Both PARP1 and PARP2 can specifically bind the centromeric proteins CENP-A, CENP-B, and BUB3 (Saxena et al., 2002b). In addition, PARP3 is localized exclusively to centrosomes (see Shall, 2002). The idea that centromere activity can be controlled by a secondary modification is attractive because it is known that centromere activity is subject to regulation and can change independent of the DNA sequence (Sullivan et al., 2001). Whether PARP has such a function at centromeres remains to be demonstrated, however. C. Episomal Chromosomes Even tiny episomal chromosomes within eukaryotic nuclei might be subject to regulation by PARP. The TRF-1 to -3 proteins that are found along with tankyrase at telomeres were shown recently to bind and regulate the replication origin of Epstein-Barr virus (EBV) (Deng et al., 2002). This herpesvirus contains a large DNA genome that replicates as a free circular plasmid under the control of both cellular and virally encoded genes, but specific host proteins had not been previously identified. The induction of EBV and other herpesviruses by stress is well known. If stress can activate tankyrase, ADPribose modification of TRF proteins might derepress the EBV replication origin, facilitating viral growth.

VIII. Concluding Thoughts In this review, we have discussed evidence linking PARP to many fundamental processes governing chromosome structure, function, and maintenance. Although the models discussed remain speculative, PARP can no longer be viewed primarily as an enzyme of DNA repair. Why, then, has PARP evolved to serve such a wide variety of critical functions involving chromatin organization? We suggest that PARP is inherently diVerent from other enzymes that modify target chromatin proteins, such as kinases, methyases, and acetylases. Unlike these proteins, PARP simultaneously undergoes automodification and generates a large network of pADPr polymers, which mimics the structure of DNA and may serve as a key protein storage site. A long list of chromatin protein has been documented to bind pADPr polymers (Gagne et al., 2003; Huletsky et al., 1989; Mathis and Althaus, 1987; Sauermann and Wesierska-Gadek, 1986).

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PARP’s ability to generate a transient DNA-like substrate may be critically important for storing and reassembling chromatin in the same developmental state in which it was removed. Chromatin proteins stripped from their normal locations may retain the developmentally specific information that has been programmed into that particular region of the chromosome by simply assembling along pADPr chains in similar complexes and relationships. Such its stored chromatin would retain the ability to dissociate from the pADPr chains and to reassemble onto DNA region of origin when the repair, transcription, or other process that had caused it to be temporarily disrupted were concluded (Althaus, 1992). This leads to the final and perhaps most intriguing possibility of all. If PARP is indeed special because of its ability to act as a transient chromatin removal and storage device, then also it is likely to be involved in the slower, developmentally mediated chromatin remodeling that constitutes a major mechanism of developmental programming in multicellular organisms. Transient loosening of chromatin proteins within a specific domain by PARP might be necessary to make histones and other chromatin proteins more accessible to enzymes that alter the ‘‘code’’ of specific modifications that control chromatin structure. Thus, PARP might be involved in setting the domains of Hox gene expression along with Polycomb group proteins, as well as in many other instances in which chromatin is remodeled. Understanding the nature of these chromatin changes and the role played by PARP remains one of the most exciting frontiers in our growing understanding of this key mediator of eukaryotic chromosomes.

Acknowledgments We thank J. Gall for providing materials. This work was supported by NIH Grant GM27875. We apologize to those whose work was not cited due to space limitations.

References Adams, M. D., Celniker, S. E., Holt, R. A., Evans, C. A., Gocayne, J. D., Amanatides, P. G., Scherer, S. E., Li, P. W., Hoskins, R. A., Galle, R. F. et al. (2000). The genome sequence of Drosophila melanogaster. Science 287, 2185–2195. Althaus, F. R. (1992). Poly ADP-ribosylation: A histone shuttle mechanism in DNA excision repair. J. Cell Sci. 102, 663–670. Althaus, F. R., Bachmann, S., Hofferer, L., Kleczkowska, H. E., Malanga, M., Panzeter, P. L., Realini, C., and Zweifel, B. (1995). Interactions of poly(ADP-ribose) with nuclear proteins. Biochimie. 77, 423–432.


77

Althaus, F. R., Hofferer, L., Kleczkowska, H. E., Malanga, M., Naegeli, H., Panzeter, P. L., and Realini, C. A. (1994). Histone shuttling by poly ADP-ribosylation. Mol. Cell. Biochem. 138, 53–59. Ame´, J. C., Rolli, V., Scureiber, V., Niedergang, C., Apiou, F., Decker, P., Muller, S., Hoger, T., Meńissier-de Murica, J., and de Murica, G. (1999). PARP-2, a novel mammalian DNA damage-dependent poly(ADP-ribose) polymerase. J. Biol. Chem. 274, 17860–17868. Amstad, P. A., Krupitza, G., and Cerutti, P. A. (1992). Mechanism of c-fos induction by active oxygen. Cancer Res. 52, 3952–3960. Aoufouchi, S., and Shall, S. (1997). Regulation by phosphorylation of Xenopus laevis poly(ADP-ribose) polymerase enzyme activity during oocyte maturation. Biochem. J. 325, 543–551. Aravind, L., and Koonin, E. V. (2000). SAP: A putative DNA-binding motif involved in chromosomal organization. Trends Biochem. Sci. 25, 112–114. Arnold, J., and Grune, T. (2002). PARP-mediated proteasome activation: A co-ordination of DNA repair and protein degradation? Bioessays 24, 1060–1065. Aubin, R. J., Frechette, A., de Murcia, G., Mandel, P., Lord, A., Grondin, G., and Poirier, G. G. (1983). Correlation between endogenous nucleosomal hyper(ADP-ribosyl)ation of histone H1 and the induction of chromatin relaxation. EMBO J. 2, 1685–1693. Babiychuk, E., Cottrill, P. B., Storozhenko, S., Fuangthong, M., Chen, Y., O’Farrell, M. K., Van Montagu, M., Inze, D., and Kushnir, S. (1998). Higher plants possess two structurally different poly(ADP-ribose) polymerases. Plant J. 15, 635–645. Bakkenist, C. J., and Kastan, M. B. (2003). DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation. Nature 421, 499–506. Bhatia, M., Kirkland, J. B., and Meckling-Gill, K. A. (1996). Over expression of poly (ADPribose) polymerase promotes cell cycle arrest and inhibits neutrophilic differentiation of NB4 acute promyelocytic leukemia cells. Cell Growth Differ. 7, 91–100. Bauer, P. I., Buki, K. G., and Kun, E. (2001). Selective augmentation of histone H1 phosphorylation sites by interaction of poly(ADP-ribose) polymerase and cdc2-kinase: Comparison with protein kinase C. Int. J. Mol. Med. 8, 691–693. Bork, P., Hofmann, K., Bucher, P., Neuwald, A. F., Altschul, S. F., and Koonin, E. V. (1997). A superfamily of conserved domains in DNA damage responsive cell cycle checkpoint proteins. FASEB J. 11, 68–76. Buki, K. G., Bauer, P. I., Hakam, A., and Kun, E. (1995). Identification of domains of poly(ADP-ribose) polymerase for protein binding and self-association. J. Biol. Chem. 270, 3370–3377. Chambon, P., Weil, J. D., and Mandel, P. (1963). Nicotinamide mononucleotide activation of a new DNA-dependent polyadenylic acid synthesizing nuclear enzyme. Biochem. Biophys. Res. Commun. 11, 39–42. Chan, P. K. (1992). Characterization and cellular localization of nucleophosmin/B23 in HeLa cells treated with selected cytotoxic agents (studies of B23-translocation mechanism). Exp. Cell. Res. 203, 174–181. Chang, W. J., and Alvarez-Gonzalez, R. (2001). The sequence-specific DNA binding of NFkappa B is reversibly regulated by the automodification reaction of poly(ADP-ribose) polymerase 1. J. Biol. Chem. 276, 47664–47670. Chi, N. W., and Lodish, H. F. (2000). Tankyrase is a gogli-associated mitogen-activated protein kinase substrate that interacts with IRAP in GLUT4 vesicles. J. Biol. Chem. 275, 38437–38444. Chiarugi, A., and Moskowitz, M. A. (2002). Cell biology. PARP-1—a perpetrator of apoptotic cell death? Science 297, 200–201.

78

Tulin et al.

Cook, B. D., Dynek, J. N., Chang, W., Shostak, G., and Smith, S. (2002). Role for the related poly(ADP-ribose) polymerases tankyrase 1 and 2 at human telomeres. Mol. Cell. Biol. 22, 332–342. D’Amours, D., Desnoyers, S., D’Silva, I., and Poirier, G. G. (1999). Poly(ADP-ribosyl)ation reactions in the regulation of nuclear functions. Biochem. J. 342, 249–268. D’Amours, D., Germain, M., Orth, K., Dixit, V. M., and Poirier, G. G. (1998). Proteolysis of poly(ADP-ribose) polymerase by caspase 3: Kinetics of cleavage of mono(ADP-ribosyl)ated and DNA-bound substrates. Radiat. Res. 150, 3–10. Dantzer, F., Nasheuer, H. P., Vonesch, J. L., de Murcia, G., and Menissier-de Murcia, J. (1998). Functional association of poly(ADP-ribose) polymerase with DNA polymerase alpha-primase complex: A link between DNA strand break detection and DNA replication. Nucleic Acids Res. 26, 1891–1898. Davidovic, L., Vodenicharov, M., Affar, E. B., and Poirier, G. G. (2001). Importance of poly(ADP-ribose) glycohydrolase in the control of poly(ADP-ribose) metabolism. Exp. Cell. Res. 268, 7–13. de Murcia, G., and Shall, S. (2000). ‘‘Poly (ADP-ribosyl)ation reactions; From DNA damage and stress signalling to cell death.’’ Oxford University Press, New York. Deng, Z., Lezina, L., Chen, C. J., Shtivelband, S., So, W., and Lieberman, P. M. (2002). Telomeric proteins regulate episomal maintenance of Epstein-Barr virus origin of plasmid replication. Mol. Cell. 9, 493–503. Desnoyers, S., Kaufmann, S. H., and Poirier, G. G. (1996). Alteration of the nucleolar localization of poly(ADP-ribose) polymerase upon treatment with transcription inhibitors. Exp. Cell. Res. 227, 146–153. Earle, E., Saxena, A., MacDonald, A., Hudson, D. F., Shaffer, L. G., Saffery, R. et al. (2000). Poly(ADP-ribose) polymerase at active centromeres and neocentromeres at metaphase. Hum. Mol. Genet. 9, 187–194. Egyhazi, E., Ossoinak, A., Filhol-Cochet, O., Cochet, C., and Pigon, A. (1999). The binding of the alpha subunit of protein kinase CK2 and RAP74 subunit of TFIIF to protein-coding genes in living cells is DRB sensitive. Mol. Cell. Biochem. 191, 149–159. Egyhazi, E., Ossoinak, A., Lee, J. M., Greenleaf, A. L., Makela, T. P., and Pigon, A. (1998). Heat-shock-specific phosphorylation and transcriptional activity of RNA polymerase II. Exp. Cell. Res. 242, 211–221. Faraone-Mennella, M. R., Gambacorta, A., Nicolaus, B., and Farina, B. (1998). Purification and biochemical characterization of a poly(ADP-ribose) polymerase-like enzyme from the thermophilic archaeon Sulfolobus solfataricus. Biochem. J. 335, 441–447. Frank, D. J., Edgar, B. A., and Roth, M. B. (2002). The Drosophila melanogaster gene brain tumor negatively regulates cell growth and ribosomal RNA synthesis. Development 129, 399–407. Futai, M., Mizuno, D., and Sugimura, T. (1968). Mode of action of rat liver phosphodiesterase on a polymer of phosphoribosyl adenosine monophosphate and related compounds. J. Biol. Chem. 243, 6325–6329. Gagne, J. P., Hunter, J., Labrecque, B., Chabot, B., and Poirier, G. G. (2003). A proteomic approach to the identification of heterogeneous nuclear ribonucleoproteins as a new family of poly(ADP-ribose)-binding proteins. Biochem. J. 371, 331–340. Gagnon, S. N., Hengartner, M. O., and Desnoyers, S. (2002). The genes pme-1 and pme-2 encode two poly(ADP-ribose) polymerases in Caenorhabditis elegans. Biochem. J. 368, 263–271. Galande, S. (2002). Chromatin (dis)organization and cancer: BUR-binding proteins as biomarkers for cancer. Curr. Cancer Drug Targets 2, 157–190. Galande, S., and Kohwi-Shigematsu, T. (2000). Linking chromatin architecture to cellular phenotype: BUR-binding proteins in cancer. J. Cell. Biochem. Suppl. 35, 36–45.


79

Ha, H. C., Hester, L. D., and Snyder, S. H. (2002). Poly(ADP-ribose) polymerase-1 dependence of stress-induced transcription factors and associated gene expression in glia. Proc. Natl. Acad. Sci. USA 99, 3270–3275. Hassa, P. O., Covic, M., Hasan, S., Imhof, R., and Hottiger, M. O. (2001). The enzymatic and DNA binding activity of PARP-1 are not required for NF-kappa B coactivator function. J. Biol. Chem. 276, 45588–45597. Hassa, P. O., and Hottiger, M. O. (1999). A role of poly (ADP-ribose) polymerase in NF-kappa B transcriptional activation. Biol. Chem. 380, 953–959. Hassa, P. O., and Hottiger, M. O. (2002). The functional role of poly(ADP-ribose) polymerase 1 as novel coactivator of NF-kappa B in inflammatory disorders. Cell. Mol. Life Sci. 59, 1534–1553. Huletsky, A., de Murcia, G., Muller, S., Hengartner, M., Menard, L., Lamarre, D., and Poirier, G. G. (1989). The effect of poly(ADP-ribosyl)ation on native and H1-depleted chromatin: A role of poly(ADP-ribosyl)ation on core nucleosome structure. J. Biol. Chem. 264, 8878–8886. Kaiser, P., Auer, B., and Schweiger, M. (1992). Inhibition of cell proliferation in Saccharomyces cerevisiae by expression of human NADþ ADP-ribosyltransferase requires the DNA binding domain (‘‘zinc fingers’’). Mol. Gen. Genet. 232, 231–239. Kameshita, I., Matsuda, M., Nishikimi, M., Ushiro, H., and Shizuta, Y. (1986). Reconstitution and poly(ADP-ribosyl)ation of proteolytically fragmented poly(ADP-ribose) synthetase. J. Biol. Chem. 261, 3863–3868. Kameshita, I., Matsuda, Z., Taniguchi, T., and Shizuta, Y. (1984). Poly(ADP-ribose) synthetase: Separation and identification of three proteolytic fragments as the substratebinding domain, the DNA-binding domain, and the automodification domain. J. Biol. Chem. 259, 4770–4776. Kaminker, P. G., Kim, S. H., Taylor, R. D., Zebarjadian, Y., Funk, W. D., Morin, G. B., Yaswen, P., and Campisi, J. (2001). TANK2, a new TRF1-associated poly(ADP-ribose) polymerase, causes rapid induction of cell death upon overexpression. J. Biol. Chem. 276, 35891–35899. Kannan, P., Yu, Y., Wankhade, S., and Tainsky, M. A. (1999). PolyADP-ribose polymerase is a coactivator for AP-2 mediated transcriptional activation. Nucleic Acids Res. 27, 866–874. Kawamura, T., Hanai, S., Yokota, T., Hayashi, T., Poltronieri, P., Miwa, M., and Uchida, K. (1998). An alternative form of poly(ADP-ribose) polymerase in Drosophila melanogaster and its ectopic expression in rat-1 cells. Biochem. Biophys. Res. Commun. 251, 35–40. Kickhoefer, V. A., Siva, A. C., Kedersha, N. L., Inman, E. M., Ruland, C., Streuli, M., and Rome, L. H. (1999). The 193-kD vault protein, VPARP, is a novel poly(ADP-ribose) polymerase. J. Cell. Biol. 146, 917–928. Koyama, Y., Katagiri, S., Hanai, S., Uchida, K., and Miwa, M. (1999). Poly(ADP-ribose) polymerase interacts with novel Drosophila ribosomal proteins, L22 and 123a, with unique histone-like amino-terminal extensions. Gene 226, 339–345. Krupitza, G., and Cerruti, P. (1988). Poly(ADP-ribosyl)ation of histones in intact human deratinocytes. Biochemistry 28, 4054–5060. Kumar, S. (2001). MEGA2: Molecular evolutionary genetics analysis software. Bioinformatics 17, 1244–1245. Kun, E., Kirsten, E., and Ordahl, C. P. (2002). Coenzymatic activity of randomly broken or intact double-stranded DNAs in auto and histone H1 trans-poly(ADP-ribosylation), catalyzed by poly(ADP-ribose) polymerase (PARP I). J. Biol. Chem. 277, 39066–39069. Lazebnik, Y. A., Kaufmann, S. H., Desnoyers, S., Poirier, G. G., and Earnshaw, W. C. (1994). Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE. Nature 371, 346–347. Lee, J. O., Rieu, P., Arnaout, M. A., and Liddington, R.(1995). Crystal structure of the A domain from the alpha subunit of integrin CR3 (CD11b/CD18). Cell 80, 631–638.

80

Tulin et al.

Leitinger, N., and Wesierska-Gadek, J. (1993). ADP-ribosylation of nucleolar proteins in HeLa tumor cells. J. Cell. Biochem. 52, 153–158. Le Page, C., Sanceau, J., Drapier, J. C., and Wietzerbin, J. (1998). Inhibitors of ADPribosylation impair inducible nitric oxide synthase gene transcription through inhibition of NF kappa B activation. Biochem. Biophys. Res. Commun. 243, 451–457. Lis, J. (1998). Promoter-associated pausing in promoter architecture and postinitiation transcriptional regulation. Cold Spring Harb. Symp. Quant. Biol. 63, 347–356. Lis, J. T., Mason, P., Peng, J., Price, D. H., and Werner, J. (2000). P-TEFb kinase recruitment and function at heat shock loci. Genes Dev. 14, 792–803. Ma, Q. (2002). Induction and superinduction of 2,3,7,8-tetrachlorodibenzo-rho-dioxininducible poly(ADP-ribose) polymerase: Role of the aryl hydrocarbon receptor/aryl hydrocarbon receptor nuclear translocator transcription activation domains and a labile transcription repressor. Arch. Biochem. Biophys. 404, 309–316. Ma, Q., Baldwin, K. T., Renzelli, A. J., McDaniel, A., and Dong, L. (2001). TCDD-inducible poly(ADP-ribose) polymerase: A novel response to 2,3,7,8-tetrachlorodibenzo-p-dioxin. Biochem. Biophys. Res. Commun. 289, 499–506. Mathis, G., and Althaus, F. R. (1987). Release of core DNA from nucleosomal core particles following (ADP-ribose)n-modification in vitro. Biochem. Biophys. Res. Commun. 143, 1049–1054. Meisterernst, M., Stelzer, G., and Roeder, R. G. (1997). Poly(ADP-ribose) polymerase enhances activator-dependent transcription in vitro. Proc. Natl. Acad. Sci. USA 94, 2261–2265. Mendoza-Alvarez, H., and Alvarez-Gonzalez, R. (1993). Poly(ADP-ribose) polymerase is a catalytic dimer and the automodification reaction is intermolecular. J. Biol. Chem. 268, 22575–22580. Mendoza-Alvarez, H., and Alvarez-Gonzalez, R. (2001). Regulation of p53 sequencespecific DNA-binding by covalent poly(ADP-ribosyl)ation. J. Biol. Chem. 276, 36425–36430. Menisser-de Murcia, J., Mark, M., Wendling, O., Wynshaw-Boris, A., and de Murcia, G. (2001). Early embryonic lethality in PARP-1 Atm double-mutant mice suggests a functional synergy in cell proliferation during development. Mol. Cell. Biol. 21, 1828–1832. Meyerowitz, E. M., and Hogness, D. S. (1982). Molecular organization of a Drosophila puff site that responds to ecdysone. Cell 28, 165–176. Miwa, M., and Sugimura, T. (1971). Splitting of the ribose-ribose linkage of poly(adenosine diphosphate-robose) by a calf thymus extract. J. Biol. Chem. 246, 6362–6364. Miyamoto, T., Kakizawa, T., and Hashizume, K. (1999). Inhibition of nuclear receptor signalling by poly(ADP-ribose) polymerase. Mol. Cell. Biol. 19, 2644–2649. Molinete, M., Vermeulen, W., Burkle, A., Menissier-de Murcia, J., Kupper, J. H., Hoeijmakers, J. H., and de Murcia, G. (1993). Overproduction of the poly(ADP-ribose) polymerase DNAbinding domain blocks alkylation-induced DNA repair synthesis in mammalian cells. EMBO J. 12, 2109–2117. Morimoto, R. I. (2002). Dynamic remodeling of transcription complexes by molecular chaperones. Cell 110, 281–284. Mossink, M., van Zon, A., Franzel-Luiten, E., Schoester, M., Scheffer, G., Scheper, R., Sonneveld, P., and Wiemer, E. (2002). The genomic sequence of the murine major vault protein and its promoter. Gene 294, 225. Neuwald, A. F., Liu, J. S., Lipman, D. J., and Lawrence, C. E. (1997). Extracting protein alignment models from the sequence database Nucleic. Acid Res. 25, 1665–1667. Nikolaev, A. Y., Li, M., Puskas, N., Qin, J., and Gu, W. (2003). Parc: A cytoplasmic anchor for p53. Cell 112, 29–40.


81

Oei, S. L., Griesenbeck, J., Schweiger, M., Babich, V., Kropotov, A., and Tomilin, N. (1997). Interaction of the transcription factor YY1 with human poly(ADP-ribosyl) transferase. Biochem. Biophys. Res. Commun. 240, 108–111. Oei, S. L., Griesenbeck, J., Schweiger, M., and Ziegler, M. (1998). Regulation of RNA polymerase II-dependent transcription by poly(ADP-ribosyl)ation of transcription factors. J. Biol. Chem. 273, 31644–31647. Oei, S. L., and Shi, Y. (2001). Transcription factor Yin Yang 1 stimulates poly(ADPribosyl)ation and DNA repair. Biochem. Biophys. Res. Commun. 284, 450–454. Oka, J., Ueda, K., Hayaishi, O., Komura, H., and Nakanishi, K. (1984). ADP-ribosyl protein lyase: Purification, properties, and identification of the product. J. Biol. Chem. 259, 986–995. Okazaki, I. J., Kim, H. J., and Moss, J. (1997). Molecular cloning and characterization of lymphocyte and muscle ADP-ribosyltransferases. Adv. Exp. Med. Biol. 419, 129–136. Oliver, F. J., Menissier-de Murcia, J., Nacci, C., Decker, P., Andriantsitohaina, R., Muller, S., de la Rubia, G., Stoclet, J. C., and de Murcia, G. (1999). Resistance to endotoxic shock as a consequence of defective NF-kappa B activation in poly(ADP-ribose) polymerase-1 deficient mice. EMBO J. 18, 4446–4454. Panzeter, P. L., Zweifel, B., and Althaus, F. R. (1992). Synthesis of poly(ADP-ribose)-agarose beads: An affinity resin for studying (ADP-ribose)n-protein interactions. Anal. Biochem. 207, 157–162. Panzeter, P. L., Zweifel, B., Malanga, M., Waser, S. H., Richard, M., and Althaus, F. R. (1993). Targeting of histone tails by poly(ADP-ribose). J. Biol. Chem. 268, 17662–17664. Pedraza-Reyes, M., and Alvarez-Gonzalez, R. (1990). Oligo(30 -deoxy ADP-ribosyl)ation of the nuclear matrix lamins from rat liver utilizing 30 -deoxyNAD as a substrate. FEBS Lett. 277, 88–92. Pellat-Deceunynck, C., Wietzerbin, J., and Drapier, J. C. (1994). Nicotinamide inhibits nitric oxide synthase mRNA induction in activated macrophages. Biochem. J. 297, 53–58. Pirrotta, V. (2003). Transcription: Puffing with PARP. Science 299, 528–529. Pleschke, J. M., Kleczkowska, H. E., Strohm, M., and Althaus, F. R. (2000). Poly(ADP-ribose) polymerase binds to specific domains in DNA damage checkpoint proteins. J. Biol. Chem. 275, 40974–40980. Poirier, G. G., de Murcia, G., Jongstra-Bilen, J., Niedergang, C., and Mandel, P. (1982). Poly(ADP-ribosyl)ation of polynucleosomes cause relaxation of chromatin structure. Proc. Natl. Acad. Sci. USA 79, 3423–3427. Rippmann, J. F., Damm, K., and Schnapp, A. (2002). Functional characterization of the poly(ADP-ribose) polymerase activity of tankyrase 1, a potential regulator of telomere length. J. Mol. Biol. 323, 217–224. Rippmann, J. F., Damm, K., and Schnapp, A. (2003). Corrigendum to Functional characterization of the poly(ADP-ribose) polymerase activity of tankyrase 1, a potential regulator of telomere length [J. Mol. Biol. (2002) 323, 217–224]. J. Mol. Biol. 325, 1107. Roger, B., Moisand, A., Amalric, F., and Bouvet, P. (2002). Repression of RNA polymerase I transcription by nucleolin is independent of the RNA sequence that is transcribed. J. Biol. Chem. 277, 10209–10219. Ruf, A., Rolli, V., de Murcia, G. M., and Schulz, G. E. (1998). The mechanism of the elongation and branching reaction of poly(ADP-ribose) polymerase as derived from crystal structures and mutagenesis. J. Mol. Biol. 278, 57–65. Sauermann, G., and Wesierska-Gadek, J. (1986). Poly(ADP-ribose) effectively competes with DNA for histone H4 binding. Biochem. Biophys. Res. Commun. 139, 523–529. Saxena, A., Saffery, R., Wong, L. H., Kalitsis, P., and Choo, K. H. (2002a). Centromere proteins Cenpa, Cenpb, and Bub3 interact with poly(ADP-ribose) polymerase-1 protein and are poly(ADP-ribosyl)ated. J. Biol. Chem. 277, 26921–26926.

82

Tulin et al.

Saxena, A., Wong, L. H., Kalitsis, P., Earle, E., Shaffer, L. G., and Choo, K. H. (2002b). Poly(ADP-ribose) polymerase 2 localizes to mammalian active centromeres and interacts with PARP-1, Cenpa, Cenpb, and Bub3, but not Cenpc. Hum. Mol. Genet. 11, 2319–2329. Sbodio, J. I., Lodish, H. F., and Chi, N. W. (2002). Tankyrase-2 oligomerizes with tankyrase1 and binds to both TRF1 (telomere-repeat-binding factor 1) and IRAP (insulin-responsive aminopeptidase). Biochem. J. 361, 451–459. Shall, S. (2002). Poly(ADP-ribos)ylation: A common control process? Bioessays 24, 197–201. Shieh, W. M., Ame, J. C., Wilson, M. V., Wang, Z. Q., Koh, D. W., Jacobson, M. K., and Jacobson, E. L. (1998). Poly(ADP-ribose) polymerase null mouse cells synthesize ADP-ribose polymers. J. Biol. Chem. 273, 30069–30072. Simbulan-Rosenthal, C. M., Rosenthal, D. S., Luo, R. B., Samara, R., Jung, M., Dritschilo, A., Spoonde, A., and Smulson, M. E. (2001). Poly(ADP-ribosyl)ation of p53 in vitro and in vivo modulates binding to its DNA consensus sequence. Neoplasia 3, 179–188. Smith, S., and de Lange, T. (2000). Tankyrase promotes telomere elongation in human cells. Curr. Biol. 10, 1299–1302. Smith, S., Giriat, I., Schmitt, A., and de Lange, T. (1998). Tankyrase, a poly(ADP-ribose) polymerase at human telomeres. Science 282, 1484–1487. Soldatenkov, V. A., Chasovskikh, S., Potaman, V. N., Trofimova, I., Smulson, M. E., and Dritschilo, A. (2002). Transcriptional repression by binding of poly(ADP-ribose) polymerase to promoter sequences. J. Biol. Chem. 277, 665–670. Soriano, F. G., Virag, L., and Szabo, C. (2001). Diabetic endothelial dysfunction: Role of reactive oxygen and nitrogen species production and poly(ADP-ribose) polymerase activation. J. Mol. Med. 79, 437–448. Spellman, P. T., and Rubin, G. M. (2002). Evidence for large domains of similarly expressed genes in the Drosophila genome. J. Biol. 1, 5. Storozhenko, S., Inze, D., Van Montagu, M., and Kushnir, S. (2001). Arabidopsis coactivator ALY-like proteins, DIP1 and DIP2, interact physically with the DNA-binding domain of the Zn-finger poly(ADP-ribose) polymerase. J. Exp. Bot. 52, 1375–1380. Sullivan, B. A., Blower, M. D., and Karpen, G. H. (2001). Determining centromere identity: Cyclical stories and forking paths. Nat. Rev. Genet. 2, 584–596. Thompson, J. D., Higgins, D. G., and Gibson, T. J. (1994). CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positionspecific gap penalties and weight matrix choice. Nucleic. Acids Res. 22, 4673–4680. Tulin, A., and Spradling, A. (2003). Chromatin loosening by poly(ADP)-ribose polymerase (PARP) at Drosophila puff loci. Science 299, 560–562. Tulin, A., Stewart, D., and Spradling, A. C. (2002). The Drosophila heterochromatic gene encoding poly(ADP-ribose) polymerase (PARP) is required to modulate chromatin structure during development. Genes Dev. 16, 2108–2119. Uchida, K., Hanai, S., Ishikawa, K., Ozawa, Y., Uchida, M., Sugimura, T., and Miwa, M. (1993). Cloning of cDNA encoding Drosophila poly(ADP-ribose) polymerase: Leucine zipper in the auto-modification domain. Proc. Natl. Acad. Sci. USA 90, 3481–3485. Ueda, K., Oka, J., Naruniya, S., Miyakawa, N., and Hayaishi, O. (1972). Poly ADP-ribose glycohydrolase from rat liver nuclei, a novel enzyme degrading the polymer. Biochem. Biophys. Res. Commun. 46, 516–523. Valenzuela, M. T., Guerrero, R., Nunez, M. I., Ruiz de Almodovar, J. M., Sarker, M., de Murcia, G., and Oliver, F. J. (2002). PARP-1 modifies the effectiveness of p53-mediated DNA damage response. Oncogene 21, 1108–1116. Vaziri, H., West, M. D., Allsopp, R. C., Davison, T. S., Wu, Y. S., Arrowsmith, C. H., Poirier, G. G., and Benchimol, S. (1997). ATM-dependent telomere loss in aging human diploid fibroblasts and DNA damage lead to the post-translational activation of p53 protein involving poly(ADP-ribose) polymerase. EMBO J. 16, 6018–6033.


83

Wang, X., Ohnishi, K., Takahashi, A., and Ohnishi, T. (1998). Poly(ADP-ribosyl)ation is required for p53-dependent signal transduction induced by radiation. Oncogene 17, 2819–2825. Wang, Z., and Lindquist, S. (1998). Developmentally regulated nuclear transport of transcription factors in Drosophila embryos enable the heat shock response. Development 125, 4841–4850. Wang, Z. Q., Stingl, L., Morrison, C., Jantsch, M., Los, M., Schulze-Osthoff, K., and Wagner, E. F. (1997). PARP is important for genomic stability but dispensable in apoptosis. Genes Dev. 11, 2347–2358. Wesierska-Gadek, J., and Schmid, G. (2001). Poly(ADP-ribose) polymerase-1 regulates the stability of the wild-type p53 protein. Cell. Mol. Biol. Lett. 6, 117–140. Wielckens, K., Schmidt, A., George, E., Bredehorst, R., and Hilz, H. (1982). DNA fragmentation and NAD depletion: Their relation to the turnover of endogenous mono(ADP-ribosyl) and poly(ADP-ribosyl) proteins. J. Biol. Chem. 257, 12872–12877. Wieler, S., Gagne, J. P., Vaziri, H., Poirer, G. G., and Benchimol, S. (2003). Poly (ADP-ribose) polymerase-1 is a positive regulator of the p53-mediated G1 arrest response following ionizing radiation. J. Biol. Chem. 278, 18914–18921. Yamanaka, H., Penning, C. A., Willis, E. H., Wasson, D. B., and Carson, D. A. (1988). Characterization of human poly(ADP-ribose) polymerase with autoantibodies. J. Biol. Chem. 263, 3879–3883. Yang, Z. (1997). PAML: A program package for phylogenetic analysis by maximum likelihood. Comput. Appl. Biosci. 13, 555–556. Ziegler, M., and Oei, S. L. (2001). A cellular survival switch: Poly(ADP-ribosyl)ation stimulates DNA repair and silences transcription. Bioessays 23, 543–548.


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Centrosomes and Kinetochores, Who Needs ’Em? The Role of Noncentromeric Chromatin in Spindle Assembly Priya Prakash Budde and Rebecca Heald Department of Molecular and Cell Biology, University of California, Berkeley, California 94720 I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. XIV. XV.

Introduction Role of Centrosomes and Kinetochores in Spindle Assembly Role of Chromatin in Spindle Assembly How Does Chromatin Stabilize Microtubules? Chromatin-Associated Kinases and Microtubules Chromatin-Associated Phosphatases and Microtubule Dynamics The Elusive Chromatin Signal: RanGTP RCC1, the Chromatin Regulator of Microtubules The RanGTP Gradient Ran and Microtubule Stabilization and Organization Ran and Microtubule Nucleation Ran and Chromosome Condensation Chromatin-Associated Microtubule-Based Motor Proteins and Spindle Assembly Centrosomes and Kinetochores Revisited Conclusions Acknowledgments References

Accurate chromosome segregation is mediated by the mitotic spindle, a bipolar structure composed primarily of microtubules. Spindle assembly and microtubule dynamics are influenced by many cellular factors. In this chapter, we discuss the prominent models of spindle assembly and focus on the recently appreciated role of noncentromeric chromatin in promoting microtubule polymerization and spindle formation. In addition to presenting the evidence for the ability of chromatin to direct spindle assembly, this chapter describes the identification and characterization of molecular components involved in this process.

I. Introduction One of the most intriguing questions in cell biology is how a cell accurately partitions its genome to two daughter cells. Arguably, this is the most important task in the cell cycle. Errors in chromosome segregation in somatic Current Topics in Developmental Biology, Vol. 56 Copyright 2003, Elsevier Inc. All rights reserved. 0070-2153/03 $35.00

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cells can lead to aneuploidy, a hallmark of many cancers. Chromosome segregation is mediated by the mitotic spindle, which is a complex and dynamic structure. The spindle is composed of microtubules and associated proteins. Microtubules are dynamic polymers of and tubulin heterodimers (Desai and Mitchison, 1997). There are two diVerent aspects of microtubules that are exploited to facilitate processes such as spindle assembly. The first is the polarity of microtubules. The asymmetry of the / tubulin heterodimer imparts an intrinsic polarity to the microtubule, with the subunits exposed at one end, termed the (minus) end, and the subunits exposed at the other end, termed the þ (plus) end. Microtubule-based motors recognize the polarity of the microtubule lattice to move directionally on microtubules. Motors perform many functions, such as microtubule organization within the spindle, chromosome movement, and vesicular traYcking. The action of such motors is indispensable for spindle assembly. Also facilitating spindle assembly are the dynamic properties of microtubules. Microtubule dynamics are defined by four parameters: growth, shrinkage, the transition from growth to shrinkage (catastrophe), and the transition from shrinkage to growth (rescue). Microtubules switch stochastically between phases of growth and shrinkage, a property called dynamic instability (Mitchison and Kirschner, 1984). Microtubule dynamics are controlled by many proteins, and in vivo microtubules polymerize more rapidly and exhibit higher transition frequencies than tubulin in vitro (Desai and Mitchison, 1997). Furthermore, there is modulation of microtubule dynamics at diVerent stages of the cell cycle. In interphase, microtubules are long and relatively stable (half-life 5–10 minutes), whereas in prophase, microtubules are extremely short and have a 10-fold increase in their catastrophe frequency, which results in a dramatic increase in turnover rate (half-life 30 seconds–1 minute) (Heald, 2000). Despite this overall increase in dynamics, microtubules are stabilized around chromosomes and organized by motors to form the spindle. In this chapter, we address the role of chromosomes, specifically noncentromeric chromatin, in stabilizing microtubules to promote spindle assembly. We describe the identification and characterization of chromatin-associated proteins that impact microtubule dynamics and organization during spindle formation.

II. Role of Centrosomes and Kinetochores in Spindle Assembly Before discussing the role of noncentromeric chromatin in spindle assembly, it is important to review the prominent theory of spindle assembly that existed before the realization of the importance of chromatin in this process.

4. The Role of Noncentromeric Chromatin in Spindle Assembly

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This model focuses on the presence of centrosomes, focal microtubule nucleating sites, and kinetochores, specific sites for microtubule attachment to the chromosome. Centrosomes are microtubule organizing centers (MTOCs) that have a pair of centrioles surrounded by pericentriolar material (PCM).

-tubulin, a component of the PCM, is thought to nucleate microtubules (Moritz et al., 1995; Zheng et al., 1995). The kinetochore is a proteinaceous, specialized trilamellar structure that forms on the centromere of a chromosome. The centromere is a heterochromatic region of DNA that appears as the primary constriction on a chromosome. Each mitotic chromosome is made up of two sister chromatids and therefore has two kinetochores. At the onset of mitosis, the duplicated centrosomes separate to form the poles of the spindle, and microtubules emanate radially from the centrosome with their minus ends anchored in the PCM and their plus ends extending outward. By a process called ‘‘search and capture’’ (Fig. 1), microtubules undergo rapid rounds of growth and shrinkage until they encounter a kinetochore. Once a microtubule encounters a kinetochore, it stably attaches and drags

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Figure 1 The search and capture model for spindle assembly. In this model, centrosomes (yellow), which are dominant microtubule nucleating sites, and kinetochores (red), which are specialized sites of microtubule attachment on the chromosomes, are crucial for spindle assembly. Microtubules (green) grow and shrink from the centrosome until they encounter a kinetochore, which stabilizes the microtubule, thereby promoting spindle assembly. (See Color Insert.)

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the chromosome poleward until a microtubule from the other pole attaches to the opposite kinetochore, and then a ‘‘tug of war,’’ or chromosome congression, ensues with the chromosome being pulled toward opposite poles. There are many unstable attachments, such as when microtubules from the same pole attach to both kinetochores, or when microtubules from both poles attach to one kinetochore, but these aberrant interactions are corrected. In this model (Kirschner and Mitchison, 1986), centrosomes are crucial for nucleating microtubules, and kinetochores are crucial for chromosome/ microtubule interactions and stabilizing the microtubules. There are several lines of evidence that support this model. First, there are cell types that are unable to form spindles in the absence of centrosomes (Zhang and Nicklas, 1995b). Second, the half-life for microtubules attached to a kinetochore is substantially longer (5–9 minutes) than that for other mitotic microtubules (Hyman and Karsenti, 1996). Third, kinetochores have been shown to stabilize and bind to microtubules in vitro (Mitchison and Kirschner, 1985a; Mitchison and Kirschner, 1985b). Fourth, spindle formation occurs in chromosome-free mouse oocyte fragments by interactions between microtubule asters (Brunet et al., 1998). Finally, it has even been shown that in the absence of chromosomes, anaphase spindle movements can occur (Zhang and Nicklas, 1996). Chromosomes were therefore thought to be mere passengers in mitosis.

III. Role of Chromatin in Spindle Assembly Over the last 20 years, the view of chromosomes as passive participants in mitosis has changed dramatically. A large amount of the evidence supporting an active role for chromatin in spindle assembly has come from female meiotic cells that lack centrosomes. For example, in oocytes of Drosophila melanogaster, chromosomes play a crucial role in spindle assembly, with spindle formation beginning with microtubules emanating from the chromosomes. These microtubules lengthen and become organized into a spindle (Theurkauf and Hawley, 1992). D. melanogaster spermatocytes contain centrosomes; however, a chromosome detached from the main centrosomal spindle assembled a spindle around it in the absence of centrosomes (Church et al., 1986). Sawin and Mitchison (1991) showed that there is a strong bias of microtubule growth toward sperm chromatin in Xenopus laevis egg extracts. Finally, removal of the entire nucleus, or the chromosomes specifically, in grasshopper spermatocytes prevented spindle formation despite the presence of centrosomes. However, the role of chromosomes in this particular system is unclear because removing centrosomes also prevented spindle assembly (Zhang and Nicklas, 1995a).


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These studies illustrate the importance of chromatin in spindle assembly, but in all these examples, the chromosomes have centromeres and, therefore, kinetochores. The eVect of chromatin on spindle assembly could be explained by the presence of kinetochores, which direct microtubule attachment and stability, thereby promoting spindle formation. It was not clear from these studies if noncentromeric chromatin had a function in spindle assembly. Some indirect evidence for the role of chromosome arms in capturing microtubules was demonstrated by the discovery of polar ejection forces. Examination of monooriented chromosomes and chromosome fragments during mitosis suggested the existence of polar ejection forces that push chromosome arms away from the spindle poles. In Newt lung cells, severing a chromatid arm resulted in the arm being pushed away radially from the spindle pole by microtubules (Rieder et al., 1986). This polar ejection force seems to be generated by chromosome arms and opposes the poleward force generated at the kinetochore (Rieder and Salmon, 1994). More compelling evidence to show that the ability of chromosomes to direct spindle assembly is not due only to the presence of kinetochores has been obtained in several systems. By extracting chromosomes by micromanipulation from grasshopper spermatocytes and measuring spindle mass, Nicklas and Gordon (1985) showed that spindle mass correlated with the number of chromosomes present. They also showed that the loss in mass on removal of chromosomes was not simply due to the loss of kinetochore microtubules, indicating that microtubule mass in the spindle depends on chromosome arms (Nicklas and Gordon, 1985). Zhang et al. showed that chromatin mass, as opposed to kinetochore number, is proportional to microtubule assembly. Additionally, Karsenti et al. (1984) showed that injection of DNA sources that do not possess centromeres, such as bacteriophage or Escherichia coli DNA, into X. laevis eggs arrested in metaphase promotes local microtubule assembly despite the absence of kinetochores. The extent of microtubule assembly was correlated with the size of the DNA molecules injected (Karsenti et al., 1984), indicating that there may be a microtubule-stabilizing activity on chromatin. In fact, microtubules have been shown to grow preferentially toward nonkinetochore chromatin in X. laevis egg extracts (Dogterom et al., 1996). Furthermore, in maize meiotic mutants that generate univalents, spindles assembled around chromosomes without the requirement for paired kinetochores (Chan and Cande, 1998). In meiosis I mouse oocytes, analysis of the interaction between microtubules and the chromosomes during spindle assembly by electron microscopy revealed that microtubules attach to the chromosome arms at sites that do not correspond to the kinetochore (Brunet et al., 1999). These experiments showed more directly that noncentromeric chromatin plays a role in spindle assembly.

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Figure 2 Local stabilization model for spindle assembly. In this model, microtubules (green) are stabilized around chromatin (DNA beads [blue]) and organized by microtubule-based motors into ordered, polar arrays leading to spindle assembly. (See Color Insert.)

To definitively address the role of noncentromeric chromatin in spindle assembly, Heald et al. (1996) introduced beads coated with plasmid DNA into X. laevis egg extracts and assayed for spindle formation. Bipolar spindles formed around the DNA-coated beads, demonstrating the importance of noncentromeric chromatin in directing spindle assembly (Heald et al., 1996). The bead spindles formed in the absence of centrosomes, indicating that the beads recruited all the proteins necessary to promote microtubule growth and stability. Additionally, due to the lack of centromeric sequences on plasmid DNA, the bead spindles formed in the absence of centromeres. It has been observed in a number of cases that in the absence of a proper centromere a neocentromere can form, upon which the kinetochore is established (Willard, 2001). By electron microscopy there are no detectable kinetochores assembled on the plasmid DNA in bead spindles. This indicates that the beads recruited the proteins required for microtubule attachment and that the centromere/kinetochore is not essential for this process. These results support a ‘‘local stabilization’’ model (Hyman and Karsenti, 1996; Karsenti and Vernos, 2001) wherein spindle assembly is a chromatin-driven


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process with chromatin possessing the ability to stabilize, attach to, and organize microtubules around DNA (Fig. 2). The plasmid-coated DNA beads are a legitimate source of chromatin by several criteria. First, a complex pattern of proteins has been shown to associate with the beads with diVerences in interphase and mitosis (Budde et al., 2001). Second, the plasmid DNA on the beads assembled into chromatin as assayed by histone association and nucleosome formation. Furthermore, characteristic chromosomal markers, such as topoisomerase II, are recruited to the beads. Spindle assembly around beads can be described in several stages. First, there is nucleation of microtubules around the beads, followed by the sorting of microtubules into polar arrays. The microtubules are then focused at the poles, which is followed by extension of the spindle (Fig. 2). Microtubule-based motor proteins are crucial for most of the steps described for the assembly of bead spindles. To form the characteristic bipolar structure, these motors are involved in the organization of microtubules into ordered polar arrays, pole focusing, and spindle extension (Walczak et al., 1998).

IV. How Does Chromatin Stabilize Microtubules? The initial models for the role of chromatin in spindle assembly were adapted from the known importance of phosphorylation in many aspects of the cell cycle. These early models predicted a phosphorylation-based local microtubule-stabilizing environment around chromatin to promote spindle assembly. For example, if there existed a soluble kinase that phosphorylated and inactivated microtubule-associated proteins (MAPs) and a chromatin-bound opposing phosphatase, this would result in dephosphorylated, active MAPs around the chromatin, resulting in a local stabilization of microtubules. Conversely, inactivating a destabilizing factor around chromatin would create the same eVect (Andersen, 1999, 2000). Presumably, a coordination of both of these activities could promote microtubule stability around chromatin. There was some evidence to support the kinase/phosphatase gradient theory, which is discussed in the following section. Additional theories hypothesized that -tubulin might be recruited to chromatin to nucleate microtubules, but this has not been substantiated.

V. Chromatin-Associated Kinases and Microtubules Protein phosphorylation is crucial in every stage of the cell cycle, with mitosis being no exception. The master regulator of mitosis is the kinase, Cdk1. It is the founding member of a family of cyclin-dependent kinases that regulates progression through the cell cycle. Cdk1 activity has been shown to

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be important in all aspects of mitosis such as nuclear envelope breakdown, chromosome condensation, spindle assembly, and Golgi fragmentation. Inactivation of Cdk1 is crucial for mitotic exit (Nigg, 2001). This kinase phosphorylates many spindle-associated proteins, including the motor Eg5 (Blangy et al., 1995). The addition of Cdk1 kinase to interphase extract mimicked the cell cycle transition to mitosis with respect to microtubule dynamics, highlighting the importance of this kinase in mitotic entry (Verde et al., 1990). In addition to Cdk1, there are several kinases that have been shown to have mitotic roles. These include Polo, Aurora kinases, CaM kinase II, Nek2, Bub1, BubR1, and Mps1, which play key roles in spindle assembly, centrosome function, the checkpoint, and cytokinesis (BischoV and Plowman, 1999; Fry et al., 1998; Giet and Prigent, 1999; Glover et al., 1998; Matsumoto and Maller, 2002; Nigg, 1998, 2001). Although there exist many mitotic centromere/kinetochore-associated kinases, Polo is one of the few kinases that has been shown, in a small number of organisms, to have a more general localization to the chromosomes. The Polo family of serine/threonine kinases are involved in mitotic progression in many organisms (Glover et al., 1998; Nigg, 1998). The founding family member is D. melanogaster Polo, which was found to be involved in centrosome function (Sunkel and Glover, 1988). Subsequently, this kinase has been shown to be conserved from yeast to humans, with roles in almost every aspect of mitosis. This kinase has been shown to localize to centrosomes, spindle microtubules, chromosomes, centromeres/kinetochores, and the midbody (Arnaud et al., 1998; Logarinho and Sunkel, 1998; Moutinho-Santos et al., 1999; Qian et al., 1998; Wianny et al., 1998). In frog embryos, Polo-like kinase in Xenopus (Plx1) is localized to centrosomes early in mitosis, on chromosomes and the spindle at metaphase, and at the midbody late in mitosis (Qian et al., 1999). A similar localization of Plx1 to chromatin and spindle microtubules has been observed in in vitro sperm spindles assembled in X. laevis egg extracts (Budde et al., 2001). In syncitial and cellularized D. melanogaster embryos, Polo has been shown to associate with condensing chromosomes (Llamazares et al., 1991). Clearly, there are organism- and tissue-specific diVerences in the localization of Polo. Members of the Polo kinase family are important in every stage of mitosis. Studies over the last 15 years have implicated Polo kinase in centrosome assembly and separation (Lane and Nigg, 1996; Sunkel and Glover, 1988), exit from mitosis by APC activation (Charles et al., 1998; Descombes and Nigg, 1998; Kotani et al., 1998; Shirayama et al., 1998), and cytokinesis (Bahler et al., 1998; Carmena et al., 1998; Glover et al., 1998; Nigg, 1998; Ohkura et al., 1995). Despite the multiple roles that Plks play in mitosis, only a handful of Plk targets have been identified. These include Cdc25 phosphatase, cyclin B1, and cohesin. Phosphorylation by Plk is important for the

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function of these proteins (Kumagai and Dunphy, 1996; Sumara et al., 2002; Toyoshima-Morimoto et al., 2001). Plk also has been shown to interact with CHO1/MKLP1, which is a motor required for establishing the spindle midzone. This interaction may be important for the role of Polo in cytokinesis (Lee et al., 1995). A key question that remains unanswered is this: What are the substrates of the chromosome-associated pool of Polo? Plx1 has been shown to be important for the dissociation of cohesin from chromosome arms (Sumara et al., 2002). This is perhaps one function that could be attributed to the chromosome-associated pool of this kinase. How does Polo aVect microtubules? Recently, Plx1 was shown to be involved in the hyperphosphorylation of Op18 in mitosis in X. laevis egg extracts (Budde et al., 2001). Op18/stathmin is a small, 17 kDa, heat-stable protein shown to depolymerize microtubules by promoting catastrophe and by sequestering tubulin (Cassimeris, 2002). Andersen et al. (1997) showed that Op18, which is negatively regulated by phosphorylation, is hyperphosphorylated in X. laevis egg extracts, but only in the presence of mitotic chromatin beads. This indicated that a factor or factors of chromosomes might promote microtubule polymerization and spindle assembly by inactivating Op18 (Andersen et al., 1997). It was later demonstrated that chromatinassociated proteins phosphorylated Op18 in vitro and that Plx1 was present in this fraction of proteins. Removal of Plx1 from X. laevis egg extracts severely aVected Op18 hyperphosphorylation (Budde et al., 2001). Also, PP

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Figure 3 Model for chromatin-associated Plx1 in the regulation of microtubule dynamics in Xenopus laevis egg extracts. The red curved lines represent Op18. P is a phosphate group. Unphosphorylated Op18 promotes microtubule depolymerization. Op18 that encounters mitotic chromatin is hyperphosphorylated in a Plx1-dependent manner, thereby promoting microtubule stabilization. Reproduced from The Journal of Cell Biology, 2001, 153, 149-157 by copyright permission of The Rockefeller University Press. (See Color Insert.)

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Plx1 depletion decreased microtubule polymerization in the extracts, which is consistent with a decrease in Op18 phosphorylation in the absence of Plx1. Additionally, Plx1 depletion caused spindle defects where the spindles were often not fully extended, and there was an eVect on chromatin–microtubule interactions where the DNA beads seemed to ‘‘fall out’’ of the spindle. It is likely that Plx1 regulates multiple proteins within the spindle. Although Op18 appears to be regulated by Plx1, it does not seem to be a direct substrate (Budde and Heald, unpublished results). Regardless, these results support the theory of a kinase on chromatin inactivating a microtubule destabilizing activity, thereby promoting spindle assembly (Fig. 3). Although this certainly contributes to spindle assembly, Op18 immunodepletion from extracts has very little eVect on spindle assembly, indicating that this is not a dominant factor regulated by chromatin (Andersen et al., 1997).

VI. Chromatin-Associated Phosphatases and Microtubule Dynamics Dephosphorylation plays an equally important part in the regulation of the cell cycle. Serine/threonine phosphatases in particular have been shown to be crucial for mitosis. For example, the phosphatase Cdc25 dephosphorylates and activates Cdk1, allowing entry into mitosis. Other serine/threonine phosphatases that are important in mitosis are protein phosphatase 2A (PP2A) and protein phosphatase 1 (PP1). Both of these phosphatases have been shown to have an eVect on microtubule dynamics. Because PP1 localizes to chromosomes and is important in mitosis, it is a likely candidate for a link between chromatin and microtubules. Immunolocalization of PP1 in mammalian fibroblasts revealed that this enzyme colocalized with chromosomes during all stages of mitosis (Fernandez et al., 1992). In HeLa cells, PP1, one of the isoforms of PP1, localized to chromosomes throughout the cell cycle by immunofluorescence with antibodies specific to the PP1 isoform (Andreassen et al., 1998). This phosphatase has been shown to have a mitotic function in multiple organisms (Axton et al., 1990; Doonan and Morris, 1989; Fernandez et al., 1992; Ohkura et al., 1989). Microinjection of function-blocking antibodies to PP1 before division blocked cells in metaphase (Fernandez et al., 1992). This study also showed a role for PP1 in exit from mitosis. Mutations in PP1 in multiple organisms resulted in aberrant mitosis with problems in sister chromatid segregation, mitotic exit, and chromosome condensation. The function of PP1 in X. laevis egg extracts has been addressed by the use of specific PP1 inhibitors. In these extracts, PP1 is thought to be important for microtubule dynamics in the transition into and out of mitosis (Tournebize et al., 1997).


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Few spindle-related targets of this phosphatase have been identified, but it seems to oppose the activity of the kinetochore-associated kinase Aurora B in worms, yeast, and frogs (Francisco et al., 1994; Murnion et al., 2001; Rogers et al., 2002; Sassoon et al., 1999). In budding yeast, both of these proteins are important for chromosome segregation, particularly kinetochore/microtubule interactions (Biggins et al., 1999). Identification of molecules that relay signals from PP1 to microtubules will shed light on the function of this phosphatase in spindle assembly. Phosphatases achieve a dynamic range of functions by association with regulatory B subunits that often determine the activity and subcellular localization of the enzyme (Virshup, 2000). Identification of the regulatory subunit that targets PP1 to chromatin would provide a reagent to remove a specific pool of PP1 and assess the importance of chromatin-associated PP1 on spindle assembly.

VII. The Elusive Chromatin Signal: RanGTP To the surprise of the entire field, a major chromatin signal for microtubule stabilization was not a kinase/phosphatase network but an unexpected factor that plays a key role in interphase: Ran, a small GTPase of the Ras superfamily. The GTP-bound form of Ran plays an important, wellcharacterized role in interphase in nucleocytoplasmic transport (Gorlich and Kutay, 1999). The guanine nucleotide exchange factor (GEF) for Ran, RCC1, is bound to chromosomes, thereby generating RanGTP in the nucleus. The low intrinsic GTPase activity of Ran is enhanced 105-fold (Klebe et al., 1995) by its GTPase activating protein (GAP), RanGAP (with the accessory protein RanBP1), which resides exclusively in the cytoplasm. Due to the localization of Ran regulators, there is a compartmentalization of Ran with the GTP form in the nucleus and the GDP form in the cytoplasm. The diVerent states of Ran bind diVerent import and export factors, thus promoting transport of cargoes into and out of the nucleus. Due to its critical role in nucleocytoplasmic transport, pleiotropic eVects of Ran mutants masked its role in mitosis. Despite this issue, there were some early studies that implicated Ran in mitosis. The first link was discovered in tsBN2 cells. These cells are mutant BHK cells that contain a temperature-sensitive allele of RCC1. tsBN2 cells enter mitosis prematurely at the restrictive temperature, resulting in premature chromosome condensation, nuclear envelope breakdown (NEB), and Cdk1 activation (Nishitani et al., 1991). In budding yeast, Saccharomyces cerevisiae, overexpression of the GEF of Ran (Prp20 in yeast) could suppress some lethal hyperstable -tubulin mutations (Kirkpatrick and Solomon, 1994). Also, temperaturesensitive mutations in a yeast Ran-binding protein, Yrb2, led to spindle

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misorientation defects (Ouspenski, 1998). Interestingly, overexpression of a Ran-binding protein, RanBPM, which is at the centrosome, caused ectopic microtubule aster formation in cells (Nakamura et al., 1998). Again, the critical role that Ran plays in nucleocytoplasmic transport made it diYcult to determine whether the eVect of these mutants on spindle assembly was direct or a consequence of disrupting nucleocytoplasmic transport. It was particularly diYcult to evaluate the data from yeast, which has a closed mitosis; i.e., the nuclear envelope does not break down during mitosis, and nucleocytoplasmic transport occurs even during the mitotic phase of the cell cycle. In higher eukaryotes, the nuclear envelope breaks down in mitosis and the contents of the nucleus and the cytoplasm mix, thereby changing the spatial separation of Ran regulatory factors. Seminal studies done in mitotically arrested X. laevis egg extracts have redefined the function of RanGTP and demonstrated that it is as crucial in mitosis as it is in interphase. This was largely due to the power of the X. laevis egg extract system. Because extracts can be prepared from eggs arrested in metaphase of meiosis II and are open to manipulation, the role of Ran could be addressed in the complete absence of any eVect that may be caused by the disruption of nucleocytoplasmic transport. Experiments done in this system clearly revealed the importance of Ran in spindle assembly. In the absence of chromosomes and centrosomes, the addition of a mutant of RanGTP that locks it in the GTPbound state (RanQ69L or RanL43E) caused microtubule assembly and even spindle formation (Kalab et al., 1999; Ohba et al., 1999; Wilde and Zheng, 1999)! Removal of Ran from X. laevis egg extracts severely impaired spindle assembly (Nachury et al., 2001). However, the eVect of Ran on microtubule polymerization is not direct because RanGTP had no eVect on pure microtubules in vitro (Wilde et al., 2001; Wilde and Zheng, 1999). Subsequently, Ran and its regulators have been shown to be important in spindle formation and chromosome positioning in worms (Askjaer et al., 2002; Bamba et al., 2002).

VIII. RCC1, the Chromatin Regulator of Microtubules By generating RanGTP, chromatin-bound RCC1 is crucial for spindle assembly. Immunodepletion of RCC1 from X. laevis egg extracts severely inhibited aster formation by sperm nuclei (Ohba et al., 1999). Similar results can be achieved by inhibiting RCC1 by the addition of RanT24N, a mutant allele of Ran that is locked in the GDP or nucleotide-free form and inhibits the GEF activity of RCC1. The addition of RCC1 alone to extracts promoted microtubule assembly in the absence of sperm, and this eVect was reversed by the addition of excess RanGAP or RanBP1, both of which promote RanGTP hydrolysis. To analyze the role of RCC1 in


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chromatin-mediated spindle assembly, Carazo-Salas et al. (1999) examined RCC1 in bead spindles. They showed that RCC1, but not RanGAP or RanBP1, was present on chromatin beads. Inhibiting RCC1 prevented bead spindle assembly, and the addition of an excess of RCC1 or RanGTP partially uncoupled spindle assembly from the chromatin beads. Interestingly, it is not clear what the role of RCC1 is in spindle formation in worms, where RNAi studies have shown in one case that RCC1 is crucial for spindle assembly but in another case that RCC1 RNAi does not aVect spindle assembly but showed anaphase chromosome segregation defects (Askjaer et al., 2002; Bamba et al., 2002). RCC1 binds directly to mononucleosomes and to histones H2A and H2B, which results in a moderate stimulation of its GEF activity (Nemergut et al., 2001). RCC1 has been shown to be on chromatin in many organisms. In live D. melanogaster embryos injected with rhodamine–RCC1 and expressing GFP histones, RCC1 colocalized with the histones throughout the cell cycle (Trieselmann and Wilde, 2002). Ran itself has been shown to localize (Nachury et al., 2001) and bind to chromatin (Bilbao-Cortes et al., 2002). Removal of RCC1 from X. laevis egg extracts caused a fourfold decrease, but not an elimination, of the binding of Ran to chromatin. This indicated that a fraction of Ran binds to chromatin independently of RCC1. These studies also demonstrated that Ran binds to sperm chromatin in vitro in the absence of RCC1. In fact, they also indicated that Ran may play a role in RCC1 binding to chromatin by showing that there is a twofold increase in the amount of RCC1 bound to chromatin in the presence of Ran (Bilbao-Cortes et al., 2002). However, the association of Ran with chromatin is controversial. A recent report demonstrated in live D. melanogaster embryos that rhodaminelabeled Ran is confined to the spindle and is not on chromatin (Trieselmann and Wilde, 2002). In worms, Ran localizes to the kinetochore in metaphase and anaphase (Bamba et al., 2002). Of course, some of this variability may be due to diVerences between organisms. Additionally, RanGAP, which associates with the spindle in Drosophila and mammalian cells (Joseph et al., 2002; Trieselmann and Wilde, 2002), has been shown in mammalian tissue culture cells to be at kinetochores (Joseph et al., 2002). Whereas it is clear that RCC1 is a bona fide chromatin protein, the mitotic localization of other members of the Ran cycle as chromatin-associated proteins has not yet been thoroughly established.

IX. The RanGTP Gradient The discovery of a role for Ran in mitosis led to the hypothesis that chromosome-bound RCC1 creates a gradient of RanGTP around the chromosomes, which promotes spindle assembly (Heald and Weis, 2000).

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The existence of the RanGTP gradient has been visualized in interphase and mitosis using FRET-based biosensors for RanGTP (Kalab et al., 2002). But how does the RanGTP gradient promote microtubule assembly? The elucidation of the role of RanGTP in mitosis was greatly aided by the excellent characterization of its interactions in interphase. Importins are a protein family that binds nuclear localization sequence (NLS)-containing proteins and translocates them into the nucleus. RanGTP, which is generated exclusively in the nucleus, causes the disruption of the importin/cargo complex, releasing the cargo in the nucleus (Weis, 1998). Similarly, in mitosis, RanGTP disrupts complexes between importins and NLS-containing proteins that promote spindle assembly (described in detail in the next section), thereby releasing them in the vicinity of chromosomes to stabilize microtubules (Fig. 4) (Dasso, 2002). This system ensures that microtubulestabilizing factors are only released in the presence of chromatin. In interphase, these factors by default reside in the nucleus. This also sequesters them away from microtubules in this stage of the cell cycle when they may not be required. Removal of Ran from X. laevis egg extracts disrupted sperm spindle formation despite the presence of centrosomes, indicating that Ran plays a general role in the mechanism of spindle assembly (Nachury et al., 2001). It remains to be determined if and to what extent the Ran gradient exists in other organisms and if it functions to create a gradient of microtubule-stabilizing activity around chromatin.

X. Ran and Microtubule Stabilization and Organization Once the role of Ran in spindle assembly was established, the next step was to identify proteins that relayed the signal between Ran and microtubules. By a series of elegant experiments, Nachury et al. (2001) and Gruss et al. (2001) showed that NuMA and TPX2, respectively, are regulated by RanGTP, the details of which are described later. Nachury et al. showed that removal of RanGTP-binding proteins from X. laevis egg extracts (RBP extracts) resulted in spontaneous aster formation. The addition of Importin , or its cargo-binding domain alone, to the ectopic asters in the RBP extracts reduced the spontaneous aster formation, demonstrating that Importin inhibited microtubule polymerization in the extracts. It appeared that Importin sequestered aster-promoting activities (APAs), thereby inhibiting microtubule polymerization in the extracts, and on the addition of RanGTP the APAs are released to promote spindle assembly. This would be analogous to the role of RanGTP in interphase where RanGTP binds to and disrupts Importin /Importin /cargo complexes, which results in the release of cargoes. In support of this theory, spontaneous aster formation in RBP extracts was abolished when proteins that were bound to the cargo-binding


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Figure 4 The Ran gradient. Chromosomally associated RCC1 (Ran GEF) and cytoplasmic RanGAP and RanBP1 generate a gradient of Ran GTP (yellow to red) around chromosomes (blue), thereby releasing spindle assembly factors (SAFs) (Dasso, 2002) from Importin / and promoting microtubule stabilization and organization in the vicinity of chromosomes. This gradient could also function to bias the growth of microtubules (green) from the centrosomes (yellow) toward the chromosomes. (See Color Insert.)

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domain of Importin (APA extracts) were removed from the extract. This eVect of Importin on aster formation was shown to be through Importin , though there also appeared to be an Importin –specific APA activity independent of Importin . NuMA was shown to be one of the APAs sequestered by Importin through Importin . NuMA is a microtubule-associated protein (MAP) that is important for microtubule organization in the spindle. Addition of the recombinantly purified C-terminal tail of NuMA to RBPAPA extracts caused spontaneous aster formation. The NuMA tail protein bound to Importin in vitro only in the presence of Importin , and this interaction was disrupted by the addition of RanGTP. NuMA as a cargo of Importin had also been identified by Wiese et al. (2001). Therefore, RanGTP causes the local release of microtubule-stabilizing cargoes around chromatin that are otherwise sequestered by Importin . Microinjection of the cargo-binding domain of Importin into Ptk1 cells resulted in abnormal mitosis (Nachury et al., 2001), and RNAi of Importins and caused spindle defects (Askjaer et al., 2002), indicating that the function of Importin is conserved. Gruss et al. (2001) identified a cargo of Importin biochemically. Aster formation by activated Ran was severely inhibited by the addition of exogenous Importin to the extracts, indicating that Importin inhibited aster formation. Also, addition of a cargo of Importin , BSA conjugated to an NLS sequence, caused ectopic microtubule polymerization in the extract, indicating that Importin was sequestering factors that promoted microtubule assembly. To identify cargoes of Importin that were important for aster formation, excess RanGTP was added to extracts, which caused the release of the cargoes of Importin and resulted in aster formation. A tagged version of Importin was then added in excess to the extracts to retrieve and remove its released cargoes. This addition of Importin abolished the Ran-induced aster formation. HeLA nuclear extracts, which contain abundant Importin cargoes, were fractionated, and the fractions were assayed for microtubule polymerization upon addition to extracts that had all Importin cargoes removed. This approach identified TPX2. TPX2 (targeting protein for the X. laevis kinesin-like protein [Xklp2]) is a MAP that mediates binding of the motor Xklp2 to microtubules. Removal of this protein from X. laevis egg extracts resulted in decreased microtubule density, indicating that it has a stabilizing eVect on microtubules (Wittmann et al., 2000). Addition of excess TPX2 to extracts caused microtubule assembly, and this activity is inhibited on the addition of excess Importin . Furthermore, in the extracts treated with activated RanGTP that have Importin cargoes removed, the addition of TPX2 rescued microtubule polymerization. Finally, activated RanGTP did not induce microtubule polymerization when TPX2 was immunodepleted from extracts. Therefore, TPX2 is required for microtubule polymerization by RanGTP.


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The function of RanGTP in mitosis to release cargoes bound to the importins is analogous to its role in interphase in nucleocytoplasmic transport. It follows that proteins that are regulated in this manner by Ran in mitosis have an NLS and are nuclear in interphase, which is true of both NuMA and TPX2. Several attractive candidates that meet this criterion and are important for microtubule stability and organization include the MAP XMAP230 and the motors MKLP1/CHO1, XKCM1, Xklp1, and XCTK2. Whereas some of the molecular components that mediate the eVect of RanGTP on microtubules have been identified, how much of these cargoes are sequestered and the precise mechanism of their inhibition by importins remains to be determined. RanGTP also aVects microtubule organization by promoting þ end directed motor activity. It has been shown that the addition of RanL43E (constitutively active RanGTP) to extracts increased the amount of motile Eg5 (Wilde et al., 2001). Eg5 is a tetrameric kinesin that has been shown to play a significant role in establishing and maintaining spindle bipolarity. Stimulating the activity of Eg5 is one way that Ran promotes microtubule organization to favor the formation of a bipolar spindle. It has also been shown that RanGTP stimulated microtubule assembly by increasing the rescue frequency of microtubules three- to eight-fold (Carazo-Salas et al., 2001; Wilde et al., 2001). Clearly, Ran is aVecting microtubule dynamics, stability, and organization by regulating a number of proteins.

XI. Ran and Microtubule Nucleation Nucleation is the rate-limiting step in microtubule assembly. Microtubules in most organisms are nucleated from the centrosome. Oakley and Oakley (1989) discovered the existence of -tubulin, a form of tubulin that localizes to centrosomes in complex eukaryotes and to spindle pole bodies of fungi. A protein complex called the -TURC ( -tubulin ring complex) was purified from X. laevis egg extracts and shown to nucleate microtubules in vitro and to cap minus ends (Moritz et al., 1995; Zheng et al., 1995). To date, other protein has clearly been shown to promote microtubule nucleation directly. RanGTP is thought to indirectly promote the microtubule nucleating activity of centrosomes (Carazo-Salas et al., 2001). In these experiments, demembranated sperm, which have an associated basal body, were incubated in extracts in the presence or absence of RanQ69L and nocodazole to prevent microtubule assembly. When these basal bodies are incubated in extracts, they recruit components from the extracts, such as the -TURCs, to become centrosomes, which are competent to nucleate microtubules. The sperm and associated centrosome were reisolated from the extracts and then

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incubated with pure tubulin. In the presence of RanQ69L, the centrosome recruited more -tubulin and promoted more microtubule assembly. Therefore RanGTP, which is a signal generated by chromatin-bound RCC1, enhances microtubule nucleation by centrosomes by an as yet unknown mechanism.

XII. Ran and Chromosome Condensation Does Ran play a role in mitosis apart from spindle assembly? Microinjection of the cargo-binding domain of Importin into somatic cells disrupted spindle assembly (Nachury et al., 2001). These injections were done in prophase and prometaphase to prevent any eVect on nucleocytoplasmic transport. In addition to the defects seen in microtubule stability and organization, over 65% of the cells injected with the cargo-binding domain of Importin had abnormal chromosomes that were decondensed and had unusual morphology. Are cargoes of Importin involved in mitotic chromosome condensation? Does RanGTP regulate these cargoes in the same manner as the microtubule-stabilizing cargoes? If such cargoes exist, how do they promote chromosome condensation? These are some of the questions that need to be addressed to determine whether RanGTP has a function in chromosome compaction.

XIII. Chromatin-Associated Microtubule-Based Motor Proteins and Spindle Assembly Motor proteins are ATPases that use the energy of ATP hydrolysis to move directionally along microtubules. In mitosis, these motors mediate three diVerent processes: cross-linking and sliding microtubules relative to other microtubules, transportation of cargo molecules along spindle microtubules, and regulation of microtubule dynamics (Sharp et al., 2000). The importance of microtubule-based motor proteins is clearest for the selforganization of microtubules into a spindle in chromatin-mediated spindle assembly. They are crucial for establishing polar arrays of microtubules and for maintaining spindle structure (Walczak et al., 1998). There are two general classes of motors, the kinesin superfamily, which includes both þ and end directed motors, and cytoplasmic dynein, which interacts with dynactin to generate end directed activity. Chromatin-associated kinesin-like motors have been identified in many organisms and have been shown to be crucial in spindle assembly. Following are descriptions of the nonkinetochore chromatin-associated motors that have been characterized.


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Xklp1: This motor is a chromokinesin with its motor domain at its amino terminus, an NLS in its stalk, and a zinc finger domain in its tail. It localizes to chromosomes throughout the cell cycle until anaphase, when it transfers to the spindle midzone and midbody. Injection of antisense oligos of Xklp1 into X. laevis oocytes that were subsequently fertilized prevented the oocytes from undergoing any cleavage, or they went through abnormal cleavages. Immunodepletion of Xklp1 from X. laevis egg extracts resulted in aberrant chromatin microtubule interactions. It is absolutely required for both sperm and bead spindle assembly in X. laevis egg extracts (Vernos et al., 1995; Walczak et al., 1998). This motor seems to serve a docking function to maintain attachment of chromosomes to the þ ends of microtubules. By attaching to microtubules and simultaneously moving toward their þ ends, Xklp1 pushes microtubules away from the chromosomes, thereby promoting microtubule/chromatin attachments and spindle extension (Fig. 5a). Xkid: This kinesin is the X. laevis homologue of human Kid (kinesin-like DNA binding protein) (Tokai et al., 1996). Removal of this protein from X. laevis egg extracts did not aVect spindle assembly but caused chromosome misalignment. This motor is responsible for pushing the chromosome arms toward the equator (polar ejection force) of the spindle to produce a tight metaphase plate (Fig. 5b). The action of this motor is required throughout metaphase because addition of function-blocking antibodies to preformed spindles also resulted in chromosome misalignment. Addition of a nondegradable version of Xkid prevented chromosome segregation, indicating that degradation of this kinesin is absolutely necessary for anaphase chromosome movement (Antonio et al., 2000; Funabiki and Murray, 2000). This is in agreement with the function of Xkid because polar ejection forces would counter the forces necessary for the poleward movement of the chromosomes during anaphase. Xkid is predictably dispensable for bead spindle assembly because the rigidity of the beads and their tendency to aggregate bypasses the requirement for polar ejection forces. In somatic cells, injection of antibodies to Xkid demonstrated that this protein is not crucial for metaphase alignment but is very important for chromosome congression (Levesque and Compton, 2001). This motor may play diVerent roles in diVerent systems due to the variation in the involvement of chromosome arms in spindle assembly. Nod: This motor is a D. melanogaster chromokinesin that does not possess a homologue in other organisms. Nod binds to (Afshar et al., 1995a; Afshar et al., 1995b) and acts along chromosome arms (Murphy and Karpen, 1995). It is important for preventing the loss of achiasmatic or nonexchange chromosomes from the meiotic spindle (Theurkauf and Hawley, 1992). It is not required for the maintenance of the exchange chromosomes, which are

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Figure 5 Chromokinesins in spindle assembly. (a) The chromokinesin Xklp1 (red) attaches to DNA (blue) and to microtubules (green) serving as a docking motor to maintain attachment between the chromosome and the microtubules. (b) The chromokinesin Xkid provides the polar ejection force to move chromosomes (blue) to the center of the spindle by attaching to microtubules (green) and moving toward their þ ends. Arrows indicate the direction of movement. (See Color Insert.)

held together at the metaphase plate by virtue of the physical connection provided by chiasmata. This motor seems to be important for polar ejection forces exerted on the nonexchange chromosomes, but it is not required for mitosis after early embryogenesis. KLP38B: KLP38B is encoded by the tiovivo (tio) gene of D. melanogaster. Mutations in this kinesin indicated a role in cytokinesis in larval mitotic divisions (Ohkura et al., 1997). It was later shown in dividing larval neuroblasts that mutations in this gene resulted in abnormal chromosome segregation and a variety of mitotic defects such as aneuploidy, circular mitotic figures (CMFs), and abnormal metaphase and anaphase figures (Molina et al., 1997; Ruden et al., 1997). KLP38B also has been shown to


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be important for mitotic chromosome condensation (Alphey et al., 1997). Cloning and domain organization of this kinesin revealed that it is a chromokinesin with the motor domain near the N-terminus of the protein and DNA binding motifs at the C-terminus. Localization of the protein by immunofluorescence showed that KLP38B colocalized with condensed chromatin during mitosis and male meiosis. KLP38B interacts by yeast two-hybrid with the catalytic subunit of PP1. These two proteins also interact in vitro. Further functional characterization of KLP38B and the relevance of its interaction with PP1 will shed light on the function of this protein in cell division.

XIV. Centrosomes and Kinetochores Revisited It was initially thought that the ability of chromosomes to direct spindle assembly was restricted to meiotic systems lacking centrosomes. Recently, the role of chromatin in spindle assembly has been corroborated in somatic cells by the observation that normal bipolar spindles form in cells even after one or both centrosomes have been laser ablated (Khodjakov et al., 2000). This indicates that in the absence of dominant microtubule nucleating sites, chromosome-mediated processes can direct spindle formation. What is the function of the centrosome? Elegant experiments in X. laevis egg extracts have demonstrated that centrosomes, when present, act as dominant focal nucleating sites for microtubules (Heald et al., 1997). In the presence of multiple centrosomes, multipolar spindles form, which can lead to aneuploidy. Therefore, centrosomes may be present to enhance the fidelity of chromosome segregation. Additionally, participation in spindle assembly ensures that each cell inherits only one centrosome, which may be essential for other functions. Astral microtubules are often a feature of centrosomal spindles and can be very important for spindle positioning (de Saint Phalle and Sullivan, 1998). In the last few years, there has been an increasing amount of evidence for an exciting and unexpected new role for centrosomes during cytokinesis (Doxsey, 2001). Whereas spindles can form when centrosomes have been removed by laser ablation, approximately half of the cells displayed cytokinesis defects (Khodjakov et al., 2000). Centrosome dynamics have been examined by labeling centriole markers such as centrin with GFP. With the use of this tool, it has been observed that the mother centriole moves to the area between the dividing cells just before abscission (Piel et al., 2001). In budding yeast, the spindle pole body is crucial for exit from mitosis and subsequent cytokinesis (Pereira and Schiebel, 2001). The centrosome also seems to be important in actin polymerization in D. melanogaster embryos during pseudocleavage (Stevenson et al., 2001). Although it is not clear what the

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role of the centrosome is in these processes, work over the next few years will hopefully identify the molecular components that are contributing to these new functions of the centrosome. What is the function of the kinetochore? In many organisms, the primary site of attachment of chromosomes to microtubules is at the kinetochore. Whereas chromatin possesses the ability to direct spindle assembly, kinetochores are involved in metaphase chromosome alignment and quality control of mitosis and are absolutely required for anaphase chromosome movements (Kapoor and Compton, 2002; Rieder and Salmon, 1998). There is an elaborate surveillance mechanism centered at the kinetochore termed the spindle assembly checkpoint. This checkpoint senses errors in chromosome attachment or spindle structure and halts mitotic progression until the error is fixed. The spindle assembly checkpoint monitors chromosome biorientation, proper tension at the kinetochore, and alignment of chromosomes at the metaphase plate. This checkpoint is extremely important to ensure accurate chromosome segregation. Kinetochores also generate force for poleward chromosome movement, which occurs during the establishment of microtubule connections from the spindle pole to the chromosome during prometaphase, as well as during anaphase (Desai and Mitchison, 1997). In maize meiotic mutants that generate univalents, spindle assembly still occurs but metaphase chromosome alignment and anaphase are aberrant (Chan and Cande, 1998), illustrating the importance of the kinetochore in these processes. The critical role of the kinetochore for chromosome alignment and segregation has been appreciated in many diVerent systems using approaches ranging from laser ablation to the molecular characterization of kinetochore components (Maney et al., 2000; Rieder and Salmon, 1998).

XV. Conclusions Spindles were initially thought to be formed primarily by a ‘‘search and capture’’ mechanism, with centrosomes and kinetochores playing a dominant role in directing spindle assembly. In this review, we have discussed evidence demonstrating the importance of chromatin in spindle formation and some of the molecular components that have been identified to mediate its role. Whereas a function for chromatin in spindle assembly is most clearly seen in female meiotic systems that do not possess centrosomes, chromosomes also have been shown to play a role in mitosis in somatic cells. Presumably, the ‘‘search and capture’’ and ‘‘local stabilization’’ mechanisms of spindle assembly are not mutually exclusive and are to some degree functionally redundant. The relative involvement of chromosomes, kinetochores, and centrosomes appears to vary significantly between organisms


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and among cell types within an organism. Clearly, the cell has developed more than one strategy to ensure that chromosome segregation proceeds accurately.

Acknowledgments The authors would like to thank Jennifer Banks and Thomas Maresca for helpful comments and suggestions during the preparation of this chapter.

References Afshar, K., Barton, N. R., Hawley, R. S., and Goldstein, L. S. (1995a). DNA binding and meiotic chromosomal localization of the Drosophila nod kinesin-like protein. Cell 81, 129–138. Afshar, K., Scholey, J., and Hawley, R. S. (1995b). Identification of the chromosome localization domain of the Drosophila nod kinesin-like protein. J. Cell Biol. 131, 833–843. Alphey, L., Parker, L., Hawcroft, G., Guo, Y., Kaiser, K., and Morgan, G. (1997). KLP38B: A mitotic kinesin-related protein that binds PP1. J. Cell Biol. 138, 395–409. Andersen, S. S. L. (1999). Balanced regulation of microtubule dynamics during the cell cycle: A contemporary view. Bioessays 21, 53–60. Andersen, S. S. L. (2000). Spindle assembly and the art of regulating microtubule dynamics by MAPs and Stathmin/Op18. Trends Cell Biol. 10, 261–267. Andersen, S. S. L., Ashford, A. J., Tournebize, R., Gavet, O., Sobel, A., Hyman, A. A., and Karsenti, E. (1997). Mitotic chromatin regulates phosphorylation of Stathmin/Op18. Nature 389, 640–643. Andreassen, P. R., Lacroix, F. B., Villa-Moruzzi, E., and Margolis, R. L. (1998). Differential subcellular localization of protein phosphatase-1 alpha, gamma1, and delta isoforms during both interphase and mitosis in mammalian cells. J. Cell Biol. 141, 1207–1215. Antonio, C., Ferby, I., Wilhelm, H., Jones, M., Karsenti, E., Nebreda, A. R., and Vernos, I. (2000). Xkid, a chromokinesin required for chromosome alignment on the metaphase plate. Cell 102, 425–435. Arnaud, L., Pines, J., and Nigg, E. A. (1998). GFP tagging reveals human Polo-like kinase 1 at the kinetochore/centromere region of mitotic chromosomes. Chromosoma 107, 424–429. Askjaer, P., Galy, V., Hannak, E., and Mattaj, I. W. (2002). Ran GTPase cycle and Importins alpha and beta are essential for spindle formation and nuclear envelope assembly in living Caenorhabditis elegans embryos. Mol. Biol. Cell 13, 4355–4370. Axton, J. M., Dombradi, V., Cohen, P. T., and Glover, D. M. (1990). One of the protein phosphatase 1 isoenzymes in Drosophila is essential for mitosis. Cell 63, 33–46. Bahler, J., Steever, A. B., Wheatley, S., Wang, Y. L., Pringle, J. R., Gould, K. L., and McCollum, D. (1998). Role of polo kinase and Mid1p in determining the site of cell division in fission yeast. J. Cell Biol. 143, 1603–1616. Bamba, C., Bobinnec, Y., Fukuda, M., and Nishida, E. (2002). The GTPase Ran regulates chromosome positioning and nuclear envelope assembly in vivo. Curr. Biol. 12, 503–507. Biggins, S., Severin, F. F., Bhalla, N., Sassoon, I., Hyman, A. A., and Murray, A. W. (1999). The conserved protein kinase Ip11 regulates microtubule binding to kinetochores in budding yeast. Genes Dev. 13, 532–544.

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Bilbao-Cortes, D., Hetzer, M., Langst, G., Becker, P. B., and Mattaj, I. W. (2002). Ran binds to chromatin by two distinct mechanisms. Curr. Biol. 12, 1151–1156. Bischoff, J. R., and Plowman, G. D. (1999). The Aurora/Ip11p kinase family: Regulators of chromosome segregation and cytokinesis. Trends Cell Biol. 9, 454–459. Blangy, A., Lane, H. A., d’Herin, P., Harper, M., Kress, M., and Nigg, E. A. (1995). Phosphorylation by p34cdc2 regulates spindle association of human Eg5, a kinesin-related motor essential for bipolar spindle formation in vivo. Cell 83, 1159–1169. Brunet, S., Maria, A. S., Guillaud, P., Dujardin, D., Kubiak, J. Z., and Maro, B. (1999). Kinetochore fibers are not involved in the formation of the first meiotic spindle in mouse oocytes, but control the exit from the first meiotic M phase. J. Cell Biol. 142, 1–12. Brunet, S., Polanski, Z., Verlhac, M. H., Kubiak, J. Z., and Maro, B. (1998). Bipolar meiotic spindle formation without chromatin. Curr. Biol. 8, 1231–1234. Budde, P. P., Kumagai, A., Dunphy, W. G., and Heald, R. (2001). Regulation of Op18 during spindle assembly in Xenopus egg extracts. J. Cell Biol. 153, 149–157. Carazo-Salas, R. E., Gruss, O. J., Mattaj, I. W., and Karsenti, E. (2001). Ran-GTP coordinates regulation of microtubule nucleation and dynamics during mitotic-spindle assembly. Nat. Cell Biol. 3, 228–234. Carazo-Salas, R. E., Guarguaglini, G., Gruss, O. J., Segref, A., Karsenti, E., and Mattaj, I. W. (1999). Generation of GTP-bound Ran by RCC1 is required for chromatin-induced mitotic spindle formation. Nature 400, 178–181. Carmena, M., Riparbelli, M. G., Minestrini, G., Tavares, A. M., Adams, R., Callaini, G., and Glover, D. M. (1998). Drosophila polo kinase is required for cytokinesis. J. Cell Biol. 143, 659–671. Cassimeris, L. (2002). The oncoprotein 18/stathmin family of microtubule destabilizers. Curr. Opin. Cell Biol. 14, 18–24. Chan, A., and Cande, W. Z. (1998). Maize meiotic spindles assemble around chromatin and do not require paired chromosomes. J. Cell Sci. 111, 3507–3715. Charles, J. F., Jaspersen, S. L., Tinker-Kulberg, R. L., Hwang, L., Szidon, A., and Morgan, D. O. (1998). The Polo-related kinase Cdc5 activates and is destroyed by the mitotic cyclin destruction machinery in S. cerevisiae. Curr. Biol. 8, 497–507. Church, K., Nicklas, R. B., and Lin, H. P. (1986). Micromanipulated bivalents can trigger minispindle formation in Drosophila melanogaster spermatocyte cytoplasm. J. Cell Biol. 103, 2765–2773. Dasso, M. (2002). The Ran GTPase: Theme and variations. Curr. Biol. 12, R502–R508. Desai, A., and Mitchison, T. J. (1997). Microtubule polymerization dynamics. Annu. Rev. Cell Dev. Biol. 13, 83–117. de Saint Phalle, B., and Sullivan, W. (1998). Spindle assembly and mitosis without centrosomes in parthenogenetic Sciara embryos. J. Cell Biol. 141, 1383–1391. Descombes, P., and Nigg, E. A. (1998). The Polo-like kinase Plx1 is required for M phase exit and destruction of mitotic regulators in Xenopus egg extracts. EMBO J. 17, 1328–1335. Dogterom, M., Felix, M. A., Guet, C. C., and Leibler, S. (1996). Influence of M-phase chromatin on the anisotropy of microtubule asters. J. Cell Biol. 133, 125–140. Doonan, J. H., and Morris, N. R. (1989). The bimG gene of Aspergillus nidulans, required for completion of anaphase, encodes a homolog of mammalian phosphoprotein phosphatase 1. Cell 57, 987–996. Doxsey, S. (2001). Re-evaluating centrosome function. Nat. Rev. Mol. Cell Biol. 2, 688–698. Fernandez, A., Brautigan, D. L., and Lamb, N. J. (1992). Protein phosphatase type 1 in mammalian cell mitosis: Chromosomal localization and involvement in mitotic exit. J. Cell Biol. 116, 1421–1430. Francisco, L., Wang, W., and Chan, C. S. (1994). Type 1 protein phosphatase acts in opposition to IpL1 protein kinase in regulating yeast chromosome segregation. Mol. Cell Biol. 14, 4731–4740.


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Fry, A. M., Mayor, T., Meraldi, P., Stierhof, Y. D., Tanaka, K., and Nigg, E. A. (1998). C-Napl, a novel centrosomal coiled-coil protein and candidate substrate of the cell cycleregulated protein kinase Nek2. J. Cell Biol. 141, 1563–1574. Funabiki, H., and Murray, A. W. (2000). The Xenopus chromokinesin Xkid is essential for metaphase chromosome alignment and must be degraded to allow anaphase chromosome movement. Cell 102, 411–424. Giet, R., and Prigent, C. (1999). Aurora/Ip11p-related kinases, a new oncogenic family of mitotic serine-threonine kinases. J. Cell Sci. 112, 3591–3601. Glover, D. M., Hagan, I. M., and Tavares, A. A. M. (1998). Polo-like kinases: A team that plays throughout mitosis. Genes Dev. 12, 3777–3787. Gorlich, D., and Kutay, U. (1999). Transport between the cell nucleus and the cytoplasm. Annu. Rev. Cell Dev. Biol. 15, 607–660. Gruss, O. J., Carazo-Salas, R. E., Schatz, C. A., Guarguaglini, G., Kast, J., Wilm, M., Le Bot, N., Vernos, I., Karsenti, E., and Mattaj, J. W. (2001). Ran induces spindle assembly by reversing the inhibitory effect of importin alpha on TPX2 activity. Cell 104, 83–93. Heald, R. (2000). A dynamic duo of microtubule modulators [news; comment]. Nat. Cell Biol. 2, E11–E12. Heald, R., Tournebize, R., Blank, T., Sandaltzopoulos, R., Becker, P., Hyman, A., and Karsenti, E. (1996). Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts [see comments]. Nature 382, 420–425. Heald, R., Tournebize, R., Habermann, A., Karsenti, E., and Hyman, A. (1997). Spindle assembly in Xenopus egg extracts: Respective roles of centrosomes and microtubule self-organization. J. Cell Biol. 138, 615–628. Heald, R., and Weis, K. (2000). Spindles get the ran around. Trends Cell Biol. 10, 1–4. Hyman, A. A., and Karsenti, E. (1996). Morphogenetic properties of microtubules and mitotic spindle assembly. Cell 84, 401–410. Joseph, J., Tan, S. H., Karpova, T. S., McNally, J. G., and Dasso, M. (2002). SUMO-1 targets RanGAP1 to kinetochores and mitotic spindles. J. Cell Biol. 156, 595–602. Kalab, P., Pu, R. T., and Dasso, M. (1999). The ran GTPase regulates mitotic spindle assembly. Curr. Biol. 9, 481–484. Kalab, P., Weis, K., and Heald, R. (2002). Visualization of a Ran-GTP gradient in interphase and mitotic Xenopus egg extracts. Science 295, 2452–2456. Kapoor, T. M., and Compton, D. A. (2002). Searching for the middle ground: Mechanisms of chromosome alignment during mitosis. J. Cell Biol. 157, 551–556. Karsenti, E., Newport, J., and Kirschner, M. (1984). Respective roles of centrosomes and chromatin in the conversion of microtubule arrays from interphase to metaphase. J. Cell Biol. 99, 47s–54s. Karsenti, E., and Vernos, I. (2001). Cell cycle: The mitotic spindle: A self-made machine. Science 294, 543–547. Khodjakov, A., Cole, R. W., Oakley, B. R., and Rieder, C. L. (2000). Centrosome-independent mitotic spindle formation in vertebrates. Curr. Biol. 10, 59–67. Kirkpatrick, D., and Solomon, F. (1994). Overexpression of yeast homologs of the mammalian checkpoint gene RCC1 suppresses the class of alpha-tubulin mutations that arrest with excess microtubules. Genetics 137, 381–392. Kirschner, M., and Mitchison, T. (1986). Beyond self-assembly: From microtubules to morphogenesis. Cell 45, 329–342. Klebe, C., Bischoff, F. R., Ponstingl, H., and Wittinghofer, A. (1995). Interaction of the nuclear GTP-binding protein Ran with its regulatory proteins RCC1 and RanGAP1. Biochemistry 34, 639–647. Kotani, S., Tugendreich, S., Fujii, M., Jorgensen, P. M., Watanabe, N., Hogg, C., Hieter, P., and Todokoro, K. (1998). PKA and MPF-activated polo-like kinase regulate anaphasepromoting complex activity. Mol. Cell 1, 371–380.

110

Budde and Heald

Kumagai, A., and Dunphy, W. G. (1996). Purification and molecular cloning of Plx1, a Cdc25regulatory kinase from Xenopus egg extracts. Science 273, 1377–1380. Lane, H. A., and Nigg, E. A. (1996). Antibody microinjection reveals as essential role for human polo-like kinase 1 (Plk1) in the functional maturation of mitotic centrosomes. J. Cell Biol. 135, 1701–1713. Lee, K. S., Yuan, Y. L., Kuriyama, R., and Erikson, R. L. (1995). Plk is an M-phase-specific protein kinase and interacts with a kinesin- like protein, CHO1/MKLP-1. Mol. Cell Biol. 15, 7143–7151. Levesque, A. A., and Compton, D. A. (2001). The chromokinesin Kid is necessary for chromosome arm orientation and oscillation, but not congression, on mitotic spindles. J. Cell Biol. 154, 1135–1146. Llamazares, S., Moreira, A., Tavares, A., Girdham, C., Spruce, B. A., Gonzalez, C., Karess, R. E., Glover, D. M., and Sunkel, C. E. (1991). Polo encodes a protein kinase homolog required for mitosis in Drosophila. Genes Dev. 5, 2153–2165. Logarinho, E., and Sunkel, C. E. (1998). The Drosophila POLO kinase localises to multiple compartments of the mitotic apparatus and is required for the phosphorylation of MPM2 reactive epitopes. J. Cell Sci. 111, 2897–2909. Maney, T., Ginkel, L. M., Hunter, A. W., and Wordeman, L. (2000). The kinetochore of higher eucaryotes: A molecular view. Int. Rev. Cytol. 194, 67–131. Matsumoto, Y., and Maller, J. L. (2002). Calcium, calmodulin, and CaMKII requirement for initiation of centrosome duplication in Xenopus egg extracts. Science 295, 499–502. Mitchison, T., and Krischner, M. (1984). Dynamic instability of microtubule growth. Nature 312, 237–242. Mitchison, T. J., and Kirschner, M. W. (1985a). Properties of the kinetochore in vitro. I. Microtubule nucleation and tubulin binding. J. Cell Biol. 101, 755–765. Mitchison, T. J., and Kirschner, M. W. (1985b). Properties of the kinetochore in vitro. II. Microtubule capture and ATP-dependent translocation. J. Cell Biol. 101, 766–777. Molina, I., Baars, S., Brill, J. A., Hales, K. G., Fuller, M. T., and Ripoll, P. (1997). A chromatin-associated kinesin-related protein required for normal mitotic chromosome segregation in Drosophila. J. Cell Biol. 139, 1361–1371. Moritz, M., Braunfeld, M. B., Sedat, J. W., Alberts, B., and Agard, D. A. (1995). Microtubule nucleation by gamma-tubulin-containing rings in the centrosome. Nature 378, 638–640. Moutinho-Santos, T., Sampaio, P., Amorim, I., Costa, M., and Sunkel, C. E. (1999). In vivo localisation of the mitotic POLO kinase shows a highly dynamic association with the mitotic apparatus during early embryogenesis in Drosophila. Biol. Cell 91, 585–596. Murnion, M. E., Adams, R. R., Callister, D. M., Allis, C. D., Earnshaw, W. C., and Swedlow, J. R. (2001). Chromatin-associated protein phosphatase 1 regulates aurora-B and histone H3 phosphorylation. J. Biol. Chem. 276, 26656–26665. Murphy, T. D., and Karpen, G. H. (1995). Interactions between the nodþ kinesin-like gene and extracentromeric sequences are required for transmission of a Drosophila minichromosome. Cell 81, 139–148. Nachury, M. V., Maresca, T. J., Salmon, W. C., Waterman-Storer, C. M., Heald, R., and Weis, K. (2001). Importin is a mitotic target of the small GTPase Ran in spindle assembly. Cell 104, 95–106. Nakamura, M., Masuda, H., Horii, J., Kuma, K., Yokoyama, N., Ohba, T., Nishitani, H., Miyata, T., Tanaka, M., and Nishimoto, T. (1998). When overexpressed, a novel centrosomal protein, RanBPM, causes ectopic microtubule nucleation similar to gammatubulin. J. Cell Biol. 143, 1041–1052. Nemergut, M. E., Mizzen, C. A., Stukenberg, T., Allis, C. D., and Macara, I. G. (2001). Chromatin docking and exchange activity enhancement of RCC1 by histones H2A and H2B. Science 292, 1540–1543.


111

Nicklas, R. B., and Gordon, G. W. (1985). The total length of spindle microtubules depends on the number of chromosomes present. J. Cell Biol. 100, 1–7. Nigg, E. A. (1998). Polo-like kinases: Positive regulators of cell division from start to finish. Curr. Opin. Cell Biol. 10, 776–783. Nigg, E. A. (2001). Mitotic kinases as regulators of cell division and its checkpoints. Nat. Rev. Mol. Cell Biol. Jan, 21–32. Nishitani, H., Ohtsubo, M., Yamashita, K., Iida, H., Pines, J., Yasudo, H., Shibata, Y., Hunter, T., and Nishimoto, T. (1991). Loss of RCC1, a nuclear DNA-binding protein, uncouples the completion of DNA replication from the activation of cdc2 protein kinase and mitosis. EMBO J. 10, 1555–1564. Oakley, C. E., and Oakley, B. R. (1989). Identification of gamma-tubulin, a new member of the tubulin superfamily encoded by mipA gene of Aspergillus nidulans. Nature 338, 662–664. Ohba, T., Nakamura, M., Nishitani, H., and Nishimoto, T. (1999). Self-organization of microtubule asters induced in Xenopus egg extracts by GTP-bound Ran [see comments]. Science 284, 1356–1358. Ohkura, H., Hagan, I. M., and Glover, D. M. (1995). The conserved Schizosaccharomyces pombe kinase plo1, required to form a bipolar spindle, the actin ring, and septum, can drive septum formation in G-1 and G-2 cells. Genes Dev. 9, 1059–1073. Ohkura, H., Kinoshita, N., Miyatani, S., Toda, T., and Yanagida, M. (1989). The fission yeast dis2þ gene required for chromosome disjoining encodes one of two putative type 1 protein phosphatases. Cell 57, 997–1007. Ohkura, H., Torok, T., Tick, G., Hoheisel, J., Kiss, I., and Glover, D. M. (1997). Mutation of a gene for a Drosophila kinesin-like protein, Klp38B, leads to failure of cytokinesis. J. Cell Sci. 110, 945–954. Ouspenski, I. I. (1998). A RanBP1 mutation which does not visibly affect nuclear import may reveal additional functions of the ran GTPase system. Exp. Cell Res. 244, 171–183. Pereira, G., and Schiebel, E. (2001). The role of the yeast spindle pole body and the mammalian centrosome in regulating late mitotic events. Curr. Opin. Cell Biol. 13, 762–769. Piel, M., Nordberg, J., Euteneuer, U., and Bornens, M. (2001). Centrosome-dependent exist of cytokinesis in animal cells. Science 291, 1550–1553. Qian, Y. W., Erikson, E., Li, C., and Maller, J. L. (1998). Activated polo-like kinase Plx1 is required at multiple points during mitosis in Xenopus laevis. Mol. Cell. Biol. 18, 4262–4271. Qian, Y. W., Erikson, E., and Maller, J. L. (1999). Mitotic effects of a constitutively active mutant of the Xenopus polo-like kinase Plx1. Mol. Cell. Biol. 19, 8625–8632. Rieder, C. L., Davison, E. A., Jensen, L. C., Cassimeris, L., and Salmon, E. D. (1986). Oscillatory movements of monooriented chromosomes and their position relative to the spindle pole result from the ejection properties of the aster and half- spindle. J. Cell Biol. 103, 581–591. Rieder, C. L., and Salmon, E. D. (1994). Motile kinetochores and polar ejection forces dictate chromosome position on the vertebrate mitotic spindle. J. Cell Biol. 124, 223–233. Rieder, C. L., and Salmon, E. D. (1998). The vertebrate cell kinetochore and its roles during mitosis. Trends Cell Biol. 8, 310–318. Rogers, E., Bishop, J. D., Waddle, J. A., Schumacher, J. M., and Lin, R. (2002). The aurora kinase AIR-2 functions in the release of chromosome cohesion in Caenorhabditis elegans meiosis. J. Cell Biol. 157, 219–229. Ruden, D. M., Cui, W., Sollars, V., and Alterman, M. (1997). A Drosophila kinesin-like protein, Klp38B, functions during meiosis, mitosis, and segmentation. Dev. Biol. 191, 284–296. Sassoon, I., Severin, F. F., Andrews, P. D., Taba, M. R., Kaplan, K. B., Ashford, A. J., Stark, M. J., Sorger, P. K., and Hyman, A. A. (1999). Regulation of Saccharomyces cerevisiae kinetochores by the type 1 phosphatase Glc7p. Genes Dev. 13, 545–555.

112

Budde and Heald

Sawin, K. E., and Mitchison, T. J. (1991). Mitotic spindle assembly by two different pathways in vitro. J. Cell Biol. 112, 925–940. Sharp, D. J., Rogers, G. C., and Scholey, J. M. (2000). Microtubule motors in mitosis. Nature 407, 41–47. Shirayama, M., Zachariae, W., Ciosk, R., and Nasmyth, K. (1998). The Polo-like kinase Cdc5p and the WD-repeat protein Cdc20p/fizzy are regulators and substrates of the anaphase promoting complex in Saccharomyces cerevisiae. EMBO J. 17, 1336–1349. Stevenson, V. A., Kramer, J., Kuhn, J., and Theurkauf, W. E. (2001). Centrosomes and the scrambled protein coordinate microtubule-independent actin reorganization. Nat. Cell Biol. 3, 68–75. Sumara, I., Vorlaufer, E., Stukenberg, P. T., Kelm, O., Redemann, N., Nigg, E. A., and Peters, J. M. (2002). The dissociation of cohesin from chromosomes in prophase is regulated by Polo-like kinase. Mol. Cell 9, 515–525. Sunkel, C. E., and Glover, D. M. (1988). Polo, a mitotic mutant of Drosophila displaying abnormal spindle poles. J. Cell Sci. 89, 25–38. Theurkauf, W. E., and Hawley, R. S. (1992). Meiotic spindle assembly in Drosophila females: Behavior of nonexchange chromosomes and the effects of mutations in the nod kinesin-like protein. J. Cell Biol. 116, 1167–1180. Tokai, N., Fujimoto-Nishiyama, A., Toyoshima, Y., Yonemura, S., Tsukita, S., Inoue, J., and Yamamota, T. (1996). Kid, a novel kinesin-like DNA binding protein, is localized to chromosomes and the mitotic spindle. EMBO J. 15, 457–467. Tournebize, R., Andersen, S. S. L., Verde, F., Doree, M., Karsenti, E., and Hyman, A. A. (1997). Distinct roles of PP1 and PP2A-like phosphatases in control of microtubule dynamics during mitosis. EMBO J. 16, 5537–5549. Toyoshima-Morimoto, F., Taniguchi, E., Shinya, N., Iwamatsu, A., and Nishida, E. (2001). Polo-like kinase 1 phosphorylates cyclin B1 and targets it to the nucleus during prophase. Nature 410, 215–220. Trieselmann, N., and Wilde, A. (2002). Ran localizes around the microtubule spindle in vivo during mitosis in Drosophila embryos. Curr. Biol. 12, 1124–1129. Verde, F., Labbe, J. C., Doree, M., and Karsenti, E. (1990). Regulation of microtubule dynamics by Cdc2 protein kinase in cell-free extracts of Xenopus eggs. Nature 343, 233–238. Vernos, I., Raats, J., Hirano, T., Heasman, J., Karsenti, E., and Wylie, C. (1995). Xklp1, a chromosomal Xenopus kinesin-like protein essential for spindle organization and chromosome positioning. Cell 81, 117–127. Virshup, D. M. (2000). Protein phosphatase 2A: A panoply of enzymes. Curr. Opin. Cell Biol. 12, 180–185. Walczak, C. E., Vernos, I., Mitchison, T. J., Karsenti, E., and Heald, R. (1998). A model for the proposed roles of different microtubule-based motor proteins in establishing spindle bipolarity. Curr. Biol. 8, 903–913. Weis, K. (1998). Importins and exportins: How to get in and out of the nucleus. Trends Biochem. Sci. 23, 185–189. Wianny, F., Tavares, A., Evans, M. J., Glover, D. M., and Zernicka-Goetz, M. (1998). Mouse polo-like kinase 1 associates with the acentriolar spindle poles, meiotic chromosomes and spindle midzone during oocyte maturation. Chromosoma 107, 430–439. Wiese, C., Wilde, A., Moore, M. S., Adam, S. A., Merdes, A., and Zheng, Y. (2001). Role of Importin- in coupling Ran to downstream targets in microtubule assembly. Science 291, 653–656. Wilde, A., Lizarraga, S. B., Zhang, L., Wiese, C., Gliksman, N. R., Walczak, C. E., and Zheng, Y. (2001). Ran stimulates spindle assembly by altering microtubule dynamics and the balance of motor activities. Nat. Cell Biol. 3, 221–227.


113

Wilde, A., and Zheng, Y. (1999). Stimulation of microtubule aster formation and spindle assembly by the small GTPase Ran. Science 284, 1359–1362. Willard, H. F. (2001). Neocentromeres and human artificial chromosomes: An unnatural act. Proc. Natl. Acad. Sci. USA 98, 5374–5376. Wittmann, T., Wilm, M., Karsenti, E., and Vernos, I. (2000). TPX2, a novel Xenopus MAP involved in spindle pole organization. J. Cell Biol. 149, 1405–1418. Zhang, D. H., and Nicklas, R. B. (1995a). Chromosomes initiate spindle assembly upon experimental dissolution of the nuclear envelope in grasshopper spermatocytes. J. Cell Biol. 131, 1125–1131. Zhang, D. H., and Nicklas, R. B. (1995b). The impact of chromosomes and centrosomes on spindle assembly as observed in living cells. J. Cell Biol. 129, 1287–1300. Zhang, D. H., and Nicklas, R. B. (1996). Anaphase and cytokinesis in the absence of chromosomes. Nature 382, 466–468. Zheng, Y., Wong, M. L., Alberts, B., and Mitchison, T. (1995). Nucleation of microtubule assembly by a gamma-tubulin-containing ring complex. Nature 378, 578–583.


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Modeling Cardiogenesis: The Challenges and Promises of 3D Reconstruction Jeffrey O. Pentecost,* Claudio Silva,{ Maurice Pesticelli, Jr.,{ and Kent L. Thornburg§ *

Department of Medical Informatics and Outcomes Research, Oregon Health and Science University, Portland, Oregon 97201. {OGI School of Science and Engineering, Oregon Health and Science University, Beaverton, Oregon 97006. { Departments of Anatomy, Cell Biology, and Surgery, University of Illinois, Chicago, Illinois 60612. §Department of Physiology/Pharmacology, Oregon Health and Science University, Portland, Oregon 97201

I. Introduction II. Embryonic Heart Development in Mammals III. Reconstruction and Modeling Techniques A. Data Sources B. Surface Modeling C. Voxel Modeling IV. The Embryonic Human Heart V. Recent Developments A. Point-Based Surface Modeling and Graphics Processing Units B. Stereolithographic Physical Models of the Embryonic Heart C. Spatial Genomics D. Optical Projection Tomography E. EpiFluorescent Stereomicroscopy F. Cellular Modeling and Simulations G. Symbolic Modeling: Anatomical Ontologies H. Fetal Origins of Adult Disease I. Visible Embryo NGI Project VI. Summary and Future Directions References

I. Introduction To bring visual reality to the silent and unseen drama of embryogenesis, anatomists and embryologists have longed to recreate the complex spatial topography of human embryos and their organs. In the late nineteenth century, His (1885) and Born (1889) used clay and wax to reconstruct enlarged serial tissue sections into physical models. Using these techniques, Osborne O. Current Topics in Developmental Biology, Vol. 56 Copyright 2003, Elsevier Inc. All rights reserved. 0070-2153/03 $35.00

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Figure 1 Wax reconstructions of the developing heart in Carnegie Collection specimen 836 made by Osborne O. Heard and H. M. Evans using methods developed by Gustav Born. RA, right atrium; OFT, outflow tract; V, common ventricle. (Images courtesy of the National Museum of Health and Medicine, Washington, DC.)

Heard created over 700 wax-based reconstructions of Carnegie Collection embryo specimens. Examples of the heart reconstructions are shown in Fig. 1. Heard’s embryo models became the foundation for modern human embryology and constitute the conceptual basis of contemporary graphical reconstruction. They also serve as the basis for an international standard by which human embryos are described and classified (O’Rahilly et al., 1986; Streeter, 1942, 1945). In the late twentieth century, it became possible to reconstruct embryos by computer. 3D reconstruction techniques have continued to evolve and now, along with improved accuracy and resolution, it is possible to link precise structure with genomic, proteomic, physiological, and biochemical information. With the advent of the Internet, models can be shared with the global scientific community. Knowledge derived from new comprehensive models is expected to provide ammunition for new techniques aimed at correcting or averting congenital heart structural and biochemical defects. Correlations between the physical structure and physiological function during cardiogenesis remain enigmatic and constitute a major focus of scientific investigation at genetic, molecular, and cellular levels. The discovery in the 1950s that two fundamental periodic patterns, the alpha helix and beta sheet, determine the tertiary (folded) structure of proteins led to the realization that tertiary structure determines the physiological function of a given protein. For an increasing number of disease-specific proteins, the relationship between tertiary structure and adverse clinical condition is becoming clear (e.g., Alzheimer’s disease and cystic fibrosis); such relationships are soon to become central in cardiac development research. Evidence showing an inverse relationship between birth weight and the risk of adult onset coronary disease is growing and suggests that prenatal development is a key determinant of heart health for an individual for life

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(Barker et al., 1989; Koupilova et al., 1997). Thus, it is now clear that insight into adult heart disease requires a broad understanding of the genetic and molecular bases of embryonic and fetal heart development. Concurrent with these advances, accelerating growth in computer technology has cultivated novel strategies for research in developmental biology. For example, in 1999 super-computer architecture discussions reached the theoretical petaflop level (1015 operations per second), which greatly enabled the complex calculations required for predicting protein folding patterns based on amino acid sequence and structure in three-dimensional space (Clark, 2000). The flood of bioinformation resulting from advances in computer technology has dramatically altered research methodologies used for understanding complex structure–function relationships, yet at each point along the molecule-to-system continuum, innumerable fundamental questions remain. This chapter covers the history, technical elements, and educational and research utility of 3D reconstructive modeling of the human heart during embryogenesis. It summarizes the challenges and potentials of modern 3D reconstruction methodologies as tools for integrating genomic, proteomic, physiological, and anatomical data. The authors write with the full knowledge that the field itself is embryonic and that present eVorts will seem primitive as technology evolves within the next few years.

II. Embryonic Heart Development in Mammals In mammalian embryos, all tissue types are derived from one of three morphologically unique germ cell layers: ectoderm, mesoderm, or endoderm. The heart, derived from the mesodermal layer, is the first mammalian organ to form and function. The early heart primordia occur as paired bilateral structures arising as a thickening of the splanchnic mesoderm at the cranial end of the flat embryonic disc. As body folds convert this disc into a cylindrical configuration, the precardiac areas move to the embryonic midline and fuse ventrally, resulting in the formation of a single tubular heart. At this point, the heart begins to beat. Figure 2 shows the truncus and conus (T, C) in the early postfusion mouse heart. Within hours, the heart tube undergoes a bending and kinking process known as looping (Fig. 3). Synthesis of cardiac-specific proteins begins concomitant with formation of the heart anlage; a variety of developmental genes are expressed, including DNA regulatory genes, transcription factors, genes guiding asymmetry, and regionalization (Harvey and Rosenthal, 1999). After looping, the interatrial and interventricular walls form to create four distinct chambers as the heart progressively acquires an adult configuration (Figs. 4 and 5) (Icardo, 1996; Vuillemin and Pexieder, 1989).

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Figures 2–5 Scanning electron microscope images of the mouse heart during development. dpc, days postconception; Ao, aorta; A-Vc, atrioventricular canal; c, conus; d, dorsal cushion; LA, left atrium; LV, left ventricle; P, pulmonary artery; RA, right atrium; RV, right ventricle; sp, septum primum; t, truncus; vs, ventricular septum.

Throughout the embryonic period the heart continually remodels, changes its metabolic and synthetic patterns, alters its electrical properties, and modifies its vascular structure. While the largest increase in structural complexity and altered appearance of the heart is occurring it continues to work, imparting kinetic energy to the blood in the face of increasing physiological demands of the developing embryo. Only a few of the complex gene expression patterns regulating these remarkable anatomical transformations are presently known.

III. Reconstruction and Modeling Techniques Common to all computer-generated 3D reconstruction techniques are certain elements: an image data source, a registration function, a segmentation process, a surface-rendering or voxelization software application, and model viewing applications. A general schematic of this process is shown on p. 119. A list of commonly used 3D modeling applications and URLs is provided in Table I.

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5. Modeling Cardiogenesis Photomicrographs from histological sections

Surface (polygonal, wire frame) graphical models

Registration

Block face photomicrographs

Segmentation (2D contours) of specific tissues

Physical models (i.e., stereolithography)

MRM, CT, confocal, OPT Voxel models

Table I Commonly Used 3D Graphics Software Applications Application

Produced by

Amira Maya

TGS AliasjWavefront

Surfdriver 3D Studio Max SolidWorks Vitrea T-Vox VoxBlast

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A. Data Sources A series of cross-sectional digital images are required for reconstructing physical and computerized graphical 3D models. Historically, image data have been derived from photomicrographs of histological sections, a method still widely and eVectively used, although several contemporary methods (e.g., laser confocal scanning microscopy [LCSM], computerized tomography X-ray [CT], and magnetic resonance microscopy [MRM]) refine this process to obtain internal anatomical data without disruption of the embryo. However, so far none of these methods oVers the high resolution of a digitized micrograph.

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1. Histological Sections Histological analyses have long been a means for gaining understanding of the organization of developing organisms and tissue structures. Frozen or embedded (e.g., resin or paraYn) embryos can be sectioned with a microtome to produce serial cross-sectional tissues for mounting on glass slides. Photomicrographs of histological sections have served as the foundation for many traditional 3D reconstruction methods. Tissue section thickness typically ranges from 15 to 40 mm, but may be as thin as 5 mm, aVording cell-level resolution and a highly accurate contour series. The major disadvantage of histological images is the long-standing problem of registration because 3D model accuracy is most dependent on proper X–Y axis realignment of the images. Even slight deviations in rotational and translational position produce orientation errors that are likely to be compounded in subsequent images. Another disadvantage is physical distortion of the histological section caused by the shearing force of the microtome blade. One early histology-based reconstruction strategy relied on bromide images of the wax block face upon which fiducial marks had been scribed in order to preserve registration (Fig. 6). Manually traced contours outlining tissues of interest in enlarged photographs were transferred to paper or some other material to become the basis for physical scale models. 2. Carnegie Collection of Human Embryos The Carnegie Collection of human embryos has served as an international source of human embryo image data. It was established in 1887 by Franklin Mall (O’Rahilly and Mu¨ller, 1987) and is now stored at the Human Developmental Anatomy Center along with six other embryo collections as part of

Figure 6 marks.

Bromide image of Carnegie Collection specimen 836 section with original registration


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the Research Collections division of the National Museum of Health and Medicine in Washington, D. C. The Carnegie Collection is among the world’s largest collections, focusing on normal human development in the first 8 weeks of life. It also contains photographs and embryo models made of plaster and acetate. A searchable online database provides histological information about a subset of 660 embryos in the collection (http:// www.natmedmuse.afip.org/embryo/html/car_form.html). Digital images of histological sections in this collection serve as the data source for the Visible Embryo NGI Project (2001), a multi-institutional collaboration funded by the National Library of Medicine aimed at creating a digital repository of this image data for broad distribution of the data for education and scientific and medical research. 3. Magnetic Resonance Microscopy Recent advances in 3D reconstruction include MRM, which has higher resolution than conventional clinical magnetic resonance imaging (MRI) systems (1 mm). Using radio frequency coils specifically designed for very small specimens (7 mm maximum) and electromagnets several times stronger (9.4 Tesla) than those used in clinical imaging, fully registered cubic 3D data sets are produced without disrupting the physical integrity of the fixed embryo (Smith et al., 1994), yet permitting ‘‘cuts’’ through the cubic voxels in any direction. Relative to conventional light microscopy, MRM’s advantage of perfectly registered images is oVset by resolution. Resolution determinates in MRM are power of magnetic field, field of view (FOV), and slice interval. Although continually improving, the resolution of images produced by these scanners is still less than standard light photomicroscopy of histological sections. When 128 serial images are captured at 256 256 resolution in 9.4-Tesla MRM scanners the FOV is 14 14 7 mm, making the smallest volume element (voxel) 25 mm in each X, Y, Z axis (Smith et al., 1999), whereas the best resolution of conventional transmission light working with a glass lens in air is approximately 200 nm (0.2 mm). Magnetic resonance histology (MRH) is a variation of MRM wherein high-resolution images detect one of many ‘‘proton stains,’’ which indicate variations in pathological tissues (Lester et al., 2000). Studies using ultra high magnetic fields (17.6 Tesla) report significant improvements in contrast–noise and signal– noise ratios in MRM-generated images of early stage chick embryos, which enhances visualization of developing vasculature (Hogers et al., 2001). These high-powered scanners also reduce acquisition time, making them more applicable for functional studies than lower-powered machines. Rapid acquisition techniques now enhance MRM such that in vivo data can be acquired where motion artifact introduced by cardiac and respiratory movement is minimal.

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4. Laser Scanning Confocal Microscopy Three-dimensional data sets of biological structures can be obtained from laser scanning confocal microscopy (LSCM), from which optical sections of the specimen produce sequential high-resolution images in register. Upon excitation by the laser beam, fluorescent labels incorporated into the specimen emit a signal in the visible light range. As the beam performs a faster sweep through the specimen at each focal plane, serial 2D images are produced. The advantages of LSCM for creating 3D data sets are high-resolution images (0.1 mm in the Z-plane), perfect registration, and the ability to detect single and multiple fluorescent labels within the specimen. However, certain limitations of LSCM currently impede its use in 3D reconstructions of the developing heart: (1) it can be destructive to tissues, resulting in death of the specimen; (2) the high-energy light may ‘‘bleach’’ the dye rather quickly, resulting in signal distortion; (3) the use of fluorescent probes (chromophores) are required for many applications; and (4) there are tissue thickness limitations. Two-photon LSCM technology has been introduced and is expanding the horizons of LSCM imaging by reducing tissue destruction and bleaching (Periasamy et al., 1999; Piston, 1999). In two-photon LSCM, two low-energy infrared photons are used to energize the chromophore instead of a high-energy visible light in single-photon LSCM. This exposes out-of-plane tissues to infrared light only, which causes neither photobleaching nor phototoxicity. Unfortunately, the physical dimensions of mid- to late-stage chick and mouse embryo specimens still impede LSCM’s utility for whole-embryo reconstructions, although long-depth single-photon experimentation in later-stage mouse embryo specimens is promising (Palmes-Saloma and Saloma, 2000).

B. Surface Modeling Most computer-generated 3D graphical models exist as some form of surface model whereby a polygonal wire frame serves as scaVolding for surfaces, or shades, to be applied. In contrast to a voxel model, discussed later, 3D surface models contain only shape and contour information. A surface model looks like a solid object, but a 3D image of the surface is all the computer retains. As the name implies, surface models are ‘‘hollow’’ with no structure inside. Familiar examples include animated dinosaurs seen in popular movies such as Jurassic Park. The process for generating surface models requires defining, or segmenting, the anatomical region of interest. Often this is accomplished manually, although there are numerous automated segmentation algorithms.


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1. Registration The diYculty of restacking images or sections in the native XYZ orientation has beleaguered 3D modeling since its beginning. This issue of registration disappears when image capture modalities such as CT, MRI, or confocal microscopy are used, but because these modalities currently lack suYcient resolution to be useful in serial reconstructions of the embryonic heart, the problem of accurate registration remains a significant obstacle. That early cardiac morphogenesis consists of a tubelike structure twisting and folding upon itself makes translational and rotational orientation particularly important for accurate graphical descriptions of the process. Automated image registration algorithms exist, many of which have been developed primarily for CT, MRI, and positron emission tomography (PET) data (Maintz and Viergever, 1998; Matsopoulos et al., 2000; Pizer et al., 1999; Treves et al., 1998; Zhilkin and Alexander, 2000; Zhu, 2002). The utility of such algorithms is dependent upon knowledge of and reference to the original morphological shape, which is often limited when using raw histology-based images (Woods, 1993; Hill et al., 1993). 2. Segmentation: Contours, Curves, and Control Vertices Numerous software packages are available for creating surface models (see Table I), most of which permit background images (e.g., histological photomicrographs) to be used as templates for tracing contours (Fig. 7). Drawing a contour around a given anatomical region of interest produces data points describing the structure’s 2D surface. The accuracy of the tracing depends upon image resolution, spacing of data points, and type of mathematical curve being used. There are several mathematical strategies used

Figure 7 Photomicrograph of histological section (transverse plane) in Carnegie Collection specimen depicting contours that trace the margins of cardiac structures (red lines). (See Color Insert.)

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by diVerent software packages to obtain contour data from a tissue section. The contours may be manually placed or automatically generated by edgedetection algorithms. Cubic splines (some of which are called Bezier curves) are very useful for developing smooth, contiguous graphical surfaces. When parametric functions are added to this fundamental splining tool, the resultant nonuniform rational B-splines (NURBS) give better control over curve continuity and smoothness. NURBS provide the flexibility needed to represent a large variety of geometrical objects, including free-form, arbitrary surfaces seen in biology, while maintaining mathematical precision and control of smoothness. They can also represent very complex shapes with very little data and are a standard curve type in 3D graphical reconstructions. Parametric curves allow quick and simple manipulations such as translation and rotation of the coordinates that lend themselves well to 3D graphical reconstructions. The parametric approach also aVords a ‘‘piecewise’’ description of the curve and surface, which is another reason it is so commonly used in computer-aided reconstructions. When a series of contours is ‘‘stacked’’ to make a 3D outline of the organ in question, the outline constitutes a framework that can be cross-linked to form a wire frame model. Wire frame modeling is an eYcient and economical means of displaying a graphical model on the computer screen. One of the earliest computerized 3D modeling methods, the wire frame has been a fundamental technique used for serial reconstructions, getting its name from the meshlike appearance of the models it produces (Fig. 8). The wire frame is generated by cross-linking control vertices above and below a given contour. This produces polygons, usually triangles or squares, between the original line contours. The smoothness of the final surface correlates directly with the number of polygons. After a continuous wire frame has been constructed, a ‘‘rendering’’ program applies a surface skin to the frame to give the object a ‘‘solid’’ appearance. Figure 9 shows a

Figure 8 Wire frame model of the heart of Carnegie Collection specimen 6517 (frontal view). Approximate height ¼ 1 mm. (Oregon Health and Science University, Heart Research Center.)


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lateral view of an embryonic human heart model after the surface has been rendered. Surfaces store visual information in the form of vertices. For NURBS surfaces, these control points allow the surfaces to be edited as a spline curve, producing changes in the surface for local refinement, a useful feature for modeling biological forms. NURBS surfaces also permit the use of lighting to clarify form and shape. Surface models can be created to show cross-sectional and internal anatomy by varying transparency of the surface, superimposing original data, or using a combination of textures to emulate biological structure (Figs. 10 and 11).

Figure 9 Left lateral view of surface model of the heart of Carnegie Collection specimen 6517. LV, left ventricle; LA, left atrium. Approximate height ¼ 1 mm. (Oregon Health and Science University, Heart Research Center.)

Figure 10 Surface model of the heart of Carnegie Collection specimen 836 showing eVect of modifying transparency at the atrium to show section of internal structures. RV, right ventricle; LV, left ventricle; A, common atrium. Metric bar ¼ 1 mm. (Oregon Health and Science University, Heart Research Center.)

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Figure 11 Composite image illustrating eVect of combining histological data data and surface model of the embryonic heart. Left lateral view of transverse section. RV, right ventricle; LV, left ventricle; SV, sinus venosus; H, histological image overlay.

C. Voxel Modeling Voxel models are comprised of cubic volume elements (voxels) as opposed to 2D polygons used in surface models. The voxel model might be thought of as a loaf of bread whose slices have been perfectly repositioned to make it appear whole. Information about the model’s internal structures is visible with arbitrary planes, and texture mapping and iso-surfaces may be applied to the voxels to highlight specific internal structural elements with polygons. Application of voxel modeling to biological data oVers a wealth of new information for medical and scientific utility. Describing spatial aspects of organogenesis is one of many such uses. At 28 days’ gestation (Carnegie stage 13), the human heart is completing the looping process and measures less than 1 mm in its greatest dimension. Wall diameters and internal structures forming at that time are measured in 10s of micrometers. The ideal 3D graphical model of this heart would resolve it to the cellular level and produce perfectly registered images, including all internal structures. Voxel modeling is the ideal approach and is accomplished by stacking 2D images of the heart. The resulting voxel size is determined by the field of view (FOV) at the time of acquisition and is dependent upon the resolution of the image (X, Y) and number of slices (Z) in the 2D series. Depending on the desired anatomical resolution, the XY matrix dimensions and slice thickness must be chosen to produce voxels of isotropic dimensions proportionate to the anatomy of interest. Histological sections viewed with light microscopy still produce the highest resolution images, but noninvasive imaging techniques that preserve registration, such as MRM, surface imaging microscopy, and others, are gaining ground. In the future, the resolution of these techniques is expected to surpass that of light


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Figure 12 Frontal view of voxel model reconstruction of mouse thorax based on frozen blockface images. Specimen is 6 hours old. Images produced with T-Vox voxel software. Metric bar ¼ 1 cm. (Oregon Health and Science University, Heart Research Center.) (See Color Insert.)

Figure 13 The same voxel model reconstruction as shown in Fig. 12, but with clipping plane (coronal) placed to reveal a four-chamber view of the heart. LV, left ventricle, RV, right ventricle, LA, left atrium; RA, right atrium. (Oregon Health and Science University, Heart Research Center.) (See Color Insert.)

microscopy. One great advantage of the use of voxel models is the opportunity to visualize microscopic anatomy in any cross-sectional plane; this allows the study of 3D spatial relationships of tissue structures by removing selected ‘‘bricks’’ of data and looking inside. Regardless of the method used to acquire the data, voxel data sets require a software interface for viewing and

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exploring the data. A comparison of some currently available voxel software packages is shown in Table I. Microscopy techniques that acquire images from block-face sections bypass the problem of registration but typically encounter challenges of low resolution, surface artifact from embedding material, and slow throughput times. Advances in computer technology, sectioning devices (optical and mechanical), and optical systems bring renewed interest to this approach (Figs. 12 and 13). Voxel models are ideal for volume calculations and automated segmentation. Within most voxel imaging software packages there are algorithms to detect contiguous voxels based on grey values and gradients. For example, chamber dimensions and volumes can be calculated in this way. When using these methods to determine volumes, it must be noted that voxel contiguity is defined as those voxels that connect at any face (6 neighbors per voxel), or those connecting at faces, edges, and vertices (26 neighbors).

IV. The Embryonic Human Heart Transformation of the lumen shape from quasilinear (Fig. 14) at 21 days to the complex curvilinear structure at 28 days (see Fig. 17) coincides with acute hemodynamic shifts within the heart. The influence of varying fluid forces on heart development has been the focus of much developmental research (Bremer, 1932; Hogers et al., 1999; Yoshida et al., 1983). The ease with which computerized graphical shapes of the heart lumen are modified makes them useful in analyzing hemodynamic characteristics

Figure 14 Surface models of human heart lumen from Carnegie Collection specimen 3710, stage 10. Left (frontal view): (a) First aortic arches; (b) bulbus cordis; (c) ventricle; (d) right horn sinus venosus. Right (left lateral view): (e) conus cordis; (f ) ventricle; (g) early atrioventricular canal; (h) sinus venosus. y dimension ¼ 0.8 mm.


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Figure 15 Surface models of human heart lumen of Carnegie Collection specimen 2053, stage 11. Left (frontal view): (a) truncus arteriosus; (b) bulbus cordis, early bulbar ridge; (c) ventricle; (d) left atrium. Right (left lateral view): (e) truncus arteriosus; (f ) bulbus cordis; (g) ventricle; (h) atrioventricular canal; (i) early left atrium. y dimension ¼ 1 mm.

Figure 16 Surface model of human heart lumen of Carnegie Collection specimen 6097, stage 12. Left (frontal view): (a) truncus arteriosus; (b) bulbus cordis; (c) ventricle; (d) fold of sinoatrial foramen. Right (left lateral view): (e) early right atrium; (f ) ventricle; (g) atrioventricular canal; (h) early left atrium. y dimension ¼ 0.8 mm.

at various stages of development using computational fluid dynamics (DeGroV et al., 2000). By 21 gestational days, the looping process has commenced and contractile motion of the heart propels blood in a cephalad direction (see bottom to top in Fig. 14). The lumen shape becomes more sigmoidal as looping progresses (see Fig. 2). The heart tube is attached to the body wall via the branchial arches at its upper pole and the septum transversum at its lower pole, leaving the tube physically free to loop in the pericardial cavity (Moore, 1982). Rudimentary

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Figure 17 Surface models of embryonic human heart lumen of Carnegie Collection specimen 5874, stage 13. Left (frontal view): (a) early right atrium; (b) bulbus cordis; (c) ventricle. Right (left lateral view); (d) ventricle; (e) atrioventricular canal; (f ) early left atrium; (g) sinus venosus; (h) truncus arteriosus; (i) cardinal vein inflow to sinus venosus. y dimension ¼ 1 mm.

bulbar and truncal ridges are seen in the outflow tract and atrioventricular canal, which has changed its orientation from vertical to horizontal at this stage (Davis, 2003). The endothelial lining of the cardiac lumen is enclosed in a sleeve of cardiac jelly, which in turn is enclosed by a mantle of contractile tissue, the myocardial primordium (O’Rahilly and Mu¨ller, 1987). Although many of the anatomical features in stage 12 embryo heart lumens remain unchanged from stage 11, we see that the radius of the bulboventricular fold is shorter, the atrium is now positioned more cephalad to the ventricle, and the sinoatrial foramen is infolding in the atrium (Fig. 16). The sinoatrial foramen eVectively marks the boundary between veins and the heart proper and serves as a valvular lip which, when the atrium contracts, acts to impede blood from escaping backward into the sinus venosus (O’Rahilly and Mu¨ller, 1987). By Carnegie stage 13, approximately 28 gestational days, the lumen volume has expanded in the atrial and ventricular chambers (Fig. 17). Trabeculations appear in the bulbus cordis and ventricle at this stage. Although the heart technically remains valveless at this stage, pulsations in the myocardium are rhythmic and coordinated contractions, progressing from the sinus to the outflow tract and eVectively propelling blood through the heart. Valvelike structures at critical sites in the cardiac tube permit this to happen. The first of these structures is the sinoatrial canal, as described previously. The second is the atrioventricular canal, where the gelatinous surrounding tissue narrows and is eVectively closed with myocardial contraction. The third constriction is the outflow tract and along the aortic trunk (see Fig. 17), which stops abruptly at the aortic sac, preventing regurgitation into the primitive ventricle.


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A voxel reconstruction of the well-known Carnegie Collection specimen 836 is demonstrated in Figs. 18–20. The data set consists of over 500 original histological photomicrographs individually processed and registered (Dr. Gasser, Louisianna State University), then restacked within a voxeliza-

Figure 18 Side views of voxel model of Carnegie Collection specimen 836, stage 13. The image on the right is a midsagittal section revealing internal anatomy impossible to visualize without voxel reconstruction. C, cranium; L, limb bud; T, tail; V, common ventricle of the heart; VL, ventricular lumen; A, atria; AVC, atrioventricular canal; B, branchial arches. Scale bar ¼ 4.0 mm. (See Color Insert.)

Figure 19 Coronal section of voxel model Carnegie Collection specimen 836. The ventricular lumen is filled with blood. Color variation within the lumen is due to inconsistency in the staining at preparation. V, common ventricle of the heart; VL, ventricular lumen; BC, bulbus cordis; C, cranium. (See Color Insert.)

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Figure 20 Transverse plane (original plane of histological sections). A, inferior regions of the common atrium; V, ventricle; BC, bulbus cordis; C, cranium; S, spinal canal. (See Color Insert.)

tion software package (T-Vox). The original plane of sectioning was transverse at 15 mm per section. This visualization tool provides cross-sectional views in arbitrary planes and removal of selected ‘‘data bricks’’ allowing unique, first-time views of anatomical relationships during embryogenesis. The eventual coupling of microarray and proteomic data with these data forms shows great promise for innovative visualization of complex gene expression patterns vital for understanding cardiogenesis.

V. Recent Developments A. Point-Based Surface Modeling and Graphics Processing Units An alternative to NURBS surface modeling techniques uses point samples specified by contours to generate a surface from the equivalent of a point cloud. Point-based modeling, a relatively new concept in surface modeling, requires a new set of mathematical functions currently being developed by the computer graphics community. The advantage of this technique is a much more versatile and flexible approach, allowing the user to skip much of the processing required for NURBS surfaces such as manually matching control vertices across contours. Also, the direct use of points allows far better control of surface detail such that each part of the biological surface gets sampled. A variation of this technique being explored is the point-set surface technique (Alexa et al., 2003). It is slightly more complicated to


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create triangulated models from point-set surfaces, which requires a surface reconstruction algorithm (Bernardini et al., 1999). Model-rendering technology has seen significant improvements in price, performance, and capabilities due to the introduction of a radically new technology initially intended to improve the capability of video games. The same technology can be applied to meet specific biological visualization and modeling needs. Graphics processing units (GPUs) have added considerable power to desktop computing, exceeding previous generations of high-end graphics hardware (Lindholm et al., 2001). One of the key features of this new generation of hardware is its unprecedented programmability. Instead of fixed function graphics pipelines, current GPUs are fully programmable, making it possible to specify one’s own functionality instead of having to rely on preexisting techniques. This has significant potential for real-time 3D visualization. It makes it possible to develop improved volume-rendering techniques targeted for particular applications. In particular, it makes it easier to develop specialized lighting and shading functionality that can be used to highlight specific features of 3D data sets. Furthermore, with GPUs having over 100 million transistors, they have the power to perform complex rendering operations in real time. It is clear that improved graphics performance will significantly enhance interactivity and model quality. In particular, such devices set the stage for the development of techniques for combining real-time imagery of volumes and surfaces, as well as improving the rendering of semitransparent voxel models.

B. Stereolithographic Physical Models of the Embryonic Heart An innovative and novel application of the 3D computer reconstructions is stereolithographical physical modeling. In the stereolithography (SLA) process, point data describing the model’s surface defines the 2D boundaries and path of ultraviolet laser light across a reservoir of liquid photopolymer resin. Energy from the laser initiates a chemical reaction to harden the resin, resulting in an exact (þ/ 125 mm) scale model of the 3D computer volume. The laser ‘‘fills in’’ serial 2D areas to produce a tangible 3D model. Variable hardness materials may be used in the SLA modeling process, depending on application needs. A series of SLA models describing the early embryonic heart surface and lumen has been produced from Carnegie Collection specimens (Pentecost et al., 1999, 2001). SLA models from some of these specimens’ lumens were used as ‘‘plugs’’ to create flexible sleeves of compliant material to study fluid dynamics in these shapes. With the use of digital particle velocimetry, computational fluid dynamics flow analyses were obtained in steady and pulsatile flow studies in early lumen forms. These models permit

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analysis of shear forces and presence/absence of streaming flow in various anatomical regions. Furthermore, shape deformations, such as narrowing the outflow tract, can easily be made in the computer and physical models to observe the eVect on fluid dynamics. Novel application of this technology illuminates cause–eVect relationships between mechanical forces and gene expression patterns in normal and anomalous heart formation (DeGroV et al., 2000) and supports recently published information about the role of hemodynamic forces on aberrant zebra fish heart development (Hove et al., 2003).

C. Spatial Genomics The gene networks responsible for controlling pattern formation during cardiogenesis remain largely unknown, but the profusion of multimodal biological data compels and aVords novel analyses of the spatial–temporal relationships between gene activity, gene product, and embryo anatomy that should enhance our ability to correlate regulatory mechanisms with developing and remodeling tissues. The practice of displaying genomic and anatomical data in 3D models is emerging, although current methods for gathering and compiling data for these models are time consuming and rudimentary. Perhaps the best example demonstrating the integration of in situ gene expression data and 3D graphical models is seen in the optical projection tomography models of whole mouse embryos where tissue distribution of the message (mRNA) and gene product are displayed (Sharpe et al., 2002). The Edinburgh mouse atlas of gene expression (EMAGE) is a prime example of emerging 3D biotechnology for spatially describing gene expression patterns during organogenesis (Davidson et al., 1997). Links with genome databases can be made from these graphical displays, oVering a new tool for advancing the understanding embryo development.

D. Optical Projection Tomography Optical projection tomography introduces high-resolution (5 mm) 3D graphical models of intact mouse embryos and the ability to map anatomical expression patterns of specific genes. This technique oVers potential solutions to several challenges facing spatial genomics by reducing throughput time, allowing multiplane visualization of fluorescent signals from intact specimens containing antibody probes, and permitting high-resolution visualization of the entire specimen. That specimens must be transparent is a significant drawback that remains to be overcome (Sharpe et al., 2002).


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E. Epifluorescent Stereomicroscopy The observation of fluorescent illumination in targeted biological specimens is becoming a standard mode of visualization in optical microscopy. It is also called incident light fluorescence, epifluorescence, or episcopic fluorescence. Images of whole mount or sectioned specimens containing fluorescent labels may be a useful source of data for 3D reconstructions. For example, in whole specimens too large for confocal microscopy, stereomicroscopic block-face imaging of specimens containing fluorescent markers can be used to avoid the problems of registration and still produce cross-sectional data for voxel modeling. Stereomicroscopy also provides a greater field of view that is needed for large specimens. Figure 21 shows an example of a chick embryo voxel model containing a fluorescent marker. Perhaps the greatest utility for this and similar techniques lies in the potential visualization of single and multigene expression patterns in a single canonical volume by superposing data sets. If this can be accomplished with the element of time precisely controlled, it is conceivable that all phases of cellular growth and diVerentiation, as well as all levels in tissue-forming gene expression cascades, could be mapped in 3D models.

Figure 21 Chick embryo (E14.5) voxel reconstruction of 5 micron sections containing a nonspecific fluorescent marker. Bricks removed to reveal internal cardiac anatomy. (Arrowhead, apex of ventricle; arrow, outflow tract and aortic arch.) The greatest dimension of this specimen is approximately 7 mm. (Oregon Health and Science University, Heart Research Center.) (See Color Insert.)

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F. Cellular Modeling and Simulations Cellular migration and cell-fate mapping studies, along with various antibody probes and immunolabeling techniques, are classical methods for studying mesodermal diversification that results in heart formation. Although it remains unknown what constitutes the message(s) for cardiac myocyte lineage specification and restriction, numerous computerized simulation algorithms have been developed to describe anatomical and spatial characteristics of protein–protein and transcription factor (TF)–gene interactions in various species (Cummings, 2000; McAdams, 1997). In addition, cell and molecular-level 3D graphical modeling is now finding purpose in many areas of biology, such as stem cell research, bioinformatics for comparison of protein tertiary structures, and genomics for molecular modeling. 3D modeling of intracellular organelles such as ribosomes and Golgi apparatus based on immunofluorescent signals in confocal microscopy and other modalities is being used to analyze a variety of signaling mechanisms, cellular aging, and cancer (Pelletier et al., 2002; Poindexter et al., 2002). With visualization detail to the subcellular level, the full scope of 3D heart modeling capabilities begins to emerge; yet we lack a single visualization mechanism through which this broad range of data can be integrated and correlated.

G. Symbolic Modeling: Anatomical Ontologies A logical anatomical terminology scheme is required to link anatomical information in 3D embryo models with gene expression data. A complete anatomical ontology for developing anatomy does not currently exist, although eVorts are underway to create such a tool. The ontology for developing tissues requires special consideration because anatomical regions change names, function, and locations during embryogenesis. This is especially true in the developing heart, where tissues undergo a complex series of physical modifications before becoming a definitive four-chambered structure. The Digital Anatomist (Brinkley and Rosse, 1997; Brinkley et al., 1997) Foundational Model (FM) for anatomical nomenclature (Rosse et al., 1998a, 1998b) is a concept-based (vs term-based) inheritance hierarchy. The most advanced and largest computer-based knowledge source of its kind, the FM contains over 70,000 concepts for adult anatomy (Rosse, 2001). It also includes an anatomical transformation abstraction (ATA) for which a developmental ontology for embryological anatomy is currently being developed. The importance of nomenclature ontology for the entire scope of embryological anatomy cannot be overstated. It is an absolute necessity if


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relationships between gene activity, embryological anatomical structure, time, and physiological function are ever to be completely understood.

H. Fetal Origins of Adult Disease One of the driving forces for studying embryo development beyond the intense intellectual curiosity that motivates the scientific quest is the new finding that prenatal development has enormous consequences for population health. As mentioned, David Barker showed that adult mortality rates due to ischemic heart disease in the United Kingdom were related to fetal undergrowth. Since that time there have been studies in more than a dozen countries that support that conclusion. Furthermore, the growth trajectory of the embryo and fetus are partially determined during the early pre- and post-implantation period (Barker et al., 1989). Thus, it is clear that environmental factors (e.g., nutritional, hemodynamic, oxygen, hormonal) strongly influence the aspects of growth of the embryo and fetus that will determine the level of susceptibility to disease during adult life. Because of the lack of information regarding the regulators of heart growth during the embryonic and fetal periods, there is now an urgent imperative to understand the role of specific nutrients, wall and shear stresses, toxicants, and receptor ligands in determining growth outcomes. Although the methods that determine the roles of single genes as regulators of cellular processes in heart diVerentiation and growth have been useful, the determination of multiple gene interactions will be required to further our understanding of heart growth and its ultimate impact on adult disease. The use of computerized models that can demonstrate multiple gene expression patterns in three dimensions will be required for understanding the patterning eVects that can be manipulated for future therapies and protection against pathology.

I. Visible Embryo NGI Project OYcially titled ‘‘Human Embryology Digital Library and Collaboratory Support Tools,’’ this project is part of the Digital Libraries Initiative and is funded by the National Library of Medicine. The project’s purpose is to demonstrate how leading-edge information technologies in computation, visualization, collaboration, and networking can expand capabilities in medical science for developmental studies, clinical work, and teaching. The project focuses on providing the capability for medical professionals to communicate detailed information about development of the human embryo in a visual form. To achieve this, the project technical team has developed a

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network of medical collaboration workstations, using high-performance oV-the-shelf networked computer systems combined with advanced software for collaboration and medical visualization. The workstations are installed at eight project locations and interconnected over high-performance networks (Internet2) operating at data rates over 100 MB per second nationwide. The Visible Embryo NGI Project (2001) has demonstrated the network of collaborative visualization workstations in three advanced applications: 1. The Annotation and Modeling application created an archive of tagged image data for visualization, where every picture element can be associated with any number of diVerent labels (e.g., the organ system to which it belongs, the structure and tissue types represented, the specimen to which it belongs). This provides the basic archive used by the other two applications. 2. The Embryology Education application makes the visualization tools available for medical student use and also creates animations of embryo organ system development. Software ‘‘morphing’’ techniques are used to make a movielike sequence of images derived from a selected set of embryos. 3. The Clinical Management Planning application makes the data available in visual form so that a geographically distributed group of physicians can look at it simultaneously, manipulate it, and discuss it. Potential gains in education, scientific investigation, and clinical management from large-scale collaboration projects such as the Visible Embryo NGI Project are oVset by the cost of connection to high bandwidth networks. Issues concerning system maintenance, management, and project oversight on a long-term basis, as well as the need to prove cost savings, scientific utility, and educational benefit, remain unanswered and, as such, pose significant hurdles. Nevertheless, without eYcient networks to empower multi-institutional collaborative research, our understanding of the complex orchestration of cardiogenesis is likely to continue growth in small increments instead of unprecedented bounds.

VI. Summary and Future Directions It is becoming increasingly important to investigate correlations between embryonic heart development and adult heart health, yet our understanding of the complex genetic, molecular, and cellular processes governing cardiogenesis is far from complete. Knowledge of cardiogenic processes continues to depend heavily on our ability to visualize anatomical structure. The enormous amount of genomic and molecular information being generated almost


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daily cannot be understood or put in its biological context without new tools capable of integrating anatomical, genomic, and molecular data in three dimensions. The recent surge of genomic information also creates an unparalleled demand for scientists with expertise in computational methods and biological/medical sciences. How we represent anatomical structure has evolved from crude restacking of various physical materials to highly accurate 3D computer models using sophisticated graphics methodologies. Tomographical data is no longer solely dependent on histological sectioning, a process that destroys the embryonic heart, but is now obtainable by nondestructive optical methods with increasing resolution and speed. The realm of descriptive embryology now overlaps that of experimental biology. Exploiting fluorescent materials for targeting, labeling, and isolating proteins via antibody probes, combined with nondestructive imaging, is providing new information about regions of gene activity in the developing heart and promises new insight into gene expression patterns. Given the microscopic dimensions of the embryonic heart, eVective resolution of nondestructive imaging methods continues to be suboptimal for voxel models in which cell-level detail can be adequately visualized and gene activity might be mapped. Extrapolating developmental cardiac gene expression knowledge from animal-based investigations to the human species, despite homology across many domains, poses significant challenges for several reasons. Among them is the sensitive temporal element of cardiogenesis. Because we can never ascertain the precise timing of fertilization in the human, we are unable to map a developmental time line of suYcient granularity to precisely describe where and when the events of heart development occur. We are able to describe anatomical transformations within a time frame of a few days at best. In the mouse, estimates of fertilization time are more precise (þ/ 12 hours), but given the rapid pace at which physical changes occur in the developing heart, even this precision is unacceptable if we are to fully understand relationships between specific gene activity and phenotypic development. Another challenge lies in anatomical variation between species. Although the mouse has been considered an excellent animal model for developmental and anatomical studies, there are morphological features in the mouse heart that diVer from those in the human heart (Icardo et al., 1993). As genomic data becomes increasingly available, cross-species comparisons using 3D visualization will require some mechanism for reconciling anatomical and temporal variations. Methods for improving computational visualization are needed. Algorithms that reduce rendering times, improve accuracy of automated image registration, and allow voxel-based 3D deformation to simulate cardiac morphogenesis will enhance the utility of graphical descriptions. Developing

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eVective Web-based visualization tools remains a research problem, although considerable progress has been made. A key challenge is guaranteeing interactive frame rates on a wide range of hardware configurations. Such a system needs to adjust the quality of the images and the frame rate depending on the performance of each stage of the visualization pipeline. On high-end devices, the system will deliver high-resolution images at realtime frame rates, whereas on lower-end devices the system will deliver smaller images and slower (still interactive) frame rates. To achieve this scalable performance over a spectrum of devices, it is necessary to develop techniques that eVectively decouple the visualization requirements from the available hardware resources. For applications that require the simultaneous use of volume and surface models, the problem becomes more complex. Current compression bitstreams for 3D data (e.g., MPEG-4) do not support volume models; in fact, only triangulated surfaces are supported. Also, although eYcient point-compression techniques exist (Fleishman et al., 2003), they also have not been integrated into current protocols. The interface required for clipping plane manipulation, zooming, and adjustment of individual voxel properties is yet too complex for Web-based applications. Limitations in network bandwidth are an impediment for transmission of most voxel-based data sets, although this issue will become less significant as more universities and research institutions become connected to Internet2 network systems. Another important need with respect to graphics technology is that of better tools for the model development interface for 3D projects. For example, the use of a 2D mouse for the tasks of generating 3D biological models is awkward and time consuming. The use of newer haptic-feedback devices has the potential to make spatially correct interaction easier and in particular, allow for more complex sculpting operations to be performed on acquired models (Ayila and Sobierajski, 1996).

References Alexa, M., Behr, J., Cohen-Or, D., Fleishman, S., Levin, D., and Silva, C. T. (2003). Computing and rendering point set surfaces. IEEE Trans. Visualization Comput. Graph. 9, 3–15. Ayila R., and Sobierajski, L. (1996). A Haptic Interaction Method for Volume Visualization. Visualization ’96 Proceedings, pp. 197–204. IEEE Computer Society Press, Los Alamitos, CA. Barker, D. J. P., Osmond, C., Winter, P., Margetts, B., and Simmonds, S. (1989). Weight in infancy and death from ischemic heart disease. Lancet 2, 577–580. Bernardini, F., Mittleman, J., Rushmeier, H., Silva, C., and Taubin, G. (1999). The ballpivoting algorithm for surface reconstruction. IEEE Trans. Visualization Comput. Graph. 5, 349–359. Born, G. (1889). Beitraege zur entwicklungsgeschichted des saugethierherzens. Archiv fur mikroskopische Anatmie und Entwicklungsmechanik 33, 284–377.


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Bremer, J. L. (1932). The presence and influence of two spiral streams in the heart of the chick embryo. Am. J. Anat. 49, 409–440. Brinkley, J. F., Bradley, S. W., Sundsten, J. W., and Rosse, C. (1997). The digital anatomist information system and its use in the generation and delivery of Web-based anatomy atlases. Comput. Biomed. Res. 30, 472–503. Brinkley, J. F., and Rosse, C. (1997). The digital anatomist distributed framework and its applications to knowledge-based medical imaging. J. Am. Med. Inform. Assoc. 4, 165–183. Clark, D. (2000). Blue gene and the race toward petaflops capacity. IEEE Concurrency 8, 5–9. Cummings, F. W. (2000). A model of pattern formation based on signaling pathways. J. Theor. Biol. 207, 107–116. Davidson, D., Bard, J., Brune, R., Burger, A., Dubreuil, C., Hill, W., Kaufman, M., Quinn, J., Stark, M., and Baldock, R. (1997). The mouse atlas and graphical gene-expression database. Semin. Cell Dev. Biol. 8, 509–517. Davis, C. L. (2003). Development of the human heart from its first appearance to the stage found in embryos of twenty paired somites. Carnegie Institution of Washington Publication 380. Contributions to Embryology 19, 245–283. DeGroff, C. G., Baptista, A., Thornburg, B., Pentecost, J., Thornburg, K., Sahn, D., and Gharib, M. (2000). Modeling applications in congenital heart disease: Flow in the embryonic human heart. Proceedings of ICMMB-11, 2000, pp. 279–281. Fleishman, S., Alexa, M., Cohen-Or, D., and Silva, C. (2003). Progressive Point Set Surfaces. ACM Transactions on Graphics. (In press.) Gasser, D. (2003). The Virtual Human Embryo: A Digital Image Database of Serially Sectioned Human Embryos from the Carnegie Collection. http://virtualhumanembryo.Isuhsc.edu/ DREM_ home.htm Harvey, R. P., and Rosenthal, N. (1999). ‘‘Heart Development.’’ Academic Press, New York. Hill, D. L., Batchelor, P. G., Holden, M., and Hawkes, D. J. (2001). Medical image registration. Phys. Med. Biol. 46, 1–45. His, W. (1885). ‘‘Anatomie Menschlicher Embryonen.’’ F. C. W., Leipzig. Hogers, B., DeRuiter, M. C., Gittenberger-de Groot, A. C., and Poelman, R. E. (1999). Extraembryonic venous obstructions lead to cardiovascular malformations and can be embryolethal. Cardiovasc. Res. 41, 87–99. Hogers, B., Gross, D., Lehmann, V., de Groot, H. J., de Roos, A., Gittenberger-de Groot, A. C., and Poelmann, R. E. (2001). Magnetic resonance microscopy at 17.6-Tesla on chicken embryos in vitro. J. Magn. Reson. Imaging 14, 83–86. Hove, J. R., Koster, R. W., Forouhar, A. S., Acevedo-Bolton, G., Fraser, S. E., and Gharib, M. (2003). Intracardiac fluid forces are an essential epigenetic factor for embryonic cardiogenesis. Nature 421, 172–177. Icardo, J. M. (1996). Developmental biology of the vertebrate heart. J. Exp. Zool. 275, 144–161. Icardo, J. M., Arrechedera, H., and Colvee, E. (1993). The atrioventricular valves of the mouse. 1. A scanning electron-microscope study. J. Anatomy 182, 87–94. Koupilova, I., Leon, D., Lithell, H., and Berglund, L. (1997). Size at birth and hypertension in longitudinally followed 50–70 year old men. Blood Pressure 6, 223–228. Lester, D. S., Pine, P. S., Delnomdedieu, M., Johannessen, J. N., and Johnson, G. A. (2000). Virtual neuropathology: Three-dimensional visualization of lesion due to toxic insult. Toxicol. Pathol. 28, 100–104. Lindholm, E., Kilgard, M., and Moreton, H. (2001). ‘‘A User-Programmable Vertex Engine.’’ Proceedings of ACM SIGGRAPH. Maintz, J. B., and Viergever, M. A. (1998). A survey of medical image registration. Med. Image Anal. 2, 1–36.

142

Pentecost et al.

Matsopoulos, G. K., Delibasis, K. K., Mouravliansky, N. A., and Nikita, K. S. (2000). Automatic medical image registration schemes using global optimization techniques. Stud. Health Technol. Inform. 79, 463–491. McAdams, H. H. (1997). Stochastic mechanisms in gene expression. Proc. Natl. Acad. Sci. USA 94, 814–819. Moore, K. L. (1982). ‘‘The Developing Human: Clinically Oriented Embryology.’’ W. B. Saunders, Philadelphia. O’Rahilly, R., and Mu¨ller, F. (1987). ‘‘Developmental Stages in Human Embryos.’’ Publication 637. Carnegie Institution of Washington, Washington, DC. O’Rahilly, R., Muller, F., and Bossy, J. (1986). Atlas of the stages of development of the external forms of the brain in the human embryo. Arch. Anat. Histol. Embryol. 69, 3–39. Palmes-Saloma, C., and Saloma, C. (2000). Long-depth imaging of specific gene expressions in whole-mount mouse embryos with single-photon excitation confocal fluorescence microscopy and FISH. J. Struct. Biol. 131, 56–66. Pelletier, L., Stern, C. A., Pypaert, M., Sheff, D., Ngo, H. M., Roper, N., He, C. Y., Hu, K., Toomre, D., Coppens, I., Roos, D. S., Joiner, K. A., and Warren, G. (2002). Golgi biogenesis in Toxoplasma gondii. Nature 418, 548–552. Pentecost, J., Icardo, J., and Thornburg, K. L. (1999). 3D computer modeling of human cardiogenesis. Comput. Med. Imaging Graph. 23, 45–49. Pentecost, J. O., Sahn, D. J., Thornburg, B. L., Gharib, M., Baptista, A., and Thornburg, K. L. (2001). Graphical and stereolithographic models of the developing human heart lumen. Comput. Med. Imaging Graph. 25, 459–463. Periasamy, A., Skoglund, P., Noakes, C., and Keller, R. (1999). An evaluation of two-photon excitation versus confocal and digital deconvolution fluorescence microscopy imaging in Xenopus morphogenesis. Microsc. Res. Tech. 47, 172–181. Piston, D. W. (1999). Imaging living cells and tissues by two-photon excitation microscopy. Trends Cell Biol. 9, 66–99. Pizer, S. M., Fritsch, D. S., Yushkevich, P. A., Johnson, V. E., and Chaney, E. L. (1999). Segmentation, registration, and measurement of shape variation via image object shape. IEEE Trans. Med. Imaging 18, 851–865. Poindexter, B., Pereira-Smith, O., Wadhwa, R., Buja, L., and Bick, R. (May 2002). 3D reconstruction and localization of mortalin by deconvolution microscopy. Microsc. Analysis. Rosse, C. (2001). Terminologia anatomica: Considered from the perspective of next-generation knowledge sources. Clin. Anat. 14, 120–133. Rosse, C., Mejino, J. L., Modayur, B. R., Jakobovits, R., Hinshaw, K. P., and Brinkley, J. F. (1998a). Motivation and organizational principles for anatomical knowledge representation: The digital anatomist symbolic knowledge base. J. Am. Med. Inform. Assoc. 5, 17–40. Rosse, C., Shapiro, L. G., and Brinkley, J. F. (1998b). The digital anatomist foundational model: Principles for defining and structuring its concept domain. Proc. AMIA. Symp. 820–824. Sharpe, J., Ahlgren, U., Perry, P., Hill, B., Ross, A., Hecksher- Sorensen, J., Baldock, R., and Davidson, D. (2002). Optical projection tomography as a tool for 3D microscopy and gene expression studies. Science 296(5567), 541–545. Smith, B. R., Huff, D. S., and Johnson, G. A. (1999). Magnetic resonance imaging of embryos: An Internet resource for the study of embryonic development. Comput. Med. Imaging Graph. 23, 33–40. Smith, B. R., Johnson, G. A., Groman, E. V., and Linney, E. (1994). Magnetic resonance microscopy of mouse embryos. Proc. Natl. Acad. Sci. USA 91, 3530–3533. Streeter, G. L. (1942). Developmental horizons in human embryos: Description of age group XI, 13 to 20 somites, and age group XII, 21 to 29 somites. Contrib. Embryol. 30, 211–245.


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Streeter, G. L. (1945). Developmental horizons in human embryos: Description of age group XIII, embryos about 4 or 5 mm long, and age group XIV, period of indentation of the lens vesicle. Contrib. Embryol. 31, 27–63. Treves, S. T., Mitchell, K. D., and Habboush, I. H. (1998). Three dimensional image alignment, registration and fusion. Q. J. Nucl. Med. 42, 83–92. Visible Embryo NGI Project. (2001). http://www.visembryo.org. Vuillemin, M., and Pexieder, T. (1989). Normal stages of cardiac organogenesis in the mouse: I. Development of the external shape of the heart. Am. J. Anat. 184, 101–113. Woods, R. P., Mazziotta, J. C., and Cherry, S. R. (1993). MRI-PET registration with automated algorithm. J. Comput. Assist. Tomogr. 17, 536–546. Yoshida, H., Manasek, F., and Archilla, R. A. (1983). Intracardiac flow patterns in early embryonic life: A reexamination. Circ. Res. 53, 363–371. Zhilkin, P., and Alexander, M. E. (2000). 3D image registration using a fast noniterative algorithm. Magn. Reson. Imaging 18, 1143–1150. Zhu, Y. M. (2002). Volume image registration by cross-entropy optimization. IEEE Trans. Med. Imaging 21, 174–180.


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Plasmid and Chromosome Traffic Control: How ParA and ParB Drive Partition Jennifer A. Surtees and Barbara E. Funnell Department of Medical Genetics and Microbiology, University of Toronto, Toronto, Ontario M5S 1A8, Canada

I. Introduction: Chromosome Dynamics in Bacterial Cells II. P1 ParA and PartB A. Gene Regulation by ParA and ParB B. ParA Action in Partition C. Physical Properties of ParA D. The ParB-IHF-parS Partition Complex E. Physical Properties of ParB F. Gene Silencing G. Importance of the Cell One Quarter and Three Quarter Positions H. ParA–ParB Interactions during Partition III. Other Plasmid Partition Systems A. F Plasmid B. P7 and pMT1 C. RK2, pTAR, and Other Plasmids with Walker-Type Partition ATPases D. R1 and the Actin-Like ATPases IV. Bacterial ParA and ParB Proteins A. Bacillus subtilis B. Caulobacter crescentus C. Streptomyces coelicolor D. Pseudomonas putida V. Concluding Remarks Acknowledgments References

I. Introduction: Chromosome Dynamics in Bacterial Cells The survival of any species requires faithful inheritance of genetic information to the oVspring. Essential events in this process are the directed movement and positioning of chromosomes in cells and nuclei, which are responsible for accurately distributing them to daughter cells at cell division. Bacterial cells and their chromosomes are no exception. Although prokaryotes do not undergo the complex mitotic steps that occur in eukaryotic cells, prokaryotic chromosomes are nevertheless dynamically arranged during the cell cycle via the action of segregation, or ‘‘partition,’’ systems. Many precise protein– DNA and protein–protein interactions coordinate the orientation and Current Topics in Developmental Biology, Vol. 56 Copyright 2003, Elsevier Inc. All rights reserved. 0070-2153/03 $35.00

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movement of these chromosomes with DNA replication and cell division. Cell biology techniques have illustrated that plasmids and chromosomes are specifically positioned and oriented inside bacterial cells (Gordon et al., 1997; Ho et al., 2002; Jensen and Gerdes, 1999; Niki and Hiraga, 1997, 1998; Webb et al., 1997, 1998). Intracellular locations have been examined by fluorescence in situ hybridization (FISH) or GFP–LacI bound to lac operator sequences in the chromosome of interest. Although the details vary somewhat during the cell cycle and from species to species, in general the bacterial chromosome is oriented so that the origin(s) of replication is/are toward the cell pole(s) and the terminus is (or termini are) centrally positioned. Also, in general, plasmids are located at the one quarter and three quarter positions (determined as position of cell length in rod-shaped bacteria) for most of the cell cycle, but at midcell in newborn cells. DiVerent types of partition and chromosomal maintenance systems are responsible for the localization patterns. One family of partition systems has emerged from numerous functional and bioinformatic studies as common to many, if not most, bacterial and plasmid species. This system is distinguished by two proteins, often called ParA and ParB, which act on one or more ‘‘centromere-like’’ partition sites to distribute plasmids and chromosomes to daughter cells. The ParA-like protein is an ATPase, and the ParB-like protein is a site-specific DNA-binding protein that recognizes the partition site(s). In plasmids, the ParA–ParB system is the primary segregation machinery and is essential for stable plasmid maintenance in growing bacterial populations. In bacterial chromosomes, this system is one of several that contribute to proper chromosome segregation. In addition, the relative importance of ParA and ParB in chromosome segregation often depends on the developmental and/or growth stage of a given bacterial species. The genes for homologues of ParAs and ParBs exist in many bacterial chromosomes and plasmids (see Bignell and Thomas, 2001; Funnell and Slavcev, 2003; Gerdes et al., 2000; Hanai et al., 1996; Hayes, 2000). The conservation of these proteins in prokaryotes argues that many of the steps in partition will be common for bacterial as well as plasmid chromosomes. One of the well-studied paradigms of the ParA–ParB partition system is that of the P1 plasmid in Escherichia coli. This chapter reviews the information on the actions of ParA and ParB that has been learned from the study of P1 and then discusses how this information has been extrapolated to and augmented by the study of other plasmid and bacterial chromosome systems.

II. P1 ParA and ParB The P1 plasmid is the prophage of bacteriophage P1 in Escherichia coli and exists as a stable, autonomously replicating, unit-copy plasmid (Prentki et al., 1977). Stable maintenance of P1 plasmids is absolutely dependent upon its

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Figure 1 The P1 partition operon. The parA and parB genes (arrows), the par promoter region, parOP (black oval), and the partition site, parS (black rectangle) are shown. An expanded view of parOP indicates the positions of transcriptional ( 35 and 10) signals, the ribosome binding site (RBS), the parA start codon (ATG), and the inverted repeat sequence recognized by ParA (arrows). The boxes in the expanded diagram of parS correspond to the boxes described in the legend to Fig. 3.

partition system, encoded by a 2.5-kb region of the plasmid (Abeles et al., 1985). The genes for ParA and ParB form an operon, and the partition site, parS, is located immediately downstream of parB (Fig. 1). All three elements are essential for partition, although only parS is required in cis. ParA and ParB are required for two distinct functions in P1 partition: (1) regulation of par gene expression and (2) physical segregation of the plasmids (Abeles et al., 1985; Davis et al., 1996; Friedman and Austin, 1988; Hayes et al., 1994). ParA’s regulatory role is as the transcriptional repressor of the par operon. ParB improves the repressor activity of ParA and is therefore a corepressor. During the partition reaction, ParB and an E. coli protein, integration host factor (IHF), bind to parS to form a nucleoprotein structure called the partition complex (Davis and Austin, 1988; Davis et al., 1990; Funnell, 1988b, 1991). The partition complex mediates localization of the plasmid (Erdmann et al., 1999), presumably through ParA–ParB and ParB–ParB interactions, as well as through interactions with specific but as yet unknown host-encoded factors. ParA is thought to couple the energy of adenosine triphosphate (ATP) binding and hydrolysis to the placement of plasmids at their specific intracellular addresses (Bouet and Funnell, 1999; Davis et al., 1996). The molecular nature of these addresses is still unknown. Candidates include the bacterial membrane and the DNA replication machinery, but their identification has proven elusive and will likely rely on further clarification of the mechanisms of the ParA and ParB proteins.

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A. Gene Regulation by ParA and ParB Regulation of parA and parB gene expression is crucial. Excessive amounts of either or both gene product(s) disrupt partition, and the nonrepressed par promoter is relatively strong (Abeles et al., 1985; Funnell, 1988a; Hayes et al., 1994). ParA represses transcription of the parAB operon by binding to a specific DNA site in the par promoter region, parOP (see Fig. 1) (Davey and Funnell, 1994; Davis et al., 1992; Friedman and Austin, 1988). DNase I footprinting has shown that DNA binding by ParA is centered over a 20-bp imperfect inverted repeat within parOP (Davis et al., 1992), implying that the inverted repeat is the ParA recognition sequence. This conclusion is supported by the eVect of several mutations in the inverted repeat, which damage or eliminate repression by ParA in vivo (Hayes et al., 1994). ParA protects about 150 bp of DNA surrounding the inverted repeat from DNase I (Davey and Funnell, 1994; Davis et al., 1992). Because ParA likely binds to the DNA as a dimer, several ParA dimers bind cooperatively to this region. ParA binding presumably interferes with the ability of RNA polymerase to interact with the promoter. ParA belongs to a superfamily of partition ATPases that contain Walker A and B nucleotide-binding motifs (Koonin, 1993; Motallebi-Veshareh et al., 1990). Consistent with the presence of these conserved sequences, ParA has a weak ATPase activity that is stimulated by ParB and by DNA of no particular sequence, length, or topology (Davis et al., 1992). The stimulatory eVects of ParB and DNA are additive. Nucleotide binding and hydrolysis by ParA modulate its site-specific DNA-binding activity in a complex manner (Bouet and Funnell, 1999; Davey and Funnell, 1994, 1997; Fung et al., 2001). First, ATP stimulates ParA binding to the operator region 5- to 10-fold, as determined by DNase I footprinting. ADP and nonhydrolyzable analogues of ATP stimulate DNA binding an additional 5- to 10-fold (Davey and Funnell, 1994). Therefore binding of ParA to adenine nucleotides stimulates its DNA-binding activities, while the act of hydrolysis is inhibitory. Second, nucleotide binding aVects the oligomeric state of ParA, pushing a monomer– dimer equilibrium toward dimer formation (Davey and Funnell, 1994). These observations suggest that ParA must be a dimer to bind to parOP. Further, they suggest that the adenosine diphosphate (ADP)-bound form of ParA is a better repressor of transcription than a ParA that is hydrolyzing ATP. Nucleotide binding also alters the conformation of ParA, increasing its percent helicity as determined by circular dichroism (Davey and Funnell, 1997). ATP and ADP alter helicity to diVerent extents, and the eVect of ATP S is similar to that of ADP. These diVerences imply that the diVerent contributions of nucleotide binding and hydrolysis to ParA activities correlate with specific conformational states of the protein.


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Mutation of the ATP-binding motifs in ParA has yielded versions of ParA that provide additional insights into the repression and partition activities of ParA. Mutations that eliminate the ability of ParA to both hydrolyze and bind ATP cause significant defects in both autoregulation and partition (Davis et al., 1996; Fung et al., 2001). Several other mutations have been shown to damage ATPase activity but not ATP binding at physiological concentrations of ATP (Fung et al., 2001). These mutations are defective for partition but behave as ‘‘super-repressors’’ of the par operon; that is, they repress transcription much more strongly than wild-type ParA. In vitro, the site-specific DNA-binding activity (to parOP) of these ParAs is still nucleotide dependent, but ATP and ADP are equally good cofactors. Therefore they act as though they adopt the ADP-bound form of ParA, regardless of the nucleotide that is bound. In addition, their conduct supports the hypothesis that ATP hydrolysis is necessary for partition but is inhibitory for repression. In vivo, ParB stimulates the repressor activity of ParA (Friedman and Austin, 1988). In vitro, ParB stimulates ParA’s site-specific DNA-binding activity in the presence of ATP but not in the absence of nucleotide or in the presence of ADP or ATP S (Davey and Funnell, 1997). Interestingly, the aYnity of ParA for parOP in the presence of both ATP and ParB is the same as its aYnity for parOP in the presence of ADP (or ATP S) only. These observations suggest that the corepressor activity of ParB somehow prevents or negates the inhibitory eVects of ATP hydrolysis on DNA binding by ParA. Because ParB also stimulates the rate of ATP hydrolysis by ParA, it may, for example, increase the amount of time that ParA spends in the ADP-bound form. In vivo, the repressor activities of the ParA superrepressor mutants are insensitive to any stimulation by ParB, and the strength of the repression by the super-repressors alone is about the same as that of wild-type ParA when accompanied by ParB. Therefore, both in vitro and in vivo results imply that the corepressor function of ParB converts ParA to its repressor form. The partition site also plays a role in the regulation of parA and parB gene expression (Hao and Yarmolinsky, 2002). The presence of parS was shown to reduce expression of the par operon, even when parS was present on a diVerent DNA molecule than parOP. The eVect depended on the presence of both ParA and ParB. Similar observations have been made in the F plasmid system (Yates et al., 1999). They suggest that ParB bound to parS is a better corepressor than free ParB (or ParB bound nonspecifically to DNA). An attractive explanation is that the ParB–parS complex influences gene regulation via the act of partition. In this scenario, for example, the partition activity of ParA at the ParB–parS partition complex requires hydrolysis of ATP, which in turn would increase the concentration of the repressor form of ParA (Bouet and Funnell, 1999; Hao and Yarmolinsky, 2002).

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B. ParA Action in Partition Whereas the absolute and relative levels of ParA and ParB in the cell must be controlled, the regulatory role of ParA can be bypassed and is therefore dispensable for plasmid stability (Davis et al., 1996). Certain mutations in parOP lead to low-level, constitutive expression of parA and parB. The resulting protein levels support partition. However, even in the presence of these mutations, ParA is required for P1 stability. Therefore, ParA is directly required for partition. The requirement for ParA in the positioning of plasmids has been established by immunofluorescent visualization of ParB and partition complexes in vivo (Erdmann et al., 1999). ParB bound to parS appears as large foci under the microscope. In the presence of ParA, most cells possess two foci that are located at the one-quarter and three-quarter positions of cell length. They sit over the bacterial nucleoids (the protein–DNA mass that contains the condensed bacterial chromosome). The number of foci and their positions correlate with total cell length. They are at midcell in the smallest, and presumably newborn, cells. The longest cells often contain four ParB foci, which are evenly distributed over the nucleoids. These positions correlate with the positions of P1 plasmids, measured by tagging with GFP–LacI or by FISH (Gordon et al., 1997; Ho et al., 2002). In the absence of ParA, the pattern of ParB foci is diVerent (Erdmann et al., 1999). There are fewer foci per cell, the number of foci does not correlate with cell length, and the foci usually localize to the ends or the middle of the cell. The latter positions are the positions not occupied by the nucleoids, as though ParB–parS complexes are somehow excluded from the same regions that the bacterial chromosomes occupy. These observations suggest that ParA helps to tether plasmids to sites that would otherwise be occluded, hidden, or out-competed by the bacterial chromosome. How it distinguishes (or helps ParB distinguish) these sites is not understood. Measured by immunofluorescence, ParA is distributed throughout the cell, although it may be more concentrated over the nucleoids (Erdmann et al., 1999). This type of pattern has also been observed for F SopA (Hirano et al., 1998). Two other ParAs, Bacillus subtilis Soj and pB171 ParA, have been observed as green fluorescent protein (GFP) fusions to dynamically associate and dissociate with nucleoids or poles of the cell and appear to oscillate from one end of the cell to the other (see later) (Ebersbach and Gerdes, 2001; Marston and Errington, 1999; Quisel et al., 1999). Although the bulk of P1 ParA–GFP in the cell has not been observed to oscillate (Z. Zhang and B. E. Funnell, unpublished results), we cannot rule out the possibility that a subpopulation of the protein does exhibit this behavior. The role of oscillation for pB171 ParA or for B. subtilis Soj is still not understood.


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In vitro, ParA interacts directly with the ParB–parS partition complex, measured in a gel-mobility shift assay (Bouet and Funnell, 1999). An interaction is supported by ATP and ATP S but not ADP, indicating that the triphosphate is necessary but hydrolysis is not. The consequence of the interaction varies with ParB concentration. At high ParB concentration, ParA promotes formation of a complex that is larger than the initial ParB–parS complex. The DNA-binding patterns measured by chemical footprinting suggest that either (1) ParA joins the complex, or (2) ParA helps to recruit further ParB dimers to the complex (or both). At low ParB concentrations, ParA interferes with ParB binding to parS. We speculate that the latter eVect is a result of the role that ParA plays in dissociating paired partition complexes. The results also indicate that nucleotide binding constitutes a molecular switch that discriminates between the partition and repressor forms of ParA. ParA–ATP is necessary for an interaction with the partition complex, whereas ParA–ADP is the transcriptional repressor. One class of parA mutants is called ‘‘propagation defective,’’ or parPD (Fung et al., 2001; Youngren and Austin, 1997). P1 plasmids encoding these mutants cannot be established or maintained in a cell. This is in direct contrast to plasmids carrying parA null mutations or other point mutations that are established and then slowly lost from a cell population by random diVusion. The dramatic phenotype of the propagation-defective parA mutants is dependent on the presence of ParB, indicating that ParA interacts with the ParB–parS complex. Interestingly, propagation-defective parA mutants cause a phenotype very much like that observed upon overexpression of parB. An excess of ParB has a strong destabilizing eVect on P1 plasmids such that they cannot be propagated in E. coli (Funnell, 1988a). Excess ParB will also destabilize medium- to high-copy plasmids that contain parS, even though such plasmids do not require a partition system for their stability. Plasmid copy number is not reduced, and thus the eVect is not due to interference with, or silencing of, the replication system. These and other results argue that too high a ratio of ParB to ParA causes plasmids to segregate in groups rather than as individual molecules (Funnell, 1988a; Radnedge et al., 1998). The phenotype of parPD parA mutants implies that ParA normally stimulates an association, or ‘‘pairing,’’ of plasmids together, but because ParB alone can sequester plasmids, plasmids are likely joined by ParB–ParB interactions. The ParPD mutant proteins likely have a defect in their ability to dissociate or separate plasmids. Alternatively, ParPD ParA proteins may fail to dissociate the plasmids from a host location. That some parPD mutations map to the ATP binding site indicates that ATP binding and hydrolysis play a role in this activity of ParA (Fung et al., 2001).

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C. Physical Properties of ParA ParA is a 44-kDa polypeptide that is 398 amino acids in length. Alignment of many ParAs from diVerent sources has revealed four main regions of similarity (Fig. 2a) (Koonin, 1993; Motallebi-Veshareh et al., 1990). Three of these regions contribute to the ATP binding site: Walker A, Walker B, and Motif A0 ; mutations in these motifs alter the ATP binding and hydrolysis properties of the protein (Davis et al., 1996; Fung et al., 2001; Libante et al., 2001). Although the structure of a ParA has not been reported, the

Figure 2 Functional domains and conserved sequences of (a) P1 ParA and (b) P1 ParB. Each protein is represented by a white rectangle. The scale in the middle of the figure shows length/ position as a number of amino acid residues. The positions of conserved motifs (described in the text) are drawn on each rectangle and labeled above it. DNA binding motifs (HTH) are grey; others are black. Thick lines below each protein show the regions for which a function and/or specificity has been defined. Grey lines correspond to DNA-binding functions and specificities; others are black.


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crystal structures of several related nonpartition ATPases are available. These include NifH (or Fe protein), ArsA, and MinD (Georgiadis et al., 1992; Hayashi et al., 2001; Sakai et al., 2001; Schindelin et al., 1997; Zhou et al., 2000). In these proteins, all three motifs contribute to binding the phosphate moieties of ATP and ADP. Their ATP binding pockets resemble those in G-proteins that are involved in signal transduction in eukaryotes. The equivalent Motif A0 and Walker B motifs have been called ‘‘switch I’’ and ‘‘switch II’’ motifs, respectively, because they are thought to transduce signals from the binding and hydrolysis of ATP to other regions of the protein and to other protein partners (Hayashi et al., 2001; Jang et al., 2000). We expect that these motifs in ParA perform similar functions and, for example, transmit signals to ParB. The Walker B/switch II sequence is adjacent to the fourth conserved motif in ParA, called Motif 3 in Fig. 2a. While the function of this motif is unknown, it is close to or within the region of ParA that is thought to interact with ParB. It also has been suggested that this region interacts with the bacterial membrane. In either case, it should be especially sensitive to conformational changes that occur during ATP hydrolysis. The N-terminus of ParA most likely contains its site-specific DNAbinding domain. It is notable that P1 and several plasmid ParAs contain an approximately 100-amino-acid N-terminal domain that is missing in many of its homologues. A common feature of these longer ParAs is that they are site-specific DNA-binding proteins, whereas the others are not. Radnedge et al. (1996, 1998) used the approach of exchanging regions of P1 and P7 partition proteins to examine the specificity of these components for their binding partners. The partition components of the P7 plasmid are similar to those of P1, but each system is specific for its own regulatory and partition components. For example, P1 ParA acts as a repressor at P1 parOP but not at P7 parOP, and vice versa (Hayes et al., 1994). By monitoring whether swapped regions of the proteins also exchanged specificity, the N-terminus of ParA was shown to contain the recognition specificity for parOP. This result is consistent with the identification of a putative helixturn-helix motif in the N-terminus of ParA. Similar domain-swapping experiments suggest that the C-terminal half of ParA contains the region that interacts with ParB (Fig. 2a), although this has not been delineated further.

D. The ParB-IHF-parS Partition Complex ParB is an unusual site-specific DNA-binding protein in that it recognizes two distinct DNA sequence motifs within the parS site (Davis and Austin, 1988; Davis et al., 1990; Funnell and Gagnier, 1993). P1 ParB and E. coli IHF bind to parS to first form a high-aYnity core complex (Funnell,

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1988b, 1991). This complex then recruits more molecules of ParB and is acted on by ParA, which is necessary to direct plasmids to their specific intracellular addresses. The complexity of the partition complex is reflected in the complexity of the parS site (Fig. 3). The parS site contains four copies of a heptamer sequence called Box A (ATTTCAC/A) and two copies of a hexamer sequence called Box B (TCGCCA) that are recognized by ParB, although not all copies are necessary for partition (Davis et al., 1990; Funnell and Gagnier, 1993, 1994; Martin et al., 1991). For the purposes of discussion, parS can be divided into three main sections: left, central, and right (Fig. 3). The right section is suYcient to mediate partition in vivo (Martin et al., 1987) and a specific interaction with ParB in vitro (Davis et al., 1990; Funnell and Gagnier, 1993). This 34-bp region contains a large 13-bp inverted repeat with a 3-bp spacer (Fig. 3). The significance of this large inverted repeat is not clear, however, because further deletion analyses narrowed the minimal region down to the leftmost 22 bp of this region (Martin et al., 1991), indicating that only the left half of this palindrome was important for function. Within the 22-bp minimal region is a smaller inverted repeat of two Box A sequences and an adjacent Box B sequence (Fig. 3). The right half of the larger palindrome contains an additional copy of Box A. The left portion of parS contains one Box A and one Box B sequence (Fig. 3) but is unable to support partition or exert incompatibility (Martin et al., 1987). In the absence of IHF, much higher ParB concentrations are required to observe binding to the left half of an intact parS site than are required for binding to the right half of parS (Funnell and Gagnier, 1993). The left and right regions of parS flank the IHF recognition sequence (Fig. 3) (Funnell, 1988b, 1991). IHF is a small, architectural protein of E. coli with site-specific DNA-binding and DNA-bending activities (Rice et al., 1996). It is involved in a number of cellular processes, including phage site-specific recombination and gene expression (reviewed in Nash, 1996).

Figure 3 DNA sequence and binding motifs of P1 parS. The grey and black boxes indicate the Box A and Box B motifs, respectively. The black line represents the region bound by IHF. The Box A repeats and the large inverted repeat in the right side of parS are designated by arrows.


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When IHF binds to parS, it creates a large bend in the DNA (Funnell, 1991). DNA bending by IHF can be partially replaced by intrinsically bent DNA (Hayes and Austin, 1994), arguing that the role of IHF is primarily to bend the DNA rather than to mediate protein–protein interactions. The IHFinduced bend brings the left and right sides of parS into close proximity (Funnell and Gagnier, 1993). In wild-type E. coli cells, IHF forms part of the partition complex and it increases ParB’s aYnity for parS approximately 10,000-fold (Funnell, 1988b, 1991). ParB and IHF binding are cooperative in that ParB also increases the aYnity of IHF for parS (Funnell and Gagnier, 1994). In the presence of IHF, both the right and left sides of parS are occupied by ParB simultaneously and with high aYnity, indicating that the bend facilitates simultaneous interactions of ParB with both sides of parS (Funnell and Gagnier, 1993). The spacing and helical phasing of the Box A and Box B sequences are critical (Funnell and Gagnier, 1993; Hayes and Austin, 1994). Insertion mutations in parS that change the spacing between the right and left halves destroy the ability of IHF to stimulate ParB binding unless the insertion is equivalent to a full turn of the DNA double helix. These observations support a model in which ParB makes simultaneous contacts with both sides of parS in the presence of an IHF-induced bend. In order for the ‘‘cross-bend’’ interactions to be made and stabilized, the two arms of parS and the ParB binding site within these arms must be properly aligned. IHF is not, however, absolutely required for P1 partition. Partition in the absence of IHF binding occurs but is slightly less eYcient because the aYnity of ParB for parS is lower (Funnell, 1991; Funnell and Gagnier, 1993). Also, without IHF, the left side of the parS site is dispensable. Therefore, there are two types of ParB partition complexes that can form at parS. The highaYnity complex forms when both ParB and IHF bind to parS, and its assembly requires the complete parS site. An ‘‘IHF-independent’’ complex forms when IHF is absent or in the presence of mutations that alter the geometry of the site. While ParB’s aYnity for parS is much lower in these circumstances, the concentration of ParB in the cell must be high enough for the minimal number of ParB molecules to bind and be suYcient for partition in vivo. Indeed, our measurements indicate that there are several thousand dimers of ParB per cell, which corresponds to approximately micromolar concentrations of the protein (Funnell and Gagnier, 1994). Partition without IHF would be more sensitive to changes in ParB concentration in vivo, which might vary with growth phase or from strain to strain, for example. Nevertheless, incompatibility phenotypes argue that IHF is always a component of the P1 partition complex at parS (Funnell, 1988b, 1991). Incompatibility is the ability to displace another plasmid that is maintained with the same par system. It is thought to represent competition between plasmids during the pairing event and is thus a function of the partition activity of parS.

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In wild-type cells, plasmids that are partitioned by a full parS site are not displaced by those partitioned by an IHF-independent one (the right side of parS, for example). However, the converse is not true; the full partition complexes displace the IHF-independent ones. In IHF mutants, both types of sites can compete with each other. These observations indicate that partition complexes in wild-type cells contain IHF and are thus insensitive to competition by the weaker ParB binding sites that lack IHF. DNase I footprinting and methylation protection experiments show that ParB contacts both the Box A and Box B motifs in parS (Davis and Austin, 1988; Funnell and Gagnier, 1993). In several diVerent studies, diVerent regions of parS were deleted and/or mutated to determine which of these boxes are required for partition in vivo (Davis et al., 1990; Funnell and Gagnier, 1993, 1994; Hayes and Austin, 1994; Martin et al., 1991). First, of the Box A motifs, only the two that form the small inverted repeat in the right of parS are necessary (Boxes A2 and A3 in Fig. 3), whether or not IHF is involved. The requirement for the Box B motifs depends on IHF. Both Box B1 and Box B2 are necessary to form the high-aYnity partition complex that depends on IHF (Davis et al., 1990; Funnell and Gagnier, 1993; Hayes and Austin, 1994). Deleting or mutating Box B1 does not, however, interfere with the slightly less eYcient partition reaction that occurs in the absence of IHF (Funnell and Gagnier, 1993; Martin et al., 1991), consistent with the idea that the left side of parS is unimportant in the absence of a bent DNA complex. Methylation interference experiments of partition complex formation in vitro were completely consistent with the in vivo results (Funnell and Gagnier, 1993). Methylation of bases in Boxes A2, A3, and B2 interfered with ParB binding in the presence and in the absence of IHF, whereas methylation of Boxes A1 and A4 showed no such interference. Methylation of Box B1 interfered with high-aYnity ParB binding; that is only in the presence of IHF. Therefore, the wild-type partition complex that contains IHF and ParB requires Boxes B1, A2, A3, and B2, and as stated earlier, these must be properly arranged with respect to each other and to the IHF bend. Boxes B1 and B2 define the outer edges of the parS site. Boxes A1 and A4 appear to be redundant in complex formation, although they may stabilize additional protein–DNA interactions in higher-order complexes. ParB is a dimer in solution (Funnell, 1991), and one dimer binds to parS to form the initial high-aYnity ParB complex with IHF (Bouet et al., 2000). The stoichiometry agrees with the assignment of required DNA sequence motifs; each monomer should contact one Box A and one Box B sequence. Following the assembly of the core complex, multiple dimers of ParB load on to create larger complexes in vitro (Bouet et al., 2000). Thus, the initial binding of ParB and IHF serve to nucleate the binding of more molecules of ParB, presumably by both protein–protein and protein–DNA interactions. The eventual size of this complex in vivo is unknown, but cell


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biology experiments argue that it is normally very large (Erdmann et al., 1999). The foci of ParB that are detected by immunofluorescence (see ParA, mentioned earlier) form only when parS is present, which suggests that many, if not most, of the thousands of ParB molecules in the cell converge on the parS site. However, the minimum number of ParB molecules that must join the partition complex for partition to occur is not known. The next step in partition is likely a pairing (or clustering) step, in which plasmids are joined together by ParB. Edgar et al. (2001) tested for pairing in vivo by adapting an assay that was originally developed by Wu and Liu (1992) to examine DNA looping between intramolecular sites. The results indicated that ParB could pair two parS sites present on the same plasmid. The ability of ParB to pair plasmids intermolecularly is supported by the number and appearance of ParB foci when visualized by immunofluorescence (Erdmann et al., 1999) and of plasmid foci when visualized by FISH (Ho et al., 2002). The number of observed ParB/plasmid foci is lower than the predicted number of copies of P1 in rapidly growing cells. (Under these conditions, cells have multiple copies of their chromosome and correspondingly of plasmids.) Most such cells contain only two foci of ParB, implying that each focus contains more than one plasmid. The simplest explanation of these results is that plasmids are joined together via their partition complexes, although we await a more direct, biochemical confirmation of this event during P1 partition.

E. Physical Properties of ParB ParB is a 38-kDa polypeptide that is 333 amino acids in length. It is extremely basic (pI ¼ 9.1) and runs anomalously (as a 45-kDa species) in SDS–polyacrylamide gels. Among ParB-like proteins in plasmids and bacterial chromosomes, the overall sequence homologies are modest, but they do share two regions of reasonable conservation (Fig. 2b). The first extends from residue 166–187 (P1 ParB numbering) and corresponds to predicted helix-turn-helix (HTH) motifs in most of these proteins (Dodd and Egan, 1990; Hanai et al., 1996; Lobocka and Yarmolinsky, 1996). A second region lies approximately between ParB residues 78 and 116 (called ‘‘B motif ’’ in Fig. 2b), but it corresponds to no known structural motif(s), and its function has not been identified. There is also little sequence similarity in the C-terminal half of these ParBs (Hanai et al., 1996). A combination of gel filtration and sedimentation analyses indicate that ParB is an asymmetric dimer in solution (Funnell, 1991). It also cross-links to a dimer-sized smear after treatment with dithiobis (succinimidyl pro˚ chemical cross-linker that reacts primarily with pionate) (DSP), a 12A lysines (Funnell, 1991). The dimerization domain is contained within the

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C-terminus of the protein. Certain amino acid substitutions in this region disrupt dimerization activity of the mutant proteins in cell lysates (Lobocka and Yarmolinsky, 1996). The dimerization domain has been narrowed to between residues 275 and 325 by deletion analyses and DSP cross-linking experiments (see Fig. 2b) (Surtees and Funnell, 1999, 2001). A second selfassociation domain was identified in the N-terminal half of the protein (Surtees and Funnell, 1999). Interestingly, this second self-association domain was not apparent unless a significant amount of the C-terminus was removed, suggesting that self-association by the N-terminal region is normally prevented in the context of full-length ParB. In addition, the N-terminus of ParB appears to be relatively flexible, measured by sensitivity to proteases. For example, limited exposure to trypsin rapidly degrades ParB from the N-terminus at a series of specific cleavage sites (Surtees and Funnell, 1999). A relatively stable proteolytic fragment that remains corresponds to the C-terminal half of ParB (from residues 142 to 333) and contains all the information necessary to bind to parS (see later). We suggest that the N-terminal self-association domain represents the oligomerization function of ParB that is necessary for higher-order complex formation but that it is normally masked until ParB undergoes a conformational change when it binds to DNA. The DNA-binding domains of ParB have been identified by biochemical analyses of protein fragments, mutagenesis, and domain-swapping experiments (Lobocka and Yarmolinsky, 1996; Radnedge et al., 1998; Surtees and Funnell, 2001). The region between residues 142 and 333 contains all the information to assemble the dimeric, high-aYnity ParB complex at parS in the presence of IHF (Surtees and Funnell, 2001). Two diVerent areas within this region are involved in sequence specificity, consistent with the fact that ParB interacts with both Box A and Box B sequences in parS. Single amino acid substitutions either near the center or within the C-terminus of ParB disrupt its DNA-binding activity (Lobocka and Yarmolinsky, 1996). The center contains a putative HTH motif (Dodd and Egan, 1990). Cocrystals of typical HTH proteins bound to their DNA sites have revealed that dimeric HTH proteins bind DNA sites containing inverted repeats (reviewed in Pabo and Sauer, 1992), and it was proposed that the ParB HTH directly contacts the Box A2–A3 inverted repeat in parS. Deletion of the HTH in ParB severely damages its DNA-binding activity, particularly to oligonucleotides containing the Box A2–A3 repeat (Surtees and Funnell, 2001). The region in ParB that interacts with the Box B motifs is near the C-terminus (Radnedge et al., 1998; Surtees and Funnell, 2001) and either overlaps or is adjacent to the dimerization domain. The picture of the P1 partition complex that emerges from these studies places the ends of parS (the Box B motifs) near or at the dimerization interface, with the DNA wrapped around an IHF–ParB protein core (Fig. 4).


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Figure 4 Model of the interactions of ParA and ParB during P1 partition. ParB binds to parS, resulting in a conformational change in the flexible N-terminus of ParB that exposes its oligomerization domain. More dimers of ParB load onto parS. A pairing reaction is mediated by ParB–ParB interactions and is stimulated by ParA–ATP. This complex may also be the substrate for an interaction with as yet undefined host signals for proper localization. ATP hydrolysis promotes separation of plasmid pairs, release of ParA–ADP, and perhaps release from the host.

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It is interesting to note that although ParB contains two DNA recognition domains, this property is not common among ParB-like proteins. A few plasmids contain parS sites with Box A and Box B motifs (see P7 and pMT1, later), but the partition sites in most other systems consist of one type of inverted repeat sequence, although it may be repeated many times. The extreme N-terminus of ParB is necessary for interaction with ParA, based on yeast two-hybrid and P1–P7 domain-swapping experiments (Radnedge et al., 1998; Surtees and Funnell, 1999). We speculate that an interaction of this region of ParB with ParA could transmit a signal to the nearby oligomerization interface and aVect the oligomerization state of ParB. Depending on other factors, such as ATP hydrolysis or interactions with host components, this signal could promote or disrupt pairing by ParB, for example. F. Gene Silencing ParB and the related SopB protein of the F plasmid have an unusual ability to silence genes up to several kitobases from their respective partition sites when the proteins are overexpressed (reviewed in Yarmolinsky, 2000). In vivo chromatin immunoprecipitation experiments in the P1 system suggested a mechanism for silencing (Rodionov et al., 1999). DNA binding by ParB was observed to extend several kitobases on both sides of parS, indicating that ParB spreads along the DNA in both directions from its nucleation site, parS. The extent of ParB spreading was reduced in the absence of IHF due to less eYcient complex formation. ParB was proposed to silence by coating the promoters to prevent access of RNA polymerase. The model for silencing is somewhat diVerent in the F system, however. Wang and colleagues have suggested that SopB sequesters DNA away from the transcription machinery, perhaps in the bacterial membrane (Kim and Wang, 1999; Lynch and Wang, 1995). The biological relevance of gene silencing by ParB is not clear because it does not appear to occur at normal intracellular concentrations of ParB (Hao and Yarmolinsky, 2002) and extensive spreading is not necessary for partition (Edgar et al., 2001). Nonetheless, these observations support a model in which ParB and IHF binding to parS stimulates the formation of a substantial nucleoprotein complex. G. Importance of the Cell One-Quarter and Three-Quarter Positions What constitutes the road signs and the stop signs that control the plasmid traYc in the cell? How the Par proteins recognize their locations inside the cell is unknown. No host factor has yet been identified as a tether. Several candidate proteins that are involved in other intracellular localization


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reactions have been ruled out. Inhibition of cell division using cephalexin, which inhibits FtsI and creates long filamentous E. coli cells (Pogliano et al., 1997), did not inhibit distribution of P1 plasmids as measured by FISH and immunofluorescence of ParB (Erdmann et al., 1999; Ho et al., 2002). Under these conditions, DNA replication and segregation continues and the bacterial chromosomes and plasmids are distributed throughout the filament. Identical results were obtained when cell division was inhibited using a temperature-sensitive allele of FtsZ (Erdmann et al., 1999), a tubulin-like protein that is one of the earliest known components of the cell division septum (Rothfield et al., 1999). Therefore, the actions of FtsI and FtsZ are not required for P1 partition. Interestingly however, distribution of a P1 plasmid tagged with GFP–LacI was prevented by cephalexin treatment (Gordon et al., 1997). This observation suggests that the GFP–LacI fusion protein interferes with the action of ParA and ParB under these conditions, but the nature of this interference is not understood. Another E. coli protein that has been examined is MukB, an SMC-like protein in E. coli that plays roles in chromosome condensation and segregation (Hiraga, 2000). P1 plasmids are stable in E. coli mukB mutants, and their stability is dependent on the P1 partition proteins. Therefore MukB is not essential for P1 partition (Funnell and Gagnier, 1995). mukB mutants produce anucleate (chromosomeless) cells at relatively high frequency compared to those produced by wild-type cells (Hiraga et al., 1989). Ezaki et al. (1991) designed an ingenious selection to isolate anucleate cells and showed that the F plasmid segregated into them. Using the same strategy, P1 plasmids were also observed to segregate into anucleate cells (Funnell and Gagnier, 1995). These studies established that the bacterial chromosome itself was not necessary for plasmid segregation at cell division. The bacterial membrane has long been proposed to be the site(s) of attachment of the replication and partition machinery (Jacob et al., 1963). The composition of the membrane is not uniform, and patches of specific phospholipids exist (Mileykovskaya and Dowhan, 2000); therefore, it is conceivable that the Par proteins are localized by association with preferred lipids. Cell fractionation experiments have found that a portion of intracellular SopA (the F plasmid ParA) copurifies with bacterial membrane fractions (Lin and Mallavia, 1998), supporting the hypothesis that ParA binds to the membrane. Bignell and Thomas (2001) have suggested a diVerent type of attachment role for the membrane: it provides a surface for facilitated diVusion of the plasmids to and from their intracellular homes. In this case, the membrane is the highway; ParA might be the vehicle, but the road signs would still be as yet unidentified proteins embedded in or associated with the cell surface. An appealing candidate for the localization signals is the replication apparatus. In E. coli and B. subtilis, components of the replication machinery appear to be grouped together in a stationary ‘‘factory,’’ through which the

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DNA travels during DNA synthesis (Lemon and Grossman, 1998, 2001). Coincidentally, the positions of replication proteins are similar to the positions of plasmids as measured by cell biology techniques. Intuitively, it could be an advantage to ‘‘hang around’’ the machinery that a plasmid may compete for and must use for its own replication, and it would be a convenient way to coordinate replication and partition. Treptow et al. (1994) showed that the act of DNA replication was not required for partition by following the segregation of plasmids with and without parS whose replication had been inhibited. However, the machinery and any potential attachment may have been maintained even if DNA synthesis did not occur. Another possibility is that the replication and partition systems respond to similar localization signals, but neither requires the other for function. The answers to these questions await the identification of host components that are required for plasmid localization in the cell.

H. ParA–ParB Interactions during Partition How mechanistically do ParA and ParB drive partition and control the plasmid traYc in the cell? We think that the available data best fit the model diagrammed in Fig. 4. The first step, assembly of a core ParB–IHF–parS partition complex, is relatively well understood. The core complex nucleates the formation of a larger ParB complex; DNA binding by ParB alters its protein conformation to expose an oligomerization interface, which is necessary to recruit more dimers of ParB. Further protein loading is stabilized by protein–protein as well as protein–DNA (nonsequence-specific) interactions. ParA, with the energy from ATP binding and hydrolysis, modulates the size of these complexes. ParA stimulates the pairing reaction, in which plasmids are joined by ParB–ParB interactions. In response to an as yet unknown signal, which may be spatial or temporal, ParA also dissociates these complexes so that plasmids can separate. We predict that the ParA–ParB interactions signal appropriate conformational changes in the partner to eVect these changes. Similarly, the interactions of ParA and ParB alter the conformation of one or both proteins so that the complex can interact with and subsequently dissociate from a host tether. The tether could be a replication component, the bacterial membrane, or another localized signal in the cell. None of these possibilities is mutually exclusive. Because the number of plasmid complexes is limited in the cell (measured as the number of plasmid or ParB foci), the partition system can count. For example, in a typical cell it recognizes that two is the magic number. The simplest way to imagine this type of traYc control is via a limited number of tethering sites in the cell. The partition system must also recognize that both tethers must be filled and join any extra copies of the plasmids in


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groups to these sites. We suggest that the arithmetic ability of the partition system is set by the ratio of ParA to ParB and by dynamic interactions between these proteins and the host. At too high a ratio of ParB to ParA, plasmids are joined in too few groups. Too much ParA dissociates the groups from each other and from their preferred host locations too rapidly or at inappropriate times in the cell cycle. In this model, therefore, the kinetics of a series of protein–protein and protein–DNA interactions adjusts the size and position of plasmid complexes inside the cell.

III. Other Plasmid Partition Systems Many diVerent plasmids encode partition systems that consist of a ParA (partition ATPase) and a ParB (centromere-binding protein). Many, but not all, components show homologies to the P1 proteins. We will not describe them in detail because they have been reviewed elsewhere (Bignell and Thomas, 2001; Funnell and Slavcev, 2003; Gerdes et al., 2000). Here we illustrate some of the important similarities and diVerences between these systems and that of P1. A. F Plasmid The partition system of the F plasmid in E. coli was one of the first to be identified (Ogura and Hiraga, 1983), and it bears many similarities in sequence and in mechanisms to the P1 partition system. Its partition proteins are called SopA and SopB (Sop ¼ stability of plasmid). SopA is an ATPase and SopB binds to the partition site, sopC, which is downstream of the sopA and sopB genes (Mori et al., 1986, 1989; Watanabe et al., 1992). SopA and SopB participate in autoregulation of their genes and in the partition reaction (Biek and Strings, 1995; Hirano et al., 1998; Mori et al., 1986). sopC is very diVerent in sequence from P1 parS and consists of 12 tandemly repeated copies of a 43-bp sequence (Helsberg and Eichenlaub, 1986; Mori et al., 1986). Each 43-bp sequence contains a short inverted repeat to which SopB binds, and SopB binds to a single inverted repeat as a dimer (Hayakawa et al., 1985; Mori et al., 1986, 1989). An HTH motif in the center of SopB is the best candidate for the DNA-binding domain of the protein, and this conclusion is consistent with DNA-binding experiments using SopB fragments (Hanai et al., 1996). It is unknown whether other parts of the protein are also involved in DNA binding. Unlike P1, however, no host factor is thought to be involved in partition complex formation. Binding of SopB to sopC in vivo alters the overall topology of the plasmid; it reduces the negative superhelicity (i.e., increases the linking number) of the DNA, indicating that the DNA is wrapped around the protein core

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(Biek and Shi, 1994; Biek and Strings, 1995; Lynch and Wang, 1994). Increased SopB expression results in increased linking numbers, even in the presence of a single copy of the 43-bp sopC repeat (Biek and Shi, 1994). This suggests that SopB bound to its site can recruit other SopB molecules and promotes wrapping of the adjacent nonspecific DNA, resulting in a large partition complex. Therefore, although the sequences of the partition sites are distinct, the evidence suggests that the architecture of the partition complexes that form on F and on P1 plasmids are similar: they resemble that of a protein core around which the DNA is specifically wrapped. Substitutions in the Walker A ATP-binding motif of SopA have also yielded super-repressor, partition-defective mutants (Libante et al., 2001). These mutations alter the ATP binding and hydrolysis properties of SopA, although not all are devoid of hydrolysis activities. They do suggest that there is a repressor form and a partition form of SopA that depend on its nucleotide binding site. In addition, their behavior supports the idea that SopB acts as a corepressor by converting SopA into its repressor form (Libante et al., 2001). By monitoring partition complex formation and integrity by its eVect on plasmid topology, Lemonnier et al. (2000) observed that SopA could disrupt partition complex formation in vivo. These results support the hypothesis that SopA is involved in modulating the SopB–sopC partition complexes and in dissociation of pairs (or groups) of plasmids during the partition reaction. SopA also is required for proper localization of SopB–sopC complexes measured in vivo by immunofluorescence, although there is some conflict in the reported localization patterns of SopB (Hirano et al., 1998; Kim and Wang, 1998). The properties of these complexes measured by immunofluorescence mirror those of P1 (Erdmann et al., 1999; Hirano et al., 1998). SopB formed fluorescent foci only in the presence of sopC. The location of these foci was dependent on the presence of SopA (Hirano et al., 1998) and coincided with the positions of the plasmids as measured by FISH (Niki and Hiraga, 1997). In contrast, however, a SopB–GFP fusion was found to be located primarily near the cell poles, irrespective of sopC (Kim and Wang, 1998). The Sop proteins were overexpressed in both studies in order to be detectable, so that it is always possible that localization patterns have been perturbed. We favor the conclusion that the patterns observed by immunofluorescence reflect those of the endogenous proteins, but more experimentation is necessary to resolve this debate. Another debate that reflects diVerences in patterns seen between F and P1 components concerns the ability of P1 ParB and F SopB to silence genes. In P1, spreading of ParB along the DNA has been observed and proposed to occlude RNA polymerase (Rodionov et al., 1999). In F however, such filamentation has not been observed, at least by electron microscopy (Lynch and Wang, 1995). Kim and Wang (1999) fused the N-terminal 82 residues of F SopB to the yeast GAL4 DNA-binding domain and showed that the


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fusion was suYcient to promote gene silencing in DNA adjacent to GAL4 DNA-binding sites. These N-terminal residues were suYcient to localize GFP to the cell poles (Kim and Wang, 1998), suggesting that these residues direct the protein–DNA complex to a specific cellular location. In the F model, sequestration at this location, e.g., in the bacterial membrane, hides the promoters from RNA polymerase. However, due to conflicting reports of SopB localization, the nature of silencing remains to be elucidated. Finally, recent FISH experiments have illustrated that F and P1 plasmids do not overlap in the cell (Ho et al., 2002), even though their general locations (at the cell quarter and midcell positions) are similar. In addition, localization of plasmid RK2 was distinct. These results imply that the tethering signals for each partition system are also distinct, which is consistent with the fact that they do not compete with each other. B. P7 and pMT1 The P7 plasmid in E. coli and the pMT1 virulence plasmid of Yersinia pestis encode partition systems that are closely related to that of P1 (Hayes and Austin, 1993; Hu et al., 1998; Ludtke et al., 1989). The organization of the par operon is virtually identical and the Par proteins are highly homologous. The sequence and arrangement of motifs, including an IHF-binding site, are almost identical in their parS sites. The pMT1 system will work in E. coli, implying that the host signals are conserved between E. coli and Y. pestis (Youngren et al., 2000). Despite the similarities, however, the par components are not interchangeable; each system is specific and only partitions plasmids containing its own site. Nevertheless, it seems very likely that the mechanisms for partition and gene regulation will be similar among these systems. Many virulence plasmids that inhabit pathogenic bacterial species (e.g., Y. pestis is the cause of bubonic plague) contain partition systems (Funnell and Slavcev, 2003), and the events that occur in E. coli with plasmids such as P1 and F are important models for their biology. For example, the Salmonella typhimurium virulence plasmid pSLT has a sequence that is almost identical to that of P1 parS and is positioned downstream of a parB-like gene (Cerin and Hackett, 1993). pMT1 is only one virulence plasmid in Y. pestis. A second, pCD1, encodes a partition system whose components more closely resemble those of F (Hu et al., 1998). C. RK2, pTAR, and Other Plasmids with Walker-Type Partition ATPases The IncC–KorB system of plasmid RK2 is a ParA–ParB–type partition system of this broad-host-range plasmid. There are several interesting similarities and diVerences between the RK2 and P1 systems. IncC (the

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ParA) and KorB (the ParB) are required for partition (Williams et al., 1998) but also regulate the expression of many genes in the RK2 genome. In this system, IncC is the corepressor that stimulates the repressor activity of KorB (Jagura-Burdzy et al., 1999). KorB binds to 12 operator sites (OB) that are distributed throughout the plasmid genome; at least one, but not all, of these sites can act as a partition site (Rosche et al., 2000; Williams et al., 1998). How many OB sites normally act as partition sites in RK2 is not known. Each site contains a 6-bp inverted repeat that is recognized by KorB, and flanking sequences influence the relative aYnity of KorB for each site (Kostelidou and Thomas, 2000). In RK2, IncC is made in a long (IncC1) and short (IncC2) version due to two diVerent translational starts in the same reading frame. Only IncC2 is necessary for partition, whereas IncC1 is required for its corepressor activities (Jagura-Burdzy et al., 1999; Williams et al., 1998). IncC2 lacks the extended N-terminus that corresponds with ParA’s putative DNA-binding domain. There is no evidence that IncC (IncC1 or IncC2) binds to DNA (Williams et al., 1998), and the role of the longer N-terminus in the gene regulation activities of IncC1 is unknown. KorB contains both dimerization and oligomerization determinants that are situated at the C-terminus and central regions of the protein, respectively (Lukaszewicz et al., 2002). Neither region shows significant homology to sequences in P1 ParB, but the dimerization domain of ParB is also at its C-terminus (Lobocka and Yarmolinsky, 1996; Surtees and Funnell, 1999). The crystal structure of the dimerization region of KorB (the C-terminal 62 residues) has been determined. It adopts a five-stranded sheet fold that strongly resembles the structure of Src homology 3 (SH3) domains (Delbruck et al., 2002). In contrast, this region of P1 ParB is thought to be extensively -helix, based on secondary structure predictions and circular dichroism experiments (Surtees and Funnell, 2001, unpublished results). RK2 KorB contains a predicted HTH motif in its center, in roughly the same place as the HTH in ParB, and the RK2 HTH presumably represents the DNA-binding domain that recognizes the OB inverted repeats (Bignell and Thomas, 2001). Finally, a region of KorB that interacts with IncC is also found in the central region of the protein, which is in contrast to the observation that the N-terminus of ParB interacts with ParA (Lukaszewicz et al., 2002; Radnedge et al., 1996; Surtees and Funnell, 1999). Although there are organizational and sequence diVerences between KorB and ParB, cell biology experiments illustrate that their overall activities in partition are likely to be very similar (Bignell et al., 1999). The pattern of localization of KorB, measured by immunofluorescence microscopy, is of protein foci that usually localize to the approximate one-quarter and threequarter positions of cell length. In larger cells, especially at higher growth rates, there were often four foci per cell, distributed evenly along the cell.


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In the absence of IncC, fewer KorB foci were present and often in only one half of the cell, consistent with the instability of the test plasmid in the absence of IncC. KorB foci also were often clumped together in the absence of IncC. The pattern of KorB localization is similar to that of the RK2 plasmid itself, as measured by FISH (Pogliano et al., 2001). It is interesting to note that there are about five copies of RK2 per cell chromosome, so multiple copies appear to be attached to a limited number of cellular sites (Bignell et al., 1999; Pogliano et al., 2001). These observations support the idea that KorB groups or pairs plasmids together; they are then separated by the action of IncC. Many other plasmids contain putative partition systems with Walker-type partition ATPases, but mechanistic details are lacking for most of them (reviewed in Bignell and Thomas, 2001; Funnell and Slavcev, 2003). One class contains very small ParBs, which bear no similarities to the larger centromere-binding proteins found in P1, F, and RK2. pTAR from Agrobacterium tumefaciens and TP228 from Salmonella newport are two whose partition functions have been tested (Gallie and Kado, 1987; Hayes, 2000; Kalnin et al., 2000). The partition site is thought to overlap the promoter of the operon for both genes, and the ParAs lack the N-terminal DNAbinding region that is present in P1 ParA. The ParBs are very small: typically less than 95 amino acids in length. As in RK2, the ParB is the transcriptional repressor and centromere-binding protein, whose activity is stimulated by ParA (Kalnin et al., 2000). One member of the pTAR group is a partition system carried by pB171, a virulence plasmid in E. coli. Its ParA has been shown to oscillate in the cell (as a ParA–GFP fusion), as though it associates with one nucleoid, dissociates, moves to the nucleoid at the other end, and so on (Ebersbach and Gerdes, 2001). E. coli MinD, a ParA-like ATPase that is involved in localization of the cell division septum, also oscillates, but from pole to pole (Raskin and deBoer, 1999). Indeed, members of the pTAR group of ParAs more closely resemble the bacterial MinD proteins than do other plasmid ATPases (Hayes, 2000). It is not known whether oscillation is a common feature among all ParAs.

D. R1 and the Actin-Like ATPases The R1 plasmid is a member of a separate class of partition systems, which also includes NR1 (R100). The organization of these partition systems is superficially like those of the par/sop partition systems, but there is very little sequence homology (Gerdes et al., 2000; Williams and Thomas, 1992). The partition site overlaps the promoter for the operon, and it is the ParBlike protein that acts as the repressor of the operon (Breuner et al., 1996;

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Jensen et al., 1994; Tabuchi et al., 1992). The partition ATPases are distinguished by ATP-binding motifs that resemble those of F-actin and the bacterial actin-like protein, MreB (Gerdes et al., 2000; Moller-Jensen et al., 2002; van den Ent et al., 2001). In R1, ParM is the ATPase and ParR is the centromere-binding protein (Jensen and Gerdes, 1997). R1 is the only system for which plasmid pairing has been demonstrated in vitro (Jensen et al., 1998). Pairing is mediated directly by the binding of ParR to the partition site, parC, and is stimulated by ParM. Recent evidence indicates that ParM polymerizes in an actin-like fashion to form a dynamic cytoskeletal filament in E. coli (Moller-Jensen et al., 2002). Its crystal structure has recently been solved and shows that ParM adopts a fold that looks like those of actin and MreB (van den Ent et al., 2002). In vitro, cycles of ATP binding and hydrolysis by ParM result in polymerization and depolymerization. In vivo, production of ParM filaments requires ParR and parC, implying that the partition complex serves to nucleate the first polymerization events. It has been proposed that the growth of the ParM filament serves to transport plasmids, via their partition complexes, to opposite ends of the cell (Moller-Jensen et al., 2002). It is unknown whether filamentation occurs with the Walker-type ATPases such as P1 ParA, although analysis of ParA and F SopA by immunofluorescence microscopy suggests that it does not (Erdmann et al., 1999; Hirano et al., 1998).

IV. Bacterial ParA and ParB Proteins Due to the rapidly expanding databases of genome sequences, homologues of plasmid ParA and ParB proteins have been detected in many diVerent bacterial species, although interestingly, not in E. coli. The genes have been identified in archaea and eubacteria and in both circular and linear chromosomes. The genetic organization of the parAB loci is similar to that of the plasmid partition systems. The bacterial par genes have been shown to play roles in chromosome segregation in B. subtilis, Caulobacter crescentus, Streptomyces coelicolor, and Pseudomonas putida (Godfrin-Estevenon et al., 2002; Ireton et al., 1994; Kim et al., 2000; Lewis et al., 2002; Mohl and Gober, 1997). The details vary from species to species, but several general themes have emerged. First, the par genes are located close to the bacterial origin of replication, oriC. Second, the sequence of the partition sites, and thus the binding specificities of the ParBs, may be conserved. Third, the partition proteins contribute to partition but are not the primary segregation machinery. Finally, the influence of the par genes often varies with the developmental or growth stage of the bacterial cells. In most species that have been tested, the par genes are not essential for cell survival. The only exception is C. crescentus (Mohl and Gober, 1997),


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and this property likely reflects the cell cycle checkpoint controls in this organism. Nevertheless, the phenotypes from mutation or overexpression of one or both par genes indicate that they contribute to faithful partition, at least under certain conditions (Godfrin-Estevenon et al., 2002; Ireton et al., 1994; Kim et al., 2000; Lewis et al., 2002; Mohl and Gober, 1997). Other maintenance systems have been shown and/or proposed to contribute to partition of bacterial chromosomes, such as topoisomerases, chromosome condensation, and DNA replication through a localized replication factory, and have been reviewed in detail elsewhere (Gordon and Wright, 2000; Hiraga, 2000; Lemon and Grossman, 2001; Moller-Jensen et al., 2000; Sawitzke and Austin, 2001). Here, we concentrate on the action of ParA- and ParB-like proteins in these organisms.

A. Bacillus subtilis The ParA and ParB proteins in B. subtilis are called Soj and Spo0J, respectively. They were the first of the chromosomal Par homologues to be shown to aVect segregation of a bacterial chromosome (Ireton et al., 1994), although the spo0J gene was identified via its requirement for sporulation (Sandman et al., 1987). A variety of evidence suggests that Spo0J, perhaps with the help of Soj, helps to organize the origin region of the chromosome, which may be a mechanism to regulate or coordinate DNA replication with partition (Glaser et al., 1997; Lee et al., 2003; Lin and Grossman, 1998; Lin et al., 1997; Marston and Errington, 1999). Mutations in spo0J increase the frequency of anucleate cells during vegetative growth (Ireton et al., 1994). Spo0J interacts with sequences near the origin of replication (Lewis and Errington, 1997; Lin et al., 1997); it specifically recognizes a 16-base pair sequence called parS (Lin and Grossman, 1998). Ten copies of parS are within the origin–proximal 20% of the chromosome and eight are bound by Spo0J in vivo, as determined by chromatin immunoprecipitation experiments. The cellular localization of Spo0J was determined by immunofluorescence microscopy in fixed cells and by following Spo0J–GFP fusions in living cells (Glaser et al., 1997; Lewis and Errington, 1997; Lin et al., 1997). Spo0J was positioned near the cell poles and was associated with the nucleoid. Following replication, two Spo0J foci separated, with one focus migrating to the opposite pole corresponding to the movement of the oriC region of the chromosome. The cell biology experiments indicate that Spo0J tracks with the origin region of the chromosome, which led to the speculation that it was required to orient the chromosome in the cell during both vegetative growth and sporulation. However, recent experiments suggest that this is not the case. The origin region was observed to be properly oriented in the absence of

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Spo0J (Wu and Errington, 2002), and Spo0J was not suYcient to promote localization of regions of the chromosome to which parS sites had been added (Lee et al., 2003). In addition, the synchrony of DNA replication and the chromosomal content were measured and were perturbed in the absence of Spo0J, suggesting that Spo0J is a negative regulator of DNA replication in B. subtilis. It was suggested that altered chromosome content, not localization defects, was responsible for the partition defects in spo0J mutants (Lee et al., 2003). The results, however, do not rule out the possibility that Spo0J plays an accessory role in chromosome organization. In this system, the ParA/Soj is not required for partition of the chromosome because soj mutants show no increased frequency of anucleate cells (Ireton et al., 1994). However, in vivo Soj does aVect the appearance of Spo0J foci. In a soj mutant, the large, discrete Spo0J foci are replaced by smaller, scattered foci. This suggests that Soj helps to coalesce or organize foci of Spo0J and thus of the origin–proximal regions of the chromosome (Marston and Errington, 1999). Genetic evidence indicates that Soj and Spo0J act together as a sporulation checkpoint that may monitor the state of chromosome partition. Mutations in spo0J greatly reduce sporulation, but this phenotype can be suppressed by mutation of soj, which indicates that Soj inhibits sporulation unless it receives a signal from Spo0J (Ireton et al., 1994). Soj is a negative regulator of transcription of several sporulation genes in B. subtilis, and this regulation is relieved in the presence of Spo0J (Cervin et al., 1998; Ireton et al., 1994; Quisel and Grossman, 2000; Quisel et al., 1999). Soj is another ParA that exhibits a dynamic oscillatory behavior inside cells (Marston and Errington, 1999; Quisel et al., 1999). Soj–GFP fusion proteins oscillate between large nucleoid-associated patches and a polar localization on a timescale of minutes. In the absence of Spo0J, Soj remains associated with the nucleoid and does not dissociate. Furthermore, point mutations in the putative ATPase domain of Soj disrupted its localization and function, indicating that ATP binding and hydrolysis by Soj is required for its activity. These point mutations also rendered Soj insensitive to regulation by Spo0J (Quisel et al., 1999). Genetic experiments suggest that the target of Soj is MinD because these localization patterns do not occur in minD mutants (Autret and Errington, 2003). B. subtilis MinD is concentrated at the cell poles and could provide the localization signals for Soj. Although Soj is not necessary for chromosome partition, it is required for plasmid partition that is promoted by the Spo0J–Soj components. Lin et al. (1997) showed that Spo0J and Soj could stabilize a plasmid in B. subtilis if the plasmid contained a copy of parS. In addition, the Spo0J–Soj duo will stabilize such plasmids in E. coli, albeit not as eYciently as other partition systems from E. coli (such as F Sop) (Yamaichi and Niki, 2000). These


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observations suggest that modulation of Spo0J–DNA complexes by Soj is more important for a plasmid than for the entire bacterial chromosome. For example, perhaps more forces are tugging the chromosome during separation, whereas the plasmid must rely on the separation activities of Soj.

B. Caulobacter crescentus The cell cycle in C. crescentus is tightly controlled (reviewed in Gober and Marques, 1995). C. crescentus is a gram-negative bacterial species in which each mother cell diVerentiates into two morphologically distinct daughter cells at cell division. One daughter cell contains a stalk (a ‘‘stalked cell’’) and the other a flagellum (a ‘‘swarmer cell’’). Only the stalked cell undergoes DNA replication and then divides again into stalked and swarmer cells. Swarmer cells eventually lose their flagella, grow stalks, and become stalked cells. These properties make it relatively easy to isolate synchronized populations in order to examine cell-cycle–related events. In C. crescentus, the parA and parB genes are essential for cell viability (Mohl and Gober, 1997). Overexpression and depletion studies indicate that viability is controlled at the level of cell division and suggest that antagonistic actions of ParA and ParB form a cell cycle checkpoint that inhibits cell division when chromosome partition is not complete (Mohl et al., 2001). Depletion of ParB or overexpression of ParA leads to long filamentous cells that have formed no septal rings, but balanced levels of both proteins show no such inhibition. In this model, ParA is the cell division inhibitor and ParB antagonizes the inhibition. The situation is likely more complicated, however, because simplistically it predicts that parB but not parA would be essential for viability. The reason parA is essential is still unknown but may be related to ParA action in chromosome organization or partition. ParB binds to an approximately 400-base-pair DNA fragment immediately downstream of parB (Mohl and Gober, 1997). Although the specific sequence of its binding site has not been reported, there are six copies of an inverted repeat similar to B. subtilis parS in the oriC region of the C. crescentus chromosome (Mohl et al., 2001). Immunofluorescence microscopy revealed cell-cycle–dependent localization of both ParA and ParB that suggests they localize with the origin of replication (Mohl and Gober, 1997). In these experiments, both proteins were dispersed throughout the cell until about 60% of the chromosome had been replicated. At this point, ParB was predominantly localized to the polar region. Upon completion of replication, both ParA and ParB exhibited bipolar localization. When DNA replication was inhibited, ParB tended to localize at midcell, suggesting that the polar localization is dependent on DNA replication and/or the subsequent movement

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of the chromosomes. Overexpression of either ParA, ParB, or both resulted in mislocalization of ParB and chromosome partition defects; at least 10% of the cells were anucleate. This suggests an active role for the Par proteins in promoting the segregation of sister chromosomes to opposite cell poles. However, because cells containing dominant negative mutants of parB did orient the origins toward the cell poles, the role of the Par proteins is not essential for, although may aid, proper chromosome orientation (Figge et al., 2003). The idea that a nucleotide switch controls the activities of ParA has received experimental support in this organism as it has in P1. ParA–ADP is the DNA-binding form and binds to single-stranded DNA (Easter and Gober, 2002). ParA–ATP interacts with ParB bound to parS, and the consequence of this interaction is that ParA removes or prevents ParB binding to parS. ParB regulates the ATP versus ADP forms of ParA by stimulating the exchange of bound nucleotide and by stimulating ParA’s ATPase activity. The in vitro behavior is supported by the observation that the ratio of ParA to ParB influences the relative amounts of ATP- and ADP-bound forms of ParA isolated directly from cells by immunoprecipitation (Easter and Gober, 2002; Figge et al., 2003). The structure–function relationships of ParB resemble those of other ParBs (Figge et al., 2003). ParB dimerizes, and dimerization requires the C-terminus of the protein. Both the HTH motif in the center of ParB and the dimerization domain are required for its DNA-binding activity, consistent with a typical HTH protein binding to an inverted repeat DNA sequence. Finally, the N-terminus of the protein is required for interactions with ParA.

C. Streptomyces coelicolor Deletion analysis of the parAB locus of S. coelicolor showed that this region is involved in proper segregation of its genome, a linear chromosome, during sporulation (Kim et al., 2000). S. coelicolor grows vegetatively as a complex mycelium of branched hyphae, which contain multiple copies of the genome. During sporulation, it develops aerial branches, distributes copies of the genome along the length of these hyphae, and then forms septa between each genome to produce chains of spores. It is this genome distribution that is perturbed in the absence of ParA and ParB. In parB or parAparB mutants, more than 13% of spore compartments did not inherit the full DNA complement (Kim et al., 2000). The S. coelicolor chromosome contains 24 copies of a sequence similar to that of B. subtilis parS, 21 of which are close to oriC. ParB has been purified and binds specifically to DNA fragments containing parS (Jakimowicz et al., 2002). Therefore, although the


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Par proteins are not essential for cell viability or for the diVerentiation regimen that produces spores, they do participate directly or indirectly in chromosome segregation during the sporulation process.

D. Pseudomonas putida The involvement of ParA and ParB in chromosome segregation in P. putida is also conditional, but in this organism it depends on the physiological state of the cell (Godfrin-Estevenon et al., 2002; Lewis et al., 2002). Mutations in either or both parA and parB showed only minor defects in partition during cell growth in rich media. However, when growth occurred more slowly in minimal media, these mutations resulted in a high frequency of anucleate cells in the population, especially during the transition from exponential to stationary phase growth. Up to 10% of the cells were anucleate under these conditions. The phenotypes were similar for both parA and parB mutations, indicating that both proteins are required for proper chromosome partition. What physiological diVerences could be responsible for their requirement? DNA replication is shutting down as cells enter the stationary phase. Other changes, such as the structure or topology of the nucleoid, may contribute. At slow growth rates, bacterial cells contain fewer chromosomal copies per cell. One or all of these factors, or other factors, must make the P. putida chromosome more susceptible to partition errors in the absence of the Par proteins.

V. Concluding Remarks There are many similarities in the actions and physical properties among the ParAs and ParBs in both plasmid and chromosomal systems, although the dependence on these proteins varies. In many cases, we do not know enough about specific details to draw precise comparisons. We expect that as more systems are examined in biochemical detail, the commonalities will increase. Many questions remain. How do ParA and ParB signal changes in each other? What are the signals that dictate plasmid localization? Does ParA oscillation contribute to plasmid localization, and if so, how? Why do diVerent chromosomes show diVerent requirements for the action of ParA in their segregation? Why do the roles of ParA and ParB vary with the physiological state of the cell? Some of these questions may be answered when we understand more about other processes that involve specific intracellular positioning, such as placement of the cell division septum and of the replication machinery. It continues to be an exciting challenge to decipher the actions of ParA and ParB in chromosome dynamics and to describe how each

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partition system has adapted these roles to the specific lifestyles of their plasmids or chromosomes.

Acknowledgments Work in our laboratory is supported by the Canadian Institutes of Health Research.

References Abeles, A. L., Friedman, S. A., and Austin, S. J. (1985). Partition of unit-copy miniplasmids to daughter cells: III. The DNA sequence and functional organization of the P1 partition region. J. Mol. Biol. 185, 261–272. Autret, S., and Errington, J. (2003). A role for division-site-selection protein MinD in regulation of internucleoid jumping of Soj (ParA) protein in Bacillus subtilis. Mol. Microbiol. 47, 159–169. Biek, D. P., and Shi, J. P. (1994). A single 43-bp sopC repeat of plasmid mini-F is sufficient to allow assembly of a functional nucleoprotein partition complex. Proc. Natl. Acad. Sci. USA 91, 8027–8031. Biek, D. P., and Strings, J. (1995). Partition functions of mini-F affect plasmid DNA topology in Escherichia coli. J. Mol. Biol. 246, 388–400. Bignell, C., and Thomas, C. M. (2001). The bacterial ParA-ParB partitioning proteins. J. Biotechnol. 91, 1–34. Bignell, C. R., Haines, A. S., Khare, D., and Thomas, C. M. (1999). Effect of growth rate and incC mutation on symmetric plasmid distribution by the IncP-1 partitioning apparatus. Mol. Microbiol. 34, 205–216. Bouet, J. Y., and Funnell, B. E. (1999). P1 ParA interacts with the P1 partition complex at parS and an ATP-ADP switch controls ParA activities. EMBO J. 18, 1415–1424. Bouet, J. Y., Surtees, J. A., and Funnell, B. E. (2000). Stoichiometry of P1 plasmid partition complexes. J. Biol. Chem. 275, 8213–8219. Breuner, A., Jensen, R. B., Dam, M., Pedersen, S., and Gerdes, K. (1996). The centromere-like parC locus of plasmid R1. Mol. Microbiol. 20, 581–592. Cerin, H., and Hackett, J. (1993). The parVP region of the Salmonella-typhimurium virulence plasmid pSLT contains 4 loci required for incompatibility and partition. Plasmid 30, 30–38. Cervin, M. A., Spiegelman, G. B., Raether, B., Ohlsen, K., Perego, M., and Hoch, J. A. (1998). A negative regulator linking chromosome segregation to developmental transcription in Bacillus subtilis. Mol. Microbiol. 29, 85–95. Davey, M. J., and Funnell, B. E. (1994). The P1 plasmid partition protein ParA: A role for ATP in site-specific DNA binding. J. Biol. Chem. 269, 29908–29913. Davey, M. J., and Funnell, B. E. (1997). Modulation of the P1 plasmid partition protein ParA by ATP, ADP and P1 ParB. J. Biol. Chem. 272, 15286–15292. Davis, M. A., and Austin, S. J. (1988). Recognition of the P1 plasmid centromere analog involves binding of the ParB protein and is modified by a specific host factor. EMBO J. 7, 1881–1888. Davis, M. A., Martin, K. A., and Austin, S. J. (1990). Specificity switching of the P1 plasmid centromere-like site. EMBO J. 9, 991–998. Davis, M. A., Martin, K. A., and Austin, S. J. (1992). Biochemical activities of the ParA partition protein of the P1 plasmid. Mol. Microbiol. 6, 1141–1147.


175

Davis, M. A., Radnedge, L., Martin, K. A., Hayes, F., Youngren, B., and Austin, S. J. (1996). The P1 ParA protein and its ATPase activity play a direct role in the segregation of plasmid copies to daughter cells. Mol. Microbiol. 21, 1029–1036. Delbruck, H., Ziegelin, G., Lanka, E., and Heinemann, U. (2002). An Src Homology 3-like domain is responsible for dimerization of the repressor protein KorB encoded by the promiscuous IncP plasmid RP4. J. Biol. Chem. 277, 4191–4198. Dodd, I. B., and Egan, J. B. (1990). Improved detection of helix-turn-helix DNA-binding motifs in protein sequences. Nucl. Acids Res. 18, 5019–5026. Easter, J., and Gober, J. W. (2002). ParB-stimulated nucleotide exchange regulates a switch in functionally distinct ParA activities. Mol. Cell 10, 427–434. Ebersbach, G., and Gerdes, K. (2001). The double par locus of virulence factor pB171: DNA segregation is correlated with oscillation of ParA. Proc. Natl. Acad. Sci. USA 98, 15078–15083. Edgar, R., Chattoraj, D. K., and Yarmolinsky, M. (2001). Pairing of P1 plasmid partition sites by ParB. Mol. Microbiol. 42, 1363–1370. Erdmann, N., Petroff, T., and Funnell, B. E. (1999). Intracellular localization of P1 ParB protein depends on ParA and parS. Proc. Natl. Acad. Sci. USA 96, 14905–14910. Ezaki, B., Ogura, T., Niki, H., and Hiraga, S. (1991). Partitioning of a mini-F plasmid into anucleate cells of the mukB null mutant. J. Bacteriol. 173, 6643–6646. Figge, R. M., Easter, J., and Gober, J. W. (2003). Productive interaction between the chromosome partitioning proteins, ParA and ParB, is required for the progression of the cell cycle in Caulobacter crescentus. Mol. Microbiol. 47, 1225–1237. Friedman, S. A., and Austin, S. J. (1988). The P1 plasmid-partition system synthesizes two essential proteins from an autoregulated operon. Plasmid 19, 103–112. Fung, E., Bouet, J. Y., and Funnell, B. E. (2001). Probing the ATP-binding site of P1 ParA: Partition and repression have different requirements for ATP binding and hydrolysis. EMBO J. 20, 4901–4911. Funnell, B. E. (1988a). Mini-P1 plasmid partitioning: Excess ParB protein destabilizes plasmids containing the centromere parS. J. Bacteriol. 170, 954–960. Funnell, B. E. (1988b). Participation of Escherichia coli integration host factor in the P1 plasmid partition system. Proc. Natl. Acad. Sci. USA 85, 6657–6661. Funnell, B. E. (1991). The P1 partition complex at parS: The influence of Escherichia coli integration host factor and of substrate topology. J. Biol. Chem. 266, 14328–14337. Funnell, B. E., and Gagnier, L. (1993). The P1 plasmid partition complex at parS: II. Analysis of ParB protein binding activity and specificity. J. Biol. Chem. 268, 3616–3624. Funnell, B. E., and Gagnier, L. (1994). P1 plasmid partition: Binding of P1 ParB protein and Escherichia coli integration host factor to altered parS sites. Biochimie 76, 924–932. Funnell, B. E., and Gagnier, L. (1995). Partition of P1 plasmids in Escherichia coli mukB chromosomal partition mutants. J. Bacteriol. 177, 2381–2386. Funnell, B. E., and Slavcev, R. (2003). Partition systems of bacterial plasmids. In ‘‘The Biology of Plasmids’’ (B. E. Funnell and G. J. Phillips, eds.). ASM Press, Washington, DC, in press. Gallie, D. R., and Kado, C. I. (1987). Agrobacterium tumefaciens pTAR parA promoter region involved in autoregulation, incompatibility, and plasmid partitioning. J. Mol. Biol. 193, 465–478. Georgiadis, M. M., Komiya, H., Chakrabarti, P., Woo, D., Kornuc, J. J., and Rees, D. C. (1992). Crystallographic structure of the nitrogenase iron protein for Azotobacter vinelandii. Science 257, 1653–1659. Gerdes, K., Moller-Jensen, J., and Jensen, R. B. (2000). Plasmid and chromosome partitioning: Surprises from phylogeny. Mol. Microbiol. 37, 455–466.

176

Surtees and Funnell

Glaser, P., Sharpe, M. E., Raether, B., Perego, M., Ohlsen, K., and Errington, J. (1997). Dynamic, mitotic-like behavior of a bacterial protein required for accurate chromosome partitioning. Genes Dev. 11, 1160–1168. Gober, J. W., and Marques, M. V. (1995). Regulation of cellular differentiation in Caulobacter crescentus. Microbiol. Rev. 59, 31–47. Godfrin-Estevenon, A. M., Pasta, F., and Lane, D. (2002). The parAB gene products of Pseudomonas putida exhibit partition activity in both P. putida and Escherichia coli. Mol. Microbiol. 43, 39–49. Gordon, G. S., Sitnikov, D., Webb, C. D., Teleman, A., Straight, A., Losick, R., Murray, A. W., and Wright, A. (1997). Chromosome and low copy plasmid segregation in E. coli: Visual evidence for distinct mechanisms. Cell 90, 1113–1121. Gordon, G. S., and Wright, A. (2000). DNA segregation in bacteria. Annu. Rev. Microbiol. 54, 681–708. Hanai, R., Liu, R. P., Benedetti, P., Caron, P. R., Lynch, A. S., and Wang, J. C. (1996). Molecular dissection of a protein SopB essential for Escherichia coli F plasmid partition. J. Biol. Chem. 271, 17469–17475. Hao, J. J., and Yarmolinsky, M. (2002). Effects of the P1 plasmid centromere on expression of P1 partition genes. J. Bacteriol. 184, 4857–4867. Hayakawa, Y., Murotsu, T., and Matsubara, K. (1985). Mini-F protein that binds to a unique region for partition of mini-F plasmid DNA. J. Bacteriol. 163, 349–354. Hayashi, I., Oyama, T., and Morikawa, K. (2001). Structural and functional studies of MinD ATPase: Implications for the molecular recognition of the bacterial cell division apparatus. EMBO J. 20, 1819–1828. Hayes, F. (2000). The partition system of multidrug resitance plasmid TP228 includes a novel protein that epitomizes an evolutionarily distinct subgroup of the ParA superfamily. Mol. Microbiol. 37, 528–541. Hayes, F., and Austin, S. (1994). Topological scanning of the P1 plasmid partition site. J. Mol. Biol. 243, 190–198. Hayes, F., and Austin, S. J. (1993). Specificity determinants of the P1 and P7 plasmid centromere analogs. Proc. Natl. Acad. Sci. USA 90, 9228–9232. Hayes, F., Radnedge, L., Davis, M. A., and Austin, S. J. (1994). The homologous operons for P1 and P7 plasmid partition are autoregulated from dissimilar operator sites. Mol. Microbiol. 11, 249–260. Helsberg, M., and Eichenlaub, R. (1986). Twelve 43-base-pair repeats map in a cis-acting region essential for partition of plasmid mini-F. J. Bacteriol. 165, 1043–1045. Hiraga, S. (2000). Dynamic localization of bacterial and plasmid chromosomes. Annu. Rev. Genet. 34, 21–59. Hiraga, S., Niki, H., Ogura, T., Ichinose, C., Mori, H., Ezaki, B., and Jaffe, A. (1989). Chromosome partitioning in Escherichia coli: Novel mutants producing anucleate cells. J. Bacteriol. 171, 1496–1505. Hirano, M., Mori, H., Onogi, T., Yamazoe, M., Niki, H., Ogura, T., and Hiraga, S. (1998). Autoregulation of the partition genes of the mini-F plasmid and the intracellular localization of their products in Escherichia coli. Mol. Gen. Genet. 257, 392–403. Ho, T. Q., Zhong, Z., Aung, S., and Pogliano, J. (2002). Compatible bacterial plasmids are targeted to independent cellular locations in Escherichia coli. EMBO J. 21, 1864–1872. Hu, P., Elliott, J., McCready, P., Skowronski, E., Garnes, J., Kobayashi, A., Brubaker, R. R., and Garcia, E. (1998). Structural organization of the virulence-associated plasmids of Yersinia pestis. J. Bacteriol. 180, 5192–5202. Ireton, K., Gunther, N. W., and Grossman, A. D. (1994). spo0J is required for normal chromosome segregation as well as the initiation of sporulation in Bacillus subtilis. J. Bacteriol. 176, 5320–5329.


177

Jacob, F., Brenner, S., and Cuzin, F. (1963). On the regulation of DNA replication in bacteria. Cold Spring Harbor Symp. Quant. Biol. 228, 329–348. Jagura-Burdzy, G., Kostelidou, K., Pole, J., Khare, D., Jones, A., Williams, D. R., and Thomas, C. M. (1999). IncC of broad-host-range plasmid RK2 modulates KorB transcriptional repressor activity in vivo and operator binding in vitro. J. Bacteriol. 181, 2807–2815. Jakimowicz, D., Chater, K., and Zakrzewska-Czerwinska, J. (2002). The ParB protein of Streptomyces coelicolor A3(2) recognizes a cluster of parS sequences within the originproximal region of the linear chromosome. Mol. Microbiol. 45, 1365–1377. Jang, S. B., Seefeldt, L. C., and Peters, J. W. (2000). Insights into nucleotide signal transduction in nitrogenase: Protein with MgADP bound. Biochemistry 39, 14745–14752. Jensen, R. B., Dam, M., and Gerdes, K. (1994). Partitioning of plasmid R1. The parA operon is autoregulated by ParR and its transcription is highly stimulated by a downstream activating element. J. Mol. Biol. 236, 1299–1309. Jensen, R. B., and Gerdes, K. (1997). Partitioning of plasmid R1. The ParM protein exhibits ATPase activity and interacts with the centromere-like ParR-parC complex. J. Mol. Biol. 269, 505–513. Jensen, R. B., and Gerdes, K. (1999). Mechanism of DNA segregation in prokaryotes: ParM partitioning protein of plasmid R1 co-localizes with its replicon during the cell cycle. EMBO J. 18, 4076–4084. Jensen, R. B., Lurz, R., and Gerdes, K. (1998). Mechanism of DNA segregation in prokaryotes: Replicon pairing by parC of plasmid R1. Proc. Natl. Acad. Sci. USA 95, 8550–8555. Kalnin, K., Stegalkina, S., and Yarmolinsky, M. (2000). pTAR-encoded proteins in plasmid partitioning. J. Bacteriol. 182, 1889–1894. Kim, H. J., Calcutt, M. J., Schmidt, F. J., and Chater, K. F. (2000). Partitioning of the linear chromosome during sporulation of Streptomyces coelicolor A3(2) involves an oriC-linked parAB locus. J. Bacteriol. 182, 1313–1320. Kim, S. K., and Wang, J. C. (1998). Localization of F plasmid SopB protein to positions near the poles of Escherichia coli cells. Proc. Natl. Acad. Sci. USA 95, 1523–1527. Kim, S. K., and Wang, J. C. (1999). Gene silencing via protein-mediated subcellular localization of DNA. Proc. Natl. Acad. Sci. USA 96, 8557–8561. Koonin, E. V. (1993). A superfamily of ATPases with diverse functions containing either classical or deviant ATP-binding motif. J. Mol. Biol. 229, 1165–1174. Kostelidou, K., and Thomas, C. M. (2000). The hierarchy of KorB binding at its 12 binding sites on the broad-host-range plasmid RK2 and modulation of this binding by IncC1 protein. J. Mol. Biol. 295, 411–422. Lee, P. S., Lin, D. C. H., Moriya, S., and Grossman, A. D. (2003). Effects of the chromosome partitioning protein Spo0J (ParB) on oriC positioning and replication initiation in Bacillus subtilis. J. Bacteriol. 185, 1326–1337. Lemon, K. P., and Grossman, A. D. (1998). Localization of bacterial DNA polymerase: Evidence for a factory model of replication. Science 282, 1516–1519. Lemon, K. P., and Grossman, A. D. (2001). The extrusion-capture model for chromosome partitioning in bacteria. Genes Dev. 15, 2031–2041. Lemonnier, M., Bouet, J. Y., Libante, V., and Lane, D. (2000). Disruption of the F plasmid partition complex in vivo by partition protein SopA. Mol. Microbiol. 38, 493–503. Lewis, P. J., and Errington, J. (1997). Direct evidence for active segregation of oriC regions of the Bacillus subtilis chromosome and co-localization with the Spo0J partitioning protein. Mol. Microbiol. 25, 945–954. Lewis, R. A., Bignell, C. R., Zeng, W., Jones, A. C., and Thomas, C. M. (2002). Chromosome loss from par mutants of Pseudomonas putida depends on growth medium and phase of growth. Microbiology 148, 537–548.

178

Surtees and Funnell

Libante, V., Thion, L., and Lane, D. (2001). Role of the ATP-binding site of SopA protein in partition of the F plasmid. J. Mol. Biol. 314, 387–399. Lin, D. C. H., and Grossman, A. D. (1998). Identification and characterization of a bacterial chromosome partitioning site. Cell 92, 675–685. Lin, D. C. H., Levin, P. A., and Grossman, A. D. (1997). Bipolar localization of a chromosome partition protein in Bacillus subtilis. Proc. Natl. Acad. Sci. USA 94, 4721–4726. Lin, Z. C., and Mallavia, L. P. (1998). Membrane association of active plasmid partitioning protein A in Escherichia coli. J. Biol. Chem. 273, 11302–11312. Lobocka, M., and Yarmolinsky, M. (1996). P1 plasmid partition: A mutational analysis of ParB. J. Mol. Biol. 259, 366–382. Ludtke, D. N., Eichorn, B. G., and Austin, S. J. (1989). Plasmid-partition functions of the P7 prophage. J. Mol. Biol. 209, 393–406. Lukaszewicz, M., Kostelidou, K., Bartosik, A. A., Cooke, G. D., Thomas, C. M., and JaguraBurdzy, G. (2002). Functional dissection of the ParB homologue (KorB) from IncP-1 plasmid RK2. Nucl. Acids Res. 30, 1046–1055. Lynch, A. S., and Wang, J. C. (1994). Use of an inducible site-specific recombinase to probe the structure of protein-DNA complexes involved in F plasmid partition in Escherichia coli. J. Mol. Biol. 236, 679–684. Lynch, A. S., and Wang, J. C. (1995). SopB protein-meditated silencing of genes linked to the sopC locus of Escherichia coli F plasmid. Proc. Natl. Acad. Sci. USA 92, 1896–1900. Marston, A. L., and Errington, J. (1999). Dynamic movement of the ParA-like Soj protein of B. subtilis and its dual role in nucleoid organization and developmental regulation. Mol. Cell 4, 673–682. Martin, K. A., Davis, M. A., and Austin, S. (1991). Fine-structure analysis of the P1 plasmid partition site. J. Bacteriol. 173, 3630–3634. Martin, K. A., Friedman, S. A., and Austin, S. J. (1987). Partition site of the P1 plasmid. Proc. Natl. Acad. Sci. USA 84, 8544–8547. Mileykovskaya, E., and Dowhan, W. (2000). Visualization of phospholipid domains in Escherichia coli by using the cardiolipin-specific fluorescent dye 10-N-nonyl acridine orange. J. Bacteriol. 182, 1172–1175. Mohl, D. A., Easter, J., and Gober, J. W. (2001). The chromosome partitioning protein, ParB, is required for cytokinesis in Caulobacter crescentus. Mol. Microbiol. 42, 741–755. Mohl, D. A., and Gober, J. W. (1997). Cell cycle-dependent polar localization of chromosome partitioning proteins in Caulobacter crescentus. Cell 88, 675–684. Moller-Jensen, J., Jensen, R. B., and Gerdes, K. (2000). Plasmid and chromosome segregation in prokaryotes. Trends Microbiol. 8, 313–320. Moller-Jensen, J., Jensen, R. B., Lowe, J., and Gerdes, K. (2002). Prokaryotic DNA segregation by an actin-like filament. EMBO J. 21, 3119–3127. Mori, H., Kondo, A., Ohshima, A., Ogura, T., and Hiraga, S. (1986). Structure and function of the F plasmid genes essential for partitioning. J. Mol. Biol. 192, 1–15. Mori, H., Mori, Y., Ichinose, C., Niki, H., Ogura, T., Kato, A., and Hiraga, S. (1989). Purification and characterization of SopA and SopB proteins essential for F plasmid partitioning. J. Biol. Chem. 264, 15535–15541. Motallebi-Veshareh, M., Rouch, D. A., and Thomas, C. M. (1990). A family of ATPases involved in active partitioning of diverse bacterial plasmids. Mol. Microbiol. 4, 1455–1463. Nash, H. A. (1996). The HU and IHF proteins: Accessory factors for complex protein-DNA assemblies. In ‘‘Regulation of Gene Expression in Escherichia coli’’ (E. C. C. Lin and A. S. Lynch, eds.), pp. 149–179. R. G. Landes Company, Austin, Texas. Niki, H., and Hiraga, S. (1997). Subcellular distribution of actively partitioning F plasmid during the cell division cycle in E-coli. Cell 90, 951–957.


179

Niki, H., and Hiraga, S. (1998). Polar localization of the replication origin and terminus in Escherichia coli nucleoids during chromosome partitioning. Genes Dev. 12, 1036–1045. Ogura, T., and Hiraga, S. (1983). Partition mechanism of F plasmid: Two plasmid geneencoded products and a cis-acting region are involved in partition. Cell 32, 351–360. Pabo, C. O., and Sauer, R. T. (1992). Transcription factors: Structural families and principles of DNA recognition. Annu. Rev. Biochem. 61, 1053–1095. Pogliano, J., Ho, T. Q., Zhong, Z. P., and Helinski, D. R. (2001). Multicopy plasmids are clustered and localized in Escherichia coli. Proc. Natl. Acad. Sci. USA 98, 4486–4491. Pogliano, J., Pogliano, K., Weiss, D. S., Losick, R., and Beckwith, J. (1997). Inactivation of FtsI inhibits constriction of the FtsZ cytokinetic ring and delays the assembly of FtsZ rings at potential division sites. Proc. Natl. Acad. Sci. USA 94, 559–564. Prentki, P., Chandler, M., and Caro, L. (1977). Replication of the prophage P1 during the cell cycle of Escherichia coli. Mol. Gen. Genet. 152, 71–76. Quisel, J. D., and Grossman, A. D. (2000). Control of sporulation gene expression in Bacillus subtilis by the chromosome partitioning proteins Soj (ParA) and Spo0J (ParB). J. Bacteriol. 182, 3446–3451. Quisel, J. D., Lin, D. C. H., and Grossman, A. D. (1999). Control of development by altered localization of a transcription factor in B-subtilis. Mol. Cell 4, 665–672. Radnedge, L., Davis, M. A., and Austin, S. J. (1996). P1 and P7 plasmid partition: ParB protein bound to its partition site makes a separate discriminator contact with the DNA that determines species specificity. EMBO J. 15, 1155–1162. Radnedge, L., Youngren, B., Davis, M., and Austin, S. (1998). Probing the structure of complex macromolecular interactions by homolog specificity scanning: The P1 and P7 plasmid partition systems. EMBO J. 17, 6076–6085. Raskin, D. M., and deBoer, P. A. J. (1999). Rapid pole-to-pole oscillation of a protein required for directing division to the middle of Escherichia coli. Proc. Natl. Acad. Sci. USA 96, 4971–4976. Rice, P. A., Yang, S. W., Mizuuchi, K., and Nash, H. A. (1996). Crystal structure of an IHF-DNA complex: A protein-induced DNA U-turn. Cell 87, 1295–1306. Rodionov, O., Lobocka, M., and Yarmolinsky, M. (1999). Silencing of genes flanking the P1 plasmid centromere. Science 283, 546–549. Rosche, T. M., Siddique, A., Larsen, M. H., and Figurski, D. H. (2000). Incompatibility protein IncC and global regulator KorB interact in active partition of promiscuous plasmid RK2. J. Bacteriol. 182, 6014–6026. Rothfield, L., Justice, S., and Garcia-Lara, J. (1999). Bacterial cell division. Annu. Rev. Genet. 33, 423–448. Sakai, N., Yao, M., Itou, H., Watanabe, N., Yumoto, F., Tanokura, M., and Tanaka, I. (2001). The three-dimensional structure of septum site-determining protein MinD from Pyrococcus horikoshii OT3 in complex with Mg-ADP. Structure 9, 817–826. Sandman, K., Losick, R., and Youngman, P. (1987). Genetic analysis of Bacillus subtilis spo mutations generated by Tn917-mediated insertional mutagenesis. Genetics 117, 603–617. Sawitzke, J., and Austin, S. (2001). An analysis of the factory model for chromosome replication and segregation in bacteria. Mol. Microbiol. 40, 786–794. Schindelin, H., Kisker, C., Schlessman, J. L., Howard, J. B., and Rees, D. C. (1997). Structure of ADP-AlF4 -stabilized nitrogenase complex and its implications for signal transduction. Nature 387, 370–376. Surtees, J. A., and Funnell, B. E. (1999). P1 ParB domain structure includes two independent multimerization domains. J. Bacteriol. 181, 5898–5908. Surtees, J. A., and Funnell, B. E. (2001). The DNA binding domains of P1 ParB and the architecture of the P1 plasmid partition complex. J. Biol. Chem. 276, 12385–12394.

180

Surtees and Funnell

Tabuchi, A., Min, Y. N., Womble, D. D., and Rownd, R. H. (1992). Autoregulation of the stability operon of incFII plasmid-NR1. J. Bacteriol. 174, 7629–7634. Treptow, N., Rosenfeld, R., and Yarmolinsky, M. (1994). Partition of nonreplicating DNA by the par system of bacteriophage P1. J. Bacteriol. 176, 1782–1786. van den Ent, F., Amos, L. A., and Lowe, J. (2001). Prokaryotic origin of the actin cytoskeleton. Nature 413, 39–44. van den Ent, F., Moller-Jensen, J., Amos, L. A., Gerdes, K., and Lowe, J. (2002). F-actin-like filaments formed by plasmid segregation protein ParM. EMBO J. 21, 6935–6943. Watanabe, E., Wachi, M., Yamasaki, M., and Nagai, K. (1992). ATPase activity of SopA, a protein essential for active partitioning of F-plasmid. Mol. Gen. Genet. 234, 346–352. Webb, C. D., Graumann, P. L., Kahana, J. A., Teleman, A. A., Silver, P. A., and Losick, R. (1998). Use of time-lapse microscopy to visualize rapid movement of the replication origin region of the chromosome during the cell cycle in Bacillus subtilis. Mol. Microbiol. 28, 883–892. Webb, C. D., Teleman, A., Gordon, S., Straight, A., Belmont, A., Lin, D. C. H., Grossman, A. D., Wright, A., and Losick, R. (1997). Bipolar localization of the replication origin regions of chromsomes in vegetative and sporulating cells of B. subilitis. Cell 88, 667–674. Williams, D. R., Macartney, D. P., and Thomas, C. M. (1998). The partitioning activity of the RK2 central control region requires only incC, korB and KorB-binding site O(B)3 but other KorB-binding sites form destabilizing complexes in the absence of O(B)3. Microbiology 144, 3369–3378. Williams, D. R., and Thomas, C. M. (1992). Active partitioning of bacterial plasmids. J. Gen. Microbiol. 138, 1–16. Wu, H. Y., and Liu, L. F. (1992). Toplogical approaches to studies of protein-mediated looping of DNA in vivo. Methods Enzymol. 212, 346–351. Wu, L. J., and Errington, J. (2002). A large dispersed chromosomal region required for chromosome segregation in sporulating cells of Bacillus subtilis. EMBO J. 21, 4001–4011. Yamaichi, Y., and Niki, H. (2000). Active segregation by the Bacillus subtilis partitioning system in Escherichia coli. Proc. Natl. Acad. Sci. USA 97, 14656–14661. Yarmolinsky, M. (2000). Transcriptional silencing in bacteria. Curr. Opin. Microbiol. 3, 138–143. Yates, P., Lane, D., and Biek, D. P. (1999). The F plasmid centromere, sopC, is required for full repression of the sopAB operon. J. Mol. Biol. 290, 627–638. Youngren, B., and Austin, S. (1997). Altered ParA partition proteins of plasmid P1 act via the partition site to block plasmid propagation. Mol. Microbiol. 25, 1023–1030. Youngren, B., Radnedge, L., Hu, P., Garcia, E., and Austin, S. (2000). A plasmid partition system of the P1-P7 par family from the pMT1 virulence plasmid of Yersinia pestis. J. Bacteriol. 182, 3924–3928. Zhou, T. Q., Radaev, S., Rosen, B. P., and Gatti, D. L. (2000). Structure of the ArsA ATPase: The catalytic subunit of a heavy metal resistance pump. EMBO J. 19, 4838–4845.

Index A Achiasmatic chromosomes, 103 Acrosomes, 29 Actin polymerization, 105 Active segregation, 8 AD. See Androstenedione Adenosine diphosphate (ADP), 148 phosphate moieties of, 153 Adenosine triphosphate (ATP) internalization and, 32 ParA and, 147 phosphate moieties of, 153 Adenovirus, 3 Adenylate cyclase, 32 activation of, 33 progesterone and, 38 ADP. See Adenosine diphosphate ADP-ribose, 56 ADP-ribosyl transferases (ARTs), 61 Adult disease, fetal origins of, 137 AYnity trapping assays, 12 Agrobacterium tumefaciens, 3, 167 Aldosterone, 26 Alpha helix, 116 Amide bonds, 2 Amplification systems of 2-m circle plasmid, 5, 6–7 Futcher model for, 6 Anatomical ontologies, 136–37 Anatomical transformation abstraction (ATA), 136 Anatomy, cross-sectional, 125 Androgen insensitivity syndrome (AIS), 30 Androgen receptor (AR) CREB and, 43–44 interactions, 25 MAP kinase pathway and, 37 in Sertoli cells, 30 Androgen(s), 26 AR-dependent actions of, 34–35 AR-independent responses to, 31–32 candidate surface receptors for, 32–34

follicular growth and, 39 granulosa cells and, 39 MAP kinase pathway and, 37 nongenomic actions of, 30–38 plasma membrane and, 35–36 second messengers and, 46 in Sertoli cells, 25–26 in spermatogenesis, 29–30 target tissues for, 26 in Xenopus oocyte maturation, 39 Androstenedione (AD), 31 Ca2+ levels and, 39 MAPK signaling cascade and, 39 Aneuploidy, 86 Angiotensin II, 33 Annotation and Modeling application, 138 Antibiotic resistance, 2 Antibodies, function-blocking, 94 Antibody probes, 136 Antisense oligos, 103 Anucleate cells, 161 Aorta, 118 Aortic arches, 128 Aortic trunk, 130 Apoptosis, 59 AR. See Androgen receptor AR antagonists, 31 AR-DNA interactions, 25, 30, 46 Arabidopsis, 71 Archaea, 61 Aromatization, of testosterone, 40 ARS plasmids, 8 ArsA, 153 ARTs. See ADP-ribosyl transferases Aster formation, 100 Aster-promoting activities (APAs), 98 ATM kinase, 68 ATP. See Adenosine triphosphate ATP hydrolysis, 102 in P1 partition, 159 partition and, 149 ATPase(s) actin-like, 167–68 181

182 ATPase(s) (cont.) F1 plasmid, 163 motor proteins and, 102 nonpartition, 153 ParA and, 148 in R1 plasmid, 168 Walker-type partition, 165–67 Atrioventricular canal, 118, 128, 129, 130 Atrium early right, 129, 130 left, 118, 129 right, 118 Aurora B kinase, 95 Aurora kinase, 92 Automated image registration algorithms, 123 B Bacillus subtilis, 169–71 par gene in, 168 Soj, 150 Bacteria chromosome dynamics in, 145–46 gram-negative, 171 insertion sequences of, 2 membrane of, 161 toxins, 61 Bacterial plasmids extrachromosomal elements and, 3 partition loci of, 14 transcription in, 14 Beta sheet, 116 Bezier curves, 124 BHK cells, 95 Biocatalysis, protein-based, 2 Bioinformation computer technology and, 117 3D reconstruction and, 116 bipartite regulators, 7 Birth weight, 116 Blood-testis barrier, 29 Branched hyphae, 172 BRCT domain, 63 Breast carcinomal cells, 33 BRN1, 13 Brn1p plasmid partition and, 15 Rep1p and, 13 Bromide imagery, 120 Bub1 kinase, 92 BubR1 kinase, 92

Index Bud emergence, 10 Bulbus cordis, 128, 129, 130 trabeculations in, 130 C C/EBP transcription factor CREB regulation and, 43–44 c-fos transcription factor CREB regulation and, 43–44 Ca2+ androstenedione (AD) and, 39 mobilization, 32 testosterone and, 31, 40 testosterone-BSA conjugates and, 35 Caenorhabditis elegans ncl-1 gene of, 70 PARP genes in, 63 Calcium, 26 Calmodulin, 37 CaM kinase II, 92 CaM kinase IV, 41–43 cAMP, 26 forskolin-induced, 32 production, 33 progesterone and, 38 response element (CRE) motifs, 41 Candidate surface receptors androgen, 32–34 signaling pathways from, 34 Cardiac morphogenesis, 123 simulating, 139 Cardiac myocyte lineage, 136 Cardiac research 3D reconstruction and, 116 Cardiac-specific proteins, 117 Cardinal vein inflow, 130 Cardiogenesis, 132 knowledge of, 138 pattern formation during, 134 temporal element of, 139 Carnegie Collection of human embryos, 120–21 specimen 836, 131 Casein kinase 2 (CKII) inhibitors, 72 Caspase 3, 59 Caulobacter crescentus, 171–72 par gene in, 168–69 Caveolin, 36

Index CBP. See CREB Cdc25, 94 Cdk1 kinase, 91–92 Cell migration, 136 positioning, 160–62 signaling cascades, 26 Cell cycle checkpoints, 56 Cell-fate mapping studies, 136 Cellular modeling and simulations, 136 Centromere activity, 74–75 composition of, 87 Centromere-binding protein in R1 plasmid, 168 Centromeric proteins, 75 Centrosomes, 105–6 activity, 74–75 focal microtubule nucleating sites and, 105

-tubulin and, 101–2 kinases and, 92 laser ablation of, 105 in mitotic spindle assembly, 86–88 RanBPM at, 96 Cephalexin, 161 Chaperone proteins heat shock genes and, 69 Checkpoint, 92 Chemokines, 33 Chiasmata, 104 Chick embryo, 135 Chloride secretion, 31–32 CHO1/MKLP1 motor protein, 93 Chromatids, 15 Chromatin beads, 97 in developing embryo, 55 loosening, 72 in microtubule regulation, 96–97 in microtubule stabilization, 91 modulation, 55 noncentromeric, 85, 89–90 organization, 13–14 PARP and, 63, 65 processes, 73–75 RCC1 binding of, 97 reassembly, 76 remodeling complex, 14 signals, 95–96 in spindle assembly, 85, 88–91, 106

183 Chromatin-associated kinases, 91–94 Chromatin immunoprecipitation assays, 15, 16, 160 Chromokinesin Nod and, 103 in spindle assembly, 104 Xklp1, 103 Chromosomal attachment, 3 Chromosome condensation, 95 mitotic, 105 MukB and, 161 Ran and, 102 Chromosome segregation, 74, 85 2-m circle segregation and, 14–15 abnormal, 104 Brn1 in, 13 cohesin and, 15 errors in, 85–86 in P. putida, 173 pathways, 1 Chromosome(s) dynamics in bacterial cells, 145–46 episomal, 75 locations, 146 microtubules and, 89 misalignment, 103 in mitosis, 88 monooriented, 89 movement, 106 ParA-ParB system in, 146 polar ejection force in, 89 Poly(ADP-ribose) polymerase distribution along,65–66 removal, 89 in spindle assembly, 105 Circular dichroism, 148 Circular mitotic figures (CMFs), 104 cis, 147 Cis-acting elements, 4 of 2-m circle plasmids, 5 CKII. See Casein kinase 2 Clinical Management Planning application, 138 Clipping plane, 127 ClustalW, 61 CMFs. See Circular mitotic figures Cohesin chromosome segregation and, 15 complex, 16 recruitment, 17

184 Cohesin (cont.) as tethering agent, 17 Composite imaging, 126 Computational fluid dynamics, 128 flow analyses, 133 Computer technology, 117 Computerized tomography X-ray (CT), 119 Condensin analysis, 15 Conjugative transfer, of bacteria, 3 Contours, 123–24 Contractile motion, 129 Control vertices, 123–25 Conus, 117, 118 Coordinately regulated genes, 72 Copy number control bipartite regulation and, 7 recombination-mediated, 4 Cornus cordis, 128 Coronary disease, birth weight and, 116 Coronary myocytes, 40 Corticosterone, 26 Covalent integration, 3 Covalent modification, of nuclear proteins, 56 CRE. See cAMP CREB activation of, 43 androgen stimulation of, 44 binding protein (CBP), 41 phosphorylation, 40–41, 42 signaling pathways and, 41 CREB-mediated transcription, 26 CREB transcription factor, 26 in Sertoli cells, 40–45 CST6/SHF1, 13 CT. See Computerized tomography X-ray Cubic splines, 124. See also Bezier curves Cyproterone acetate, 32 Cytokinesis centrosomes in, 105 kinases and, 92 Plk and, 93 Cytoplasmic dynein, 102 events, 56 Cytoskeletal elements, 32 D DAG formation, 31 testosterone-BSA conjugates and, 35 DAPI staining, 9

Index Deletion analysis, 172 Density shift experiments, 8 Dephosphorylation, 94 DHT. See 5-dihydrotestosterone, 26 DHT-mediated activation of MAP kinases, 34 Diaclyglycerol (DAG), 26 Diazoxide, 40 Dictyostelium discoideum PARP2/3-like proteins of, 60 PARP genes in, 63 Digital Anatomist, 136 Digital Libraries Initiative, 137 Dihybrid assays of Rep1p mutants, 15 in vivo, 12 5-dihydrotestosterone (DHT), 26, 34 Erk phosphorylation and, 37 Dimerization, 60 BRCT domain-mediated, 70 KorB and, 166 Par A and, 148 of ParB, 158, 172 Dimethylnortestosterone, 34 Dithiobis succinimidyl propionate (DSP), 157 DNA-binding domains, 59–60 ParA and, 148 of ParB, 158 site-specific, 153 DNA damage p53 and, 68 PARP1 activation and, 63 DNA ligase III enzymes, 59 DNA regulatory genes, 117 DNA repair, 55 PARP action during, 64 PARP activation and, 71 PARP1 response in, 66–67 DNA replication, 170 DNase I footprinting, 148 ParB and, 156 Dorsal cushion, 118 DRB, 72 Drosphila melanogaster brat gene of, 70 chromosomes in, 88 coordinately regulated genes in, 72 heat shock genes in, 69 immune response in, 66 Nod and, 103

185

Index PARG gene of, 59 PARP, 63–65 PARP-e and, 73 PARP-I, 59 Polo kinases and, 92 polytene chromosome, 65 RCC1 colocalization in, 97 tiovivo gene of, 104–5 DSP. See Dithiobis succinimidyl propionate E E element, 14 EBV. See Epstein-Barr virus Ecdysone, 69 Ectoderm, 117 Ectopic microtubule aster formation, 96 Edinburgh mouse atlas of gene expression (EMAGE), 134 Eg5, 101 motor proteins, 92 EGFR. See Epidermal growth factor receptor EGTA, 41 Embryo development adult disease and, 137 environmental factors and, 137 Embryogenesis, 115–16 Embryology Education application, 138 Embryonic heart development, 117–18 Embryo(s) histological database of, 121 models, 116 sectioned, 120 Endoderm, 117 Endothelial lining, 130 Environmentally responsive genes, 67 Epidermal growth factor, heparin-bound (HB-EGF), 33–34 Epidermal growth factor receptor (EGFR), 33 EpiFluorescent stereomicroscopy, 135 Episomal viral genomes, 18 Episomes, nonintegrated, 3 Epithelia, 31 Epstein-Barr virus (EBV), 75 ER. See Estrogen receptor Erk CREB phosphorylation and, 41 DHT and, 37 phosphorylation, 42

Eschericia coli cell division inhibition of, 161 F plasmid in, 163 IHF, 153–54 P1 plasmid in, 146 P7 plasmid of, 165 pB171 plasmid in, 167 R1 plasmid in, 168 Esp1 protease, 15 Estradiol, 31 GPR30 and, 33 LNCaP cells and, 37 receptors, 37 SHBG and, 32–33 Estrogen, 26 plasma membrane and, 35–36 Estrogen receptor (ER), 33–34 plasma membrane (mER), 35–36 Eukaryota, 61 Eukaryotes repeat families in, 2 selfish nucleic acid elements of, 2 Extrachromosomal entities autonomously replicating, 2 bacterial plasmids and, 3 F F-actin, 168 F plasmid(s) in anucleate cells, 161 partition system, 163–65 SopB protein of, 160 system, 149 F SobA, 150 FACS analysis, 16 Fibrillarin, 70 Fibroblasts, human genital skin, 34 Field of view (FOV), 121 FISH. See Fluorescence in situ hybridization Flagellum, of spermatids, 29 Flp acquisition of, 18 recombination, 6 FLP gene bipartite regulation of, 7 expression, 19 Flp-mediated resolution, 6 Flp recombination targets (FRT), 4, 5–6

186

Index

Fluorescence in situ hybridization (FISH), 146 of plasmid foci, 157 Fluorescence tagging, 12 of STB-containing plasmids, 8 Fluorescent materials, 139 Flutamide, 32 AR and, 39 CREB phosphorylation and, 43 Focal microtubule nucleating sites, 87 centrosomes and, 105 Follicle-stimulating hormone (FSH), 29 spermatazoa production and, 44–45 in spermatogenesis regulation, 29–30 stimulation, 39 Follicular growth androgens and, 39 Foundational Model (FM), 136 FOV. See Field of view FRET-based biosensors, 98 FRT. See Flp recombination targets FSH. See Follicle-stimulating hormone FtsI inhibition, 161 FtsZ, 161 FUN30, 13 Fun30p, 13 Futcher model, 19

in 3d models, 134 3D reconstruction and, 116 Genotoxic stress, 56 Germ cells development of, 25 regulation of, 38–45 GFP. See Green fluorescent protein (GFP) GFP-LacI, 161 lac operator sequences and, 146 GFP-LacI/LacO interaction, 8 Glutamic acid (Glu) COOH residue of, 58 residues, 60 Golgi aparatus, 60 GPCR. See G protein coupled receptor GPR30, 33 GPUs. See Graphics processing units Granulosa cells, 31 androgens and, 39 Graphics processing units (GPUs), 132–33 Grasshopper spermatocytes, 89 Green fluorescent protein (GFP), 150 GTPase, 95 Guanine nucleotide exchange factors (GEFs), 95 androgen and, 37 GVBD, 38

G

H

G1-arrest, -factor-induced, 10 G-protein identification, 38 SHBG and, 33 G-protein coupled receptor (GPCR), 29 localization of, 32 SHBG and, 33 GAL4, 164–65 G scavengers, 38 GEF. See Guanine nucleotide exchange factor Gene expression AR-mediated, 26 3D models of, 134 ER-mediated, 38 extrapolating, 139 Gene expression data patterns, 135 Gene silencing, of ParB, 160 Gene transcription, 55 Genome databases, 134, 168 Genomics

H-89, 41 Haptic-feedback devices, 140 HB-EGF. See Epidermal growth factor Heart anlage, 117 chambers of, 117 composite image of, 126 congenital defects, 116 embryonic human, 128–32 embryonic vs. adult, 138–39 growth regulators, 137 looping process of, 129 stereolithographical physical models of, 133–34 surface model of, 125 transverse section of, 126 wire frame model of, 124 Heat shock factor (Hsf), 69 Heat shock genes, 69 HeLa nuclear extracts, 100

187

Index PP1 and, 94 Helicity, 148 Helix-turn-helix (HTH) motifs, 59, 157–58 Hemodynamic forces, 128, 134 Heterochomatin condensation, 73–74 high-aYnity core complex, 153–54 hippocampal pyramidal neurons, depolarized, 41 Histological sections, 120 photomicrograph of, 123 Histone, 65–66 Histone H1 pADPr aYnity of, 63 PARP1 interaction with, 72 Homologous recombination, 6 Hormone response elements (HREs), 27 Horn sinus venosus, 128 Hox gene expression, 76 Hsf. See Heat shock factor Hst3 protein, 14 Human Developmental Anatomy Center, 120 Human Embryology Digital Library and Collaboratory Support Tools, 137 Hydrolysis, by Par A, 148 Hydroxyflutamide, 34 Hyperphosphorylation, 93 I IC-21 macrophages, 32 IGF-1. See Insulin-like growth factor 1 IHF, 153–57 Image data sources, 118–19 Immune response genes PARP and, 66, 67–68 Immunofluorescence of HeLa cells, 94 of KLP38B, 105 Immunohistochemistry studies, 35–36 Immunostaining, 8 Importin identification, 100 Importin , 98 chromosome condensation and, 102 Importins, 98 spindle defects and, 100 IncC, 166 IncC-KorB system, 165

Incident light fluorescence. See EpiFluorescent stereomicroscopy Insulin-like growth factor 1 (IGF-1), 39 Interatrial walls, 117 Intermolecular recombination, 6–7 Internet model sharing over, 116 visualization tools on, 139–40 Internet2, 138 Interphase microtubules during, 86 RanGTP gradient in, 98 Interventricular walls, 117 Intracellular organelles, 3d modeling of, 136 Intracellular receptor proteins, 26 Inverted repeats (IRs), 4 IP3 formation, 31 levels, 35 Ischemic heart disease, 137 K K+ channels, in Sertoli cells, 40 Kinase/phosphatase gradient theory, 91 Kinases, in mitosis, 92 Kinesin-like DNA binding protein, 103 Kinesin motor proteins, 102 Kinetochore(s), 105–6 absence of, 89 composition of, 87 microtubule interactions, 95 in mitotic spindle assembly, 86–88 proteins, 75 KLP38B KorB, 166 L L-type channels, 40 lac operator sequences GFP-LacI and, 146 Lactate, 29 Lamin, 74 Laser ablation, 105 Laser scanning confocal microscopy (LSCM), 119, 122 distortion in, 122 limitations of, 122 two photon, 122 LDH-A gene, 44

188 Leydig cells, 28, 29 Liquid photopolymer resin, 133 LNCaP prostate cancer cells, 34 inhibition of, 37 Localization in C. crescentus, 171–72 signals, 161–62 Loop formation, 74 LSCM. See Laser scanning confocal microscopy Lumen shapes development of, 129 model of, 128 SLA of, 133 surface models of, 129 transformation of, 128 Lysogenic phage, 2 M Macrophages, 35 Magnetic resonance histology (MRH), 121 Magnetic resonance imagery (MRI), 121 Magnetic resonance microscopy (MRM), 119, 121, 126 vs. light microscopy, 121 vs. MRI, 121 Mammalian embryos, 117 MAP kinase androgens and, 39 DHT-mediated activation of, 34 Erk, 37, 51 in Sertoli cells, 40–45 signaling cascade, 43 testosterone-BSA conjugates and, 35 MAP kinase kinase kinase (MAPKKK), 37 MAP kinase pathways activation of, 33–34 androgen and, 37 FSH and, 45 progesterone and, 38–39 testosterone and, 45 MARs, 74 Matrix metaloprotease (MMP), 33–34 Mcd1 protein, 15 Mcd1p cleavage, 17 Mcd1p/Scc1p cleavage, 15 Meiosis, in spermatocytes, 29 MEK MAPKK, 39 Membrane-associated receptors, 38 mER. See Estrogen receptor

Index Mesoderm, 117 Mesodermal diversification, 136 Metaphase chromosome alignment, 106 Xkid in, 103 Metazoan organisms PARP activity in, 61 PARP-related proteins and, 56 Methylation protection experiments, 156 Methyltrienolone, 35 Mibolerone, 34 Microtome, 120 Microtubule assembly, 96 TPX2 and, 100 Microtubule-associated proteins (MAPs), 91 Microtubule-based motors, 86 chromatin-associated, 102–5 Microtubule organizing centers (MTOCs), 87 Microtubule(s) chromatin-associated kinases and, 91–94 chromosome interaction with, 89 dynamics, 86, 94–95 half-life of, 88 nucleation, 87–88, 101–2 Plk and, 93 polarity of, 86 polymerization, 85, 100 polymers, 86 PP1 and, 94 Ran and, 96 RanGTP gradient and, 98 regulation, 96–97 stabilization, 91, 98–101 MinD, 153 proteins, 167 Missegregation of chromosomes, 12 circumventing, 3 events, 1, 6 Mitogen-activated protein (MAP) kinase signaling pathway, 26, 37–38 Mitosis chromosomes in, 88 kinetochores in, 106 M phase of, 74 microtubule-based motors in, 102 phosphatases in, 94 prokaryotic vs. eukaryotic, 145 protein phosphorylation in, 91–92 Ran in, 95, 102 RanGTP gradient in, 98

189

Index RanGTP in, 96, 101 of spermatocytes, 29 spindle formation in, 87–88 Mitotic exit, 92 MMP. See Matrix metaloprotease, 33 MNAR, 38 Model viewing applications, 118–19 Moieties, chromosomally integrated, 2 Monohybrid assays, 15 Mononucleosomes, 97 Morphing, 138 Motifs bacterial actin-like, 168 Box A, 154–57 Box B, 154–57 helix-turn-helix (HTH), 157–58 Motion artifacts, 121 Motor proteins, 102–5 cytoplasmic dynein, 102 kinesin superfamily of, 102 Mouse oocytes, 89 Mps1 kinase, 92 MRH. See Magnetic resonance histology MRI. See Magnetic resonance imagery MRM. See Magnetic resonance microscopy MTOCs. See Microtubule organizing centers MukB, 161 Mutational analysis, of Rep1 protein, 13 Mutations, 14–15 Mycelium, 172 Myocardial primordium, 130 N NAD, 56 National Library of Medicine, 137 National Museum of Health and Medicine, 121 NEB. See Nuclear envelope breakdown Negative regulatory mechanism, 1 Nek2 kinase, 92 Neomycin, 31 Neurospora crassa PARP2/3-like proteins of, 60 PARP genes in, 63 Newt lung cells, 89 NF-B binding of, 68 in PARP1 mice, 67–68 transcription factors, 66

Nib1 genotype, 19–20 NifH, 153 NLS. See Nuclear localization sequence NLS-containing proteins, 98 Nocodazole, 101 Nod, 103–4 Nongenomic actions, 26 Noninvasive imaging techniques, 126 Nonuniform rational B-splines (NURBS), 124 alternatives to, 132 NOP140, 70 NR1 plasmid, 167 Nuclear envelope breakdown (NEB), 95 Nuclear localization sequence (NLS), 98 Nuclear location signal (NLS), 60 Nuclear pore complexes, 60 Nuclear proteins, 56 Nucleocytoplasmic transport, 95 Ran in, 96 Nucleolar proteins, 70 Nucleoli, 69–70 Nucleotide binding by Par A, 148, 151 Nucleotide synthesis, 2 NuMA regulation of, 98 sequestering of, 100 NURBS. See Nonuniform rational B-splines O Oligomerization KorB and, 166 of ParB, 158 Oligoribonucleotide entities, self-replicating, 2 Oocyte maturation, 38–39 Op18, 93–94 Optical projection tomography, 134 Organogenesis, 134 ORI. See Replication origin oriC, 168 Origin recognition complex (ORC), 14 Osteoblast cells, 31 Osteroblasts, 35 P p53, 68 P-CREB, 44 p38 kinase inhibitor, 41

190 P1 partition operon, 147 P7 plasmid, 165 P1 plasmid(s) cephalexin and, 161 in Eschericia coli, 146 ParA-ParB system and, 146–47 ParB excess and, 151 partition, 155, 159 vs. F, 164 vs. RK2, 165–66 P-TEPb, 72 pADPr. See Poly(ADP-ribose) pADPr glycohydrolase (PARG), 59 DNA damage and, 56 pADPr lyase, 59 DNA damage and, 56 par gene, 168 operon, 165 super-repressors of, 149 ParA, 146 bacterial, 168–73 in C. crescentus, 171–72 conserved sequences of, 152 functional domains of, 152 gene regulation, 148–49 GFP-LacI and, 161 modulation of ParB-IHF-parS, 162 motifs of, 153 mutations of, 149 nucleotide bonding by, 148 oscillation, 170, 173 in P. putida, 173 in P1 partition, 147, 159 ParB-parS interaction, 151 in partition, 150–51 pB171, 150 physical properties of, 152–53 signals, 173 parA gene expression, 149 mutants, 151 parPD, 151 regulation, 148 parAB operon transcription of, 148 Parametric functions, 124 ParB, 146 bacterial, 168–73 in C. crescentus, 171–72 conserved sequences of, 152

Index corepressor activity of, 149 dimerization domain of, 157–58 DNA-binding domains of, 158 DNA recognition domains of, 160 eVect on P1 plasmids of, 151 foci, 150, 157 function of, 149 functional domains of, 152 gene regulation, 148–49 gene silencing of, 160 GFP-LacI and, 161 immunofluorescent visualization of, 150 oligomerization function of, 158 in P. putida, 173 during P1 partition, 159 in P1 partition, 147 during partition, 162 physical properties of, 157–60 signals, 173 stimulatory eVects of, 148 structure-function relationships of, 172 vs. KorB, 166 parB gene expression, 149 regulation, 148 ParB-IHF-parS partition complex, 153–57 action of, 162 PARG. See pADPr glycohydrolase Parlsop partition system, 167 ParM, 168 parOP, 148 mutations of, 150 PARP action, 64 activation, 70–72 automodification, 75 branching, 57, 59 catalytic domain of, 60 chromatin aYnity of, 60 dimerization, 60 distribution of, 65–66 DNA aYnity of, 60 DNA damage and, 56 domains, 59–61 elongation, 57–58 in environmentally responsive genes, 67 enzymatic reactions catalyzed by, 56–58 evolutionary relationships of, 61–63 heterochromatin and, 73 Hsf and, 69

191

Index initiation, 57–58 mechanisms of action, 63–66 metabolism, 56–59 in mitosis, 75 nonenzymatic role of, 73, 74 in nucleoli, 70 resistance, 72 signature (PS), 60 structural role of, 73 structure of, 58 transcription and, 66–70, 72–73 in undamaged cells, 56 vault (V-PARP), 60 PARP1. See Poly(ADP-ribose) polymerase 1 PARP-e, 73 PARP-GFP protein, 65 PARP-I, 73 PARP protein family, 59–61 PARP-related genes, 63 PARP-related proteins, 62 PARP2s, 58 ParR, 168 parS, 147 binding of, 153–54 Box A of, 154–56 Box B of, 154–56 insertion mutations in, 155 motifs of, 153–56 in P1 partition, 159 in S. coelicolor, 172 Partition ATP hydrolysis and, 149 in B. subtilis, 170 bacterial membrane and, 161 cohesin-mediated, 20 complex formation, 164 errors, 173 localization and, 161–62 loci, 4, 6, 14 ParA in, 150–51 ParA-ParB interactions during, 162–63 in RK2 plasmids, 165–66 of subtelomeric repeats, 14 Partition systems, 1, 10–12 of 2-m circle plasmid, 5, 6–7 arithmetic ability of, 162–63 of bacterial cells, 145 F plasmid, 163–65 host factors for, 13–14 parlsop, 167 plasmid, 163–68

pB171 plasmid, 167 pCD1 plasmid, 165 PCM. See Pericentriolar material PD98059, 41 Pericentriolar material (PCM), 87 Pertussis toxin, 31 chloride secretion and, 32 PET. See Positron emission tomography Phosphatases chromatin-associated, 94–95 serine/threonine, 94–95 Phospholipase inhibitor U73122, 32 Phospholipase C, 26 androgen and, 37 androgen eVects and, 39 Phospholipase proteins internalization and, 32 Phospholipids, 161 Phosphorylation Op18, 93–94 by Plk, 92–93 Photomicrographs, 119 of histological section, 123 Physical structure, vs. physiological function, 116 Physiological information, 116 PI3-kinase, 36 inhibitor, 41 Pince-nez (PN) structures, 6–7, 19 PKA. See Protein kinase A PKC. See Protein kinase C Plasma membrane androgen and, 35–36 hormones and, 26 Plasmid amplification, 19 chromosome attachment, 17 clusters, 17 copy number, 1 foci, 157 gene expression, 7–15 localization, 173 locations, 146 ParA-ParB system in, 146 positioning, 150 segregation, 17–18 stability, 12–13, 20 topology, 164 transport, 168 virulence, 167

192 Plasmid partition condensin in, 15 mechanisms for, 8 Rep protein interaction in, 13 requirements for, 8 Soj and, 170 systems, 15 yeast cohesin complex in, 14–15 Plasmid residence zone (PRZ), 8–10 Pleiotropic eVects, 95 Plks. See Polo kinases Plx1 kinase, 92–93 PMC42 breast cancer cells, 37 pMT1 plasmid, 165 Point-set surface modeling, 132–33 Polo kinases (Plks), 92 Poly(ADP-ribose) catabolism, 57 cleaving, 59 lamin and, 74 modification, 63 p53 aYnity for, 68 (pADPr), 56 phosphodiesterase, 59 polymerase distribution, 65–66 Poly(ADP-ribose) polymerase 1 (PARP1), 55, 59 activation, 70–71, 72 MARs and, 74 secondary modification of, 72 Polycomb group proteins, 76 Polycystic ovary syndrome, 39 Polygonal wire frame, 122 Positron emission tomography (PET), 123 PP1. See Protein phosphatase 1 PP2A. See Protein phosphatase 2A Predicting-protein-folding-patterns, 117 PROBE, 61 Progesterone, 26 adenylate cyclase and, 38 antagonists, 39 GPR30 and, 33 MAP kinase pathway and, 38–39 in oocyte maturation, 38 receptors, 38 Prokaryotes, selfish nucleic acid elements of, 2 Prometaphase, kinetochores and, 106 Prophase, microtubules during, 86 Protein kinase A (PKA), 33–34

Index androgen and, 37 cAMP concentrations and, 38 Protein kinase C (PKC) androgen and, 37 Protein phosphatase 1 (PP1), 94 Protein phosphatase 2A (PP2A), 94 Protein(s) kinases, 26 phosphorylation, 91–92 tertiary structure of, 116 tethers, 3 Proteomic information, 116 Pseudomonas putida, 168, 173 pSLT plasmid, 165 pTAR, 165–67, 167 Pulmonary artery, 118 R R1881, 37 R1 plasmid, 16–17 Raf-1, inhibition of, 33 RAF1 gene, 7 Raf1p, 7 Ran, 95 chromosome condensation and, 102 localization of, 97 microtubule nucleation and, 101–2 in nucleocytoplasmic transport, 96 organization, 98–101 rhodamine-labeled, 97 RanBP1, 96–97 Ran gradient and, 99 RanBPM, 96 Random segregation, 8 RanGAP, 96–97 Ran gradient and, 99 RanGTP, 95–96 function of, 101 gradient, 97–98 hydrolysis, 96 microtubule nucleation and, 101 protein regulation of, 98 RanQ69L, 101 Rap1 protein, 14 RCC1 allele, 95 chromatin-bound, 102 in microtubule regulation, 96–97 mononucleosomes and, 97 RanGTP gradient and, 97–98

193

Index rDNA genes, 69–70 Real-time imagery, 133 Recombinase Flp, 5 Recombination site-specific, 5–6 Registration function, 118–19 histological sections and, 120 image acquisition and, 128 of LSCM, 122 of surface modeling, 123 Rendering, 124–25 accelerating, 139 REP1 gene, 7 REP2 gene, 7 Rep1 protein, 13 bipartite regulators and, 7 Rep proteins, 6 eVect of, 19 Fun30p and, 13 organization of, 8 Plasmid stability and, 20 STB DNA and, 12–13 in STB plasmids, 12 visualization of, 9 Rep2 proteins, 7 Rep-STB system, 6, 8 evolution of, 18 yeast cohesin complex and, 15–17 Replication apparatus, 161–62 Replication origin (ORI), 4, 5–6 Replication proteins, 162 Rep1p, 5 Brn1p and, 13 mutants, 15 Rep2p, 5 DNA binding activity of, 13 Resolution of MRM, 121 of voxel models, 126 Retrotransposons, 3 Ribosome production, 69 Ribozymes, 2 RK2 plasmids, 165–67 RNA polymerase ParB blocking of, 160 PARP resistance of, 72–73 RNA Z, 3 RNAi, 97 rRNA synthesis, 69 Rsc2 protein, 14

RSHBG, 32–33 RU486, 39 S Saccharomyces cerevisiae, 5 overexpression of GEF of Ran, 95–96 SAFs. See Spindle assembly Salmonella newport, 167 Salmonella typhimurium, 165 ScaVold proteins, 36 receptor-interacting, 38 SDS-polyacrylamide gels, 157 Second messengers, 26, 46 Sectioning devices, 128 Segmentation, 123–25 algorithms, 122 automated, 128 process, 118–19 Segregation of 2-m plasmid, 10 of bacterial chromosome, 169 MukB and, 161 of P1 plasmid, 147 pathways, 12 patterns, 11 Selfish nucleic acid elements, 2 Selfishness evolutionary success of, 2 of yeast plasmids, 5 Seminferous tubules, 27, 28 testosterone in, 30 Septal rings, 171 Septum primum, 118 Serine 133 activating kinases and, 41 androgen stimulation and, 40 Serine/threonin kinases, 92 Sertoli cells, 28 AR actions of androgen in, 34–35 ARs in, 30 Ca2+ levels in, 40 CREB phosphorylation in, 44 CREB transcription factor in, 40–45 depolarization of, 40 gene expression in, 25 GPCR on, 29 MAP kinase in, 40–45 spermatogenesis and, 29 testosterone-BSA conjugates and, 35

194 Sex hormone-binding globulin (SHBG), 32–33 Shc, 37 Shf1 protein STB and, 13 Signal transduction, 153 Silencer elements, 14 Sinoatrial foramen, 129, 130 Sinus venosus, 128 Sir proteins, 14 SLA. See Stereolithography Soj, 169–71 SopA, 163–65 SopB, 163–65 HTH motif of, 163 protein, 160 sopC, 163–64 Southwestern assay, 12 Spacial genomics, 134 Spermatogenesis, 25 hormonal regulation of, 27–30 process of, 27–29 regulation of, 29–30 Spermatogonial germ cells, 27–29 Spermatozoa maturation of, 27–29 testosterone and, 25 Spindle assembly, 85, 86 chromatin in, 88–91 chromokinesins in, 104 factors (SAFs), 99 kinases and, 92 kinetochores and, 86–88 local stabilization model for, 90 mechanisms of, 106–7 motor proteins in, 102–5 Ran and, 96 RCC1 in, 96–97 RNAi and, 97 theory of, 86–87 Spindle poles, 8 depolymerization of, 10 Spindle(s) bead, 90–91, 97 centrosomes and, 86–88 in chromosome segregation, 85 extension, 103 formation, 88, 106 microtubules, 74–75 misorientation defects, 95–96 NuMA and, 100

Index Spo0J, 169–71 spo0J, 169–70 Sporulation, 170 in S. coelicolor, 172 Src, 37 kinase, 43 Stalked cell, 171 STB binding of, 13 Mcd1 and, 15 nucleosome pattern at, 14 plasmids, 12 Rep proteins and, 12–13 Shf1 protein and, 13 silencer elements and, 14 Steady state copy number, 3 regulating, 5 Stereolithographical physical models, 133–34 Stereolithography (SLA), 133 Stereomicroscopic block-face imaging, 135 Steroid action, 26, 27 nongenomic, 38 Steroid hormone-dependent genes, 69 Steroid-receptor complex, 26 Steroidogenic enzyme CYP17, 39 Streptomyces coelicolor, 172–73 par gene in, 168 Stress, 75 Structural homology of GPR30, 33 Subtelomeric repeats, 14 Sulfolobus solfataricus, 61 Surface modeling, 122–25 cross-sectional anatomy and, 125 point-based, 132–33 Surface-rendering, 118–19. See also Voxel modeling Swarmer cell, 171 Symbolic modeling, 136–37 T T-cells splenic, 32 testosterone-BSA conjugates and, 35 T-Vox, 132 Tankyrase(s), 58 activation, 63 ankyin repeats of, 60 Golgi aparatus and, 60 nuclear pore complexes and, 60 stress and, 75

195

Index TRF proteins and, 75 TATA-binding protein, 72 TCDD. See Tetrachlorodibenzo-p-dioxin treatment Telomere elongation, 66–67 maintenance, 56 Tesla MRM scanners, 121 Testicular feminization mutation (tfm), 30, 43 Testis, 27, 28 Testosterone, 25 androgens and, 26 aromatization of, 40 Ca2+ levels and, 40 CREB transcription factor and, 40–45 MAP kinase and, 40–45 MAPK signaling cascade and, 39 nongenomic actions of, 25 in osteoblasts, 31 paradoxical actions of, 30 production of, 28 Sertoli cells and, 40–45 SHBG and, 32 signaling pathways, 45 spermatazoa production and, 44–45 in spermatogenesis regulation, 29–30 Testosterone-BSA conjugates, 35 Tetrachlorodibenzo-p-dioxin treatment (TCDD), 62 3D data compression, 140 3D modeling applications, 118–19 3D reconstruction cross-species comparison and, 139 development of, 116 developments in, 132–38 scalability of, 140 techniques of, 118–28 ti PARP. See Tetrachlorodibenzo-p-dioxin treatment (TCDD) Time-lapse microscopy, 10 Tiovivo (tio) gene, 104–5 Tissue models, 115–16 Tobacco ringspot virus, 3 Tomographical data, 139 Topoisomerase II, 91 TP288, 167 TPX2 identification, 100

microtubule assembly and, 100 regulation of, 98 Trabeculations, 130 Transcription in bacterial plasmids, 14 p53 dependent, 68 PARP and, 56, 66–70 rDNA, 69 Transcription factors ATF/bZIP class of, 13 cardiac, 117 gene interactions, 136 ligand-inducible, 26 Myc, 30 Pem, 30 Transcriptional activation, 72–73 Transcriptional repressors, 70 Transferrin, 29, 44 Transmembrane phosphoproteins, 36 Transposons, 2 TRF-1, 632 TRF proteins, 75 Truncus, 117, 118 arteriosus, 129 TS mutations, 15 tsBN2 cells, 95 Tubulin heterodimers, 86 -tubulin, 95

-tubulin, 87 localization of, 101

-TURC, 101 2-m plasmid(s), 1, 5 amplification systems of, 6–7 cluster duplication, 15 cohesin complex and, 16 copy number control of, 4 gene expression of, 7 localization of, 9 organization of, 4, 5–6, 9 partition systems of, 6–7 replication of, 6 segregation, 14–15 segregation kinetics of, 10 segregation patterns of, 11 selfishness of, 18 in yeast nucleus, 8–12 Tyrosine kinases activation of, 33 LNCaP cells and, 37 Src, 37

196

Index

U

X

Ultraviolet laser light, 133

Xenopus laevis oocyte maturation, 72 oocytes, 38 Polo-like kinase in, 92 RCC1 removal in, 97 sperm chromatin in, 88 spindle formation in, 90 Xklp1 and, 103 Xkid, 103 Xklp1, 103 XPR. See Progesterone

V Vas deferens, 31 Ventricle, 128, 129, 130 right, 118 Ventricular septum, 118 Verapamil, 40 Vertebrates, PARP activity in, 61–63 Visible embryo NGI project, 121, 137–38 Vitamin D, 26 Voltage-sensitive calcium channels (VSCC), 40 Volume calculations, 128 Volume models, 140 von Willebrand factor (vWF) of vPARP, 62 Voxel data sets, 126 transmitting, 140 Voxel modeling, 118–19, 126–28 of Carnegie Collection specimen 836, 131 of mouse thorax, 127 semitransparent, 133 vPARP, 61 vertebrate-specific subclass, 62 VSCC. See Voltage-sensitive calcium channels vWF. See von Willebrand factor

Y Yeast cells nucleus of, 8–12 synchronously dividing, 12 Yeast cohesin complex in partition, 17 in plasmid partition, 14–15 Rep-STB system and, 15–17 Yeast condensin, 13 Yeast plasmids, 5 amplification system of, 1 stability of, 14 Yersinia pestis, 165 Yrb2 protein, 95–96 YY1 transcription factor, 72

W

Z

Walker nucleotide bonding motifs, 148, 153 Walker-type partition ATPases, 165–67 Wax-based tissue models, 115–16 Wire frame modeling, 124 Wortmanin, 41

Zn fingers, 59 DNA binding, 62 DNA repair and, 63 PARP/LigIII-type, 70–71 Zoligomerization interface, 160

Contents of Previous Volumes Volume 47 1 Early Events of Somitogenesis in Higher Vertebrates: Allocation of Precursor Cells during Gastrulation and the Organization of a Moristic Pattern in the Paraxial Mesoderm Patrick P. L. Tam, Devorah Goldman, Anne Camus, and Gary C. Shoenwolf

2 Retrospective Tracing of the Developmental Lineage of the Mouse Myotome Sophie Eloy-Trinquet, Luc Mathis, and Jean-François Nicolas

3 Segmentation of the Paraxial Mesoderm and Vertebrate Somitogenesis Olivier Pourqule´

4 Segmentation: A View from the Border Claudio D. Stern and Daniel Vasiliauskas

5 Genetic Regulation of Somite Formation Alan Rawls, Jeanne Wilson-Rawls, and Eric N. Olsen

6 Hox Genes and the Global Patterning of the Somitic Mesoderm Ann Campbell Burke

7 The Origin and Morphogenesis of Amphibian Somites Ray Keller

8 Somitogenesis in Zebrafish Scott A. Halley and Christiana Nu¨sslain-Volhard

9 Rostrocaudal Differences within the Somites Confer Segmental Pattern to Trunk Neural Crest Migration Marianne Bronner-Fraser

197

198

Contents of Previous Volumes

Volume 48 1 Evolution and Development of Distinct Cell Lineages Derived from Somites Beate Brand-Saberi and Bodo Christ

2 Duality of Molecular Signaling Involved in Vertebral Chondrogenesis Anne-He´le`ne Monsoro-Burq and Nicole Le Douarin

3 Sclerotome Induction and Differentiation Jennifer L. Docker

4 Genetics of Muscle Determination and Development Hans-Henning Arnold and Thomas Braun

5 Multiple Tissue Interactions and Signal Transduction Pathways Control Somite Myogenesis Anne-Gae¨lle Borycki and Charles P. Emerson, Jr.

6 The Birth of Muscle Progenitor Cells in the Mouse: Spatiotemporal Considerations Shahragim Tajbakhsh and Margaret Buckingham

7 Mouse–Chick Chimera: An Experimental System for Study of Somite Development Josiane Fontaine-Pe´rus

8 Transcriptional Regulation during Somitogenesis Dennis Summerbell and Peter W. J. Rigby

9 Determination and Morphogenesis in Myogenic Progenitor Cells: An Experimental Embryological Approach Charles P. Ordahl, Brian A. Williams, and Wilfred Denetclaw

Volume 49 1 The Centrosome and Parthenogenesis Thomas Ku¨ntziger and Michel Bornens

2 g-Tubulin Berl R. Oakley


199

3 g-Tubulin Complexes and Their Role in Microtubule Nucleation Ruwanthi N. Gunawardane, Sofia B. Lizarraga, Christiane Wiese, Andrew Wilde, and Yixian Zheng

4 g-Tubulin of Budding Yeast Jackie Vogel and Michael Snyder

5 The Spindle Pole Body of Saccharomyces cerevisiae: Architecture and Assembly of the Core Components Susan E. Francis and Trisha N. Davis

6 The Microtubule Organizing Centers of Schizosaccharomyces pombe Iain M. Hagan and Janni Petersen

7 Comparative Structural, Molecular, and Functional Aspects of the Dictyostelium discoideum Centrosome Ralph Gra¨f, Nicole Brusis, Christine Daunderer, Ursula Euteneuer, Andrea Hestermann, Manfred Schliwa, and Masahiro Ueda

8 Are There Nucleic Acids in the Centrosome? Wallace F. Marshall and Joel L. Rosenbaum

9 Basal Bodies and Centrioles: Their Function and Structure Andrea M. Preble, Thomas M. Giddings, Jr., and Susan K. Dutcher

10 Centriole Duplication and Maturation in Animal Cells B. M. H. Lange, A. J. Faragher, P. March, and K. Gull

11 Centrosome Replication in Somatic Cells: The Significance of the G1 Phase Ron Balczon

12 The Coordination of Centrosome Reproduction with Nuclear Events during the Cell Cycle Greenfield Sluder and Edward H. Hinchcliffe

13 Regulating Centrosomes by Protein Phosphorylation Andrew M. Fry, Thibault Mayor, and Erich A. Nigg

14 The Role of the Centrosome in the Development of Malignant Tumors Wilma L. Lingle and Jeffrey L. Salisbury

15 The Centrosome-Associated Aurora/lpl-like Kinase Family T. M. Goepfert and B. R. Brinkley

200


16 Centrosome Reduction during Mammalian Spermiogenesis G. Manandhar, C. Simerly, and G. Schatten

17 The Centrosome of the Early C. elegans Embryo: Inheritance, Assembly, Replication, and Developmental Roles Kevin F. O’Connell

18 The Centrosome in Drosophila Oocyte Development Timothy L. Megraw and Thomas C. Kaufman

19 The Centrosome in Early Drosophila Embryogenesis W. F. Rothwell and W. Sullivan

20 Centrosome Maturation Robert E. Palazzo, Jacalyn M. Vogel, Bradley J. Schnackenberg, Dawn R. Hull, and Xingyong Wu

Volume 50 1 Patterning the Early Sea Urchin Embryo Charles A. Ettensohn and Hyla C. Sweet

2 Turning Mesoderm into Blood: The Formation of Hematopoietic Stem Cells during Embryogenesis Alan J. Davidson and Leonard I. Zon

3 Mechanisms of Plant Embryo Development Shunong Bai, Lingjing Chen, Mary Alice Yund, and Zinmay Rence Sung

4 Sperm-Mediated Gene Transfer Anthony W. S. Chan, C. Marc Luetjens, and Gerald P. Schatten

5 Gonocyte–Sertoli Cell Interactions during Development of the Neonatal Rodent Testis Joanne M. Orth, William F. Jester, Ling-Hong Li, and Andrew L. Laslett

6 Attributes and Dynamics of the Endoplasmic Reticulum in Mammalian Eggs Douglas Kline

7 Germ Plasm and Molecular Determinants of Germ Cell Fate Douglas W. Houston and Mary Lou King


201

Volume 51 1 Patterning and Lineage Specification in the Amphibian Embryo Agnes P. Chan and Laurence D. Etkin

2 Transcriptional Programs Regulating Vascular Smooth Muscle Cell Development and Differentiation Michael S. Parmacek

3 Myofibroblasts: Molecular Crossdressers Gennyne A. Walker, Ivan A. Guerrero, and Leslie A. Leinwand

4 Checkpoint and DNA-Repair Proteins Are Associated with the Cores of Mammalian Meiotic Chromosomes Madalena Tarsounas and Peter B. Moens

5 Cytoskeletal and Ca21 Regulation of Hyphal Tip Growth and Initiation Sara Torralba and I. Brent Heath

6 Pattern Formation during C. elegans Vulval Induction Minqin Wang and Paul W. Sternberg

7 A Molecular Clock Involved in Somite Segmentation Miguel Maroto and Olivier Pourquie´

Volume 52 1 Mechanism and Control of Meiotic Recombination Initiation Scott Keeney

2 Osmoregulation and Cell Volume Regulation in the Preimplantation Embryo Jay M. Baltz

3 Cell–Cell Interactions in Vascular Development Diane C. Darland and Patricia A. D’Amore

4 Genetic Regulation of Preimplantation Embryo Survival Carol M. Warner and Carol A. Brenner

202


Volume 53 1 Developmental Roles and Clinical Significance of Hedgehog Signaling Andrew P. McMahon, Philip W. Ingham, and Clifford J. Tabin

2 Genomic Imprinting: Could the Chromatin Structure Be the Driving Force? Andras Paldi

3 Ontogeny of Hematopoiesis: Examining the Emergence of Hematopoietic Cells in the Vertebrate Embryo Jenna L. Galloway and Leonard I. Zon

4 Patterning the Sea Urchin Embryo: Gene Regulatory Networks, Signaling Pathways, and Cellular Interactions Lynne M. Angerer and Robert C. Angerer

Volume 54 1 Membrane Type-Matrix Metalloproteinases (MT-MMP) Stanley Zucker, Duanqing Pei, Jian Cao, and Carlos Lopez-Otin

2 Surface Association of Secreted Matrix Metalloproteinases Rafael Fridman

3 Biochemical Properties and Functions of Membrane-Anchored Metalloprotease-Disintegrin Proteins (ADAMs) J. David Becherer and Carl P. Blobel

4 Shedding of Plasma Membrane Proteins Joaquıń Arribas and Anna Merlos-Sua´rez

5 Expression of Meprins in Health and Disease Lourdes P. Norman, Gail L. Matters, Jacqueline M. Crisman, and Judith S. Bond

6 Type II Transmembrane Serine Proteases Qingyu Wu

7 DPPIV, Seprase, and Related Serine Peptidases in Multiple Cellular Functions Wen-Tien Chen, Thomas Kelly, and Giulio Ghersi


203

8 The Secretases of Alzheimer’s Disease Michael S. Wolfe

9 Plasminogen Activation at the Cell Surface Vincent Ellis

10 Cell-Surface Cathepsin B: Understanding Its Functional Significance Dora Cavallo-Medved and Bonnie F. Sloane

11 Protease-Activated Receptors Wadie F. Bahou

12 Emmprin (CD147), a Cell Surface Regulator of Matrix Metalloproteinase Production and Function Bryan P. Toole

13 The Evolving Roles of Cell Surface Proteases in Health and Disease: Implications for Developmental, Adaptive, Inflammatory, and Neoplastic Processes Joseph A. Madri

14 Shed Membrane Vesicles and Clustering of Membrane-Bound Proteolytic Enzymes M. Letizia Vittorelli

Volume 55 1 The Dynamics of Chromosome Replication in Yeast Isabelle A. Lucas and M. K. Raghuraman

2 Micromechanical Studies of Mitotic Chromosomes M. G. Poirier and John F. Marko

3 Patterning of the Zebrafish Embyro by Nodal Signals Jennifer O. Liang and Amy L. Rubinstein

4 Folding Chromosomes in Bacteria: Examining the Role of Csp Proteins and Other Small Nucleic Acid-Binding Proteins Nancy Trun and Danielle Johnston

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