INTERNATIONAL REVIEW OF CYTOLOGY V130, Volume 130

VOLUME 130

SERIES EDITORS Geoffrey H. Bourne James F. Danielli Kwang W. Jeon Martin Friedlander

1949-1 988 1949-1 984 19671984-

ADVl SORY EDITORS Howard A. Bern Dean Bok Stanley Cohen Rene Couteaux Marie A. DiBerardino Donald K. Dougall Charles J. Flickinger Nicholas Gillham M. Nelly Golarz De Bourne Elizabeth D. Hay Mark Hogarth H. R. Kaback Keith E. Mostov Audrey Muggleton-Harris

Andreas Oksche Muriel J. Ord Valdimir R. Pantic M. V. Parthasarathy Lionel I. Rebhun Jean-Paul Revel L. Evans Roth Jozef St. Schell Hiroh Shibaoka Wilfred Stein Ralph M. Steinrnan D. L. Taylor M. Tazawa Alexander L. Yudin

Edited by Kwang W. Jeon Department of Zoology The University of Tennessee Knoxville, Tennessee

Martin Friedlander

Jules Stein Eye Institute UCLA School of Medicine Los Angeles, California

VOLUME 130

W Academic Press, Inc. Harcourt Brace Jovanovich, Publishers San Diego New York Boston London

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This book is printed on acid-free paper. @ Copyright 0 1991 BY ACADEMIC PRESS, 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.

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Library of Congress Catalog Card Number: 52-5203 ISBN 0-12-364530-1

(alk. paper)

PRINTED IN THE UNITED STATES OFAMERICA 91929394 9 8 7 6 5 4 3 2 1

CONTENTS

Contributors .............................................................................................................

ix

Immunoglobulin Transport in B Cell Development Shiv Pillai Introduction .................................................................................................... Overview of B Lymphocyte Ontogeny .......................................................... lntracellular Retention of Secretory Immunoglobulins .............................. Membrane Immunoglobulin Transport during B Cell Ontogeny ............... Immunoglobulin Secretion in Plasma Cells ................................................

I. II. 111. IV. V. VI. Summary: Choices between RetentionlDegradation and Transport of Immunoglobulins Are Dictated by Function ................................................ References ......................................................................................................

1 2 13 20 29

31 34

The Cytoskeletal System of Nucleated Erythrocytes William D. Cohen Introduction .................................................................................................... Nucleated Erythrocytes: A Phylogenetic and Physiological Portrait ........ The Marginal Band of Mature Erythrocytes ................................................. Marginal Band Biogenesis and Function during Erythrocyte Morphogenesis ............................................................................................... V. The Membrane Skeleton ...............................................................................

1. II. 111. IV.

37 39 43 59 67 V

Vi

CONTENTS

VI . Intermediate Filaments .................................................................................. VII. The Cytoskeletal System of Mammalian Primitive Erythrocytes ................ VIII. Concluding Remarks ..................................................................................... References ......................................................................................................

Structure of the Mouse Egg Extracellular Coat. the Zona Pellucida Paul M. Wassarman and Steven Mortillo I. Introduction .................................................................................................... II. 111. IV . V.

Functions of the Zona Pellucida ................................................................... Characteristics of the Zona Pellucida .......................................................... Ultrastructure of the Zona Pellucida ............................................................ Concluding Remarks ..................................................................................... References ......................................................................................................

75 76 79 80

85 86 89 96 105 108

The Male Germ Cell Protective Barrier along Phylogenesis Mordechai Abraham I. Introduction .................................................................................................... II . Background .................................................................................................... 111 . IV. V. VI .

The Male Germ Cell Protective Barrier along Phylogenesis ...................... Structure of the Male Germ Cell Protective Barrier .................................... Function of the Male Germ Cell Protective Barrier ..................................... Concluding Remarks ..................................................................................... References ......................................................................................................

111 112 119 143 159 173 177

Mitochondria-Rich Cells in the Gill Epithelium of Teleost Fishes: An Ultrastructural Approach M. Pisam and A . Rambourg I. II. 111. IV.

Introduction .................................................................................................... Gill Morphology ............................................................................................. General Ultrastructural Features of Chloride Cells ..................................... Mitochondria-Rich Cells and Modifications of the Environment ..............

191 192 193 204

CONTENTS

V . Mitochondria-Rich Cells and Smoltification in Salmonids ........................ VI . Hormones and Chloride Cells ....................................................................... VII . Concluding Remarks ..................................................................................... References ......................................................................................................

Vii

221 225 227 228

Structure and Function of Plant Cell Walls: Immunological Approaches Takayuki Hoson I. II. Ill. IV . V. VI . VII.

Introduction .................................................................................................... Antibodies as Probes for the Study of Plant Cell Walls .............................. Location and Metabolism of Cell Wall Polymers ........................................ Growth Regulation ......................................................................................... Selective Breakdown of Plant: Cell Walls ..................................................... Other Aspects of Plant Cell Walls ................................................................. Conclusions and Future Prospects .............................................................. References ......................................................................................................

233 234 240 246 254 259 261 263

Biologically Localized Firefly Luciferase: A Tool t o Study Cellular Processes Claude Aflalo Introduction .................................................................................................... Cellular Biogenesis and Organization ......................................................... Firefly Luciferase: An Overview .................................................................... A Local Probe for ATP in Model Systems .................................................... Leading Luciferase into Cellular Compartments ........................................ Light Emission by Luciferase in Biological Systems .................................. Experimental Approaches and Perspectives ............................................... References ......................................................................................................

269 270 280 287 296 300 309 318

Index .........................................................................................................................

325

I. II. 111. IV. V. VI . VII .

This Page Intentionally Left Blank

CONTRIBUTORS

Numbers in parentheses indicate the pages on which the author’s contributions begin.

Mordechai Abraham (1 1 l ) , Department of Zoology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel Claude Aflalo (269),Department of Biochemistry, The Weizmann Institute of Science, Rehovot 76100, Israel William D. Cohen (37),Department of Biological Sciences, Hunter College of CUNY, New York, New York 10021; and The Marine Biological Laboratory, Woods Hole, Massachusetts 02543 Takayu ki Hoson (233),Department of Biology, Faculty of Science, Osaka City University, Osaka 558, Japan Steven Mortillo (85),Department of Cell and Developmental Biology, Roche lnstitute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110 Shiv Pillai (l), Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02129 M. Pisam (191),Service de Biologie Cellulaire, Departement de Biologie Cellulaire et Moleculaire, Centre d ’ h d e s Nucleaires de Saclay, 91 191 Gif-sur-Yvette Cedex, France

ix

X

CONTRIBUTORS

A. Rambourg (191), Service de Biologie Cellulaire, Departement de Biologie Cellulaire et Moleculaire, Centre d’Etudes Nucleaires de Saclay, 91 191 Gif-sur-Yvette Cedex, France

Paul M. Wassarman (85), Department ofCell andtDevelopmentalBiology, Roche institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 071 10

INTERNATIONAL REVIEW OF CYTOLOGY. VOL. 130

Immunoglobulin Transport in B Cell Development SHIVPILLAI Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02129

I. Introduction The B lymphocyte differentiation pathway is one of the best-studied models of eukaryotic differentiation. While B cells are of obvious interest to students of immunology, the study of these cells has also yielded numerous insights into cellular processes that are common to many other cell types in higher eukaryotes. Examples of such processes include tissue-specific transcriptional regulation, alternative splicing to yield mRNAs for membrane and secretory proteins, and differential polyadenylation of genes during development. There are a number of extremely interesting cellular and molecular processes unique to immune cells, which make the study of B lymphocytes particularly exciting. In pre-B lymphocytes, unusual molecular events include the rearrangement of immunoglobulin genes and the processes of allelic and isotypic exclusion [which ensure that a given B cell expresses only one rearranged heavy- and one light-chain (H- and L-chain, respectively) allele]. In early B cells, exposure to antigen may lead to clonal deletion, one of the mechanisms by which self-non-self discrimination is achieved. The exposure of slightly more differentiated B cells to antigen leads to the activation of a number of molecular events, including two that are unique to the B lineage: A rearrangement process at the H-chain locus leads to isotype switching, and the process of somatic mutation of rearranged immunoglobulin genes further diversifies the immune repertoire. The transport of immunoglobulin molecules is exquisitely regulated during B lymphocyte differentiation and closely parallels the vastly different functions subserved by membrane and secretory immunoglobulins during B cell ontogeny. The study of the processes by which immunoglobulin transport is regulated, apart from yielding useful insights into protein trafficking in general, also provides a paradigm for the regulation of cellular differentiation at the level of protein transport. In this chapter I begin by providing an overview of the process of B cell differentiation, highlighting features of the pathway which are particularly relevant from the viewpoint of immunoglobulin transport. I then describe the regulation of trans1

Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

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SHIV PILLAI

port during the process of differentiation in some detail, discussing at each step mechanisms of broad relevance to protein trafficking and cell biology in general. 11. Overview of B Lymphocyte Ontogeny

The goal of B lymphocyte differentiation is to generate a wide range of antigen-responsive cells which will respond to antigen by secreting specific antibodies. Each B cell clone must express a single cell surface antigen receptor in order to maintain clonal specificity; self-reactive B cell clones must be either eliminated or rendered anergic. Immunoglobulin H-chain proteins are synthesized in two forms, the membrane (m) and secretory (s) forms, which are products of alternative splicing at the H-chain locus (reviewed by Wall and Kuehl, 1983). A schematic view of the p H-chain locus is provided in Fig. 1. Many hundred variable region (V) immunoglobulin genes are separated by a large distance from a small group of diversity (D) genes. Downstream of the D cluster is a small set of joining (J) genes separated by the J-C intron from the first exon of the constant region (C) of the p H-chain gene. In the case of the p chain (the other isotypes downstream of p have broadly similar structural features), the C region is made up of four common exons, respectively encoding the c p l to cp4 domains. Two membrane exons, pMl and pM2, encode a total of 42 amino acids, which include 13 amino acids that form an extracellular negatively charged region, 26 amino acids that form a relatively hydrophilic membrane-spanning domain (nine of the 26 amino acids are serines and threonines), and three amino acids that constitute a cytoplasmic tail. Two polyadenylation sites, one immediately downstream of the cp4 exon and the other downstream of the pM2 exon, permit the generation of two mRNAs from the p gene (similar polyadenylation and splice sites characterize every H-chain isotype). A splice donor site in the coding region of the cp4 domain leads to the generation of an alternatively spliced version of the p gene which includes the two membrane exons and encodes the membrane form of the immunoglobulin H chain. The secretory form of the p H chain (ps) includes a tailpiece distal to the splice site in the cp4 exon, but excludes the relatively hydrophobic pMl and pM2 exons. An extremely simplified view of B cell differentiation is presented in Fig. 2. In this scheme the ontogenic process is considered to be made up of three stages, each having a well-defined and -demarcated function; the immunoglobulin molecule at each of these broadly defined stages subserves a distinct function and is transported to a specific subcellular loca-

3

IMMUNOGLOBULIN TRANSPORT

V Genes 100-1000

D Cluster 5 10 genes

-

-

J C lntron

Constant Region

p m Messenger RNA

s Messenger RNA

FIG.1. A schematic view of the p heavy-chain locus and its products after rearrangement and splicing. V, Variable region; D, diversity region; J, joining region; L, leader exon.

tion in order to achieve this function. In very general terms, the pre-B lymphocyte represents the period of differentiation at which the stage is set for the synthesis of immunoglobulins; stepwise rearrangement of immunoglobulin genes leads to the generation of cells which first express intracellular p H chains. Intracellular membrane immunoglobulin at this stage plays a feedback role in differentiation, a role that is expanded on later in this chapter. The B cell stage corresponds to the antigenresponsive portion of ontogeny ; membrane immunoglobulin is expressed on the cell surface, where it functions as the antigen receptor. Antigenactivated B cells differentiate into plasma cells, whose sole function is to

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Pre-B lntraceliular Pm lntracellular Ps

B Cell surface

Plasma cell

Prn 2 L2

Secretion of

lntracellular Ps 2 L2 No J chain synthesis No sulfydryl oxidase

pentamers

FIG. 2. A highly simplified view of the process of B cell differentiation.

secrete antibody, thus serving as the effector end cells of the humoral immune system. In the following subsections a more detailed description of all the defined cellular stages in B cell ontogeny is provided. These stages are schematically represented in Fig. 3. A. PRO-BLYMPHOCYTES

The sites of B lymphocyte generation are the spleen and the liver before birth and the bone marrow in adult life. The earliest identifiable precursor of a B cell is a pro-B lymphocyte; a pro-B cell is committed to the B lineage, but has not yet undergone any rearrangements at the H-chain locus. A number of genes of functional significance in the B lineage are known to be expressed at this stage. These include the A5 (Sakaguchi and Melchers, 1986) and uPreB (Kudo and Melchers, 1987) genes, which encode the o and L surrogate immunoglobulin L chains; the RAG-I and RAG-2 genes (Schatz et af.,1989; Oettinger et d.,1990), which encode the V-D-J recombinase; and the rnb-1 (Sakaguchi et al., 1988) and B29 (Her-

IMMUNOGLOBULIN TRANSPORT Pro - B no rearrangement

5

-

Pre B lntracellular

pIwl1

FIG. 3. A detailed view of B lymphocyte ontogeny.

manson et al., 1988) genes, which encode presumed signal transduction/ transport-relatedproteins that associate with membrane immunoglobulin. Rearrangement of immunoglobulin genes in pre-B cells depends on the activity of a site-specific V-D-J recombinase, which is presumably the product of the pre-B and pre-T-specific RAG-I and RAG-2 genes, and other stage-specific DN A-binding factors that bind to specific regulatory

6

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motifs in immunoglobulin genes. These DNA-binding proteins are transacting factors that selectively “open” specific immunoglobulin loci, making these regions accessible to the V-D-J recombinase, which, in turn, recognizes the conserved heptamer-nonamer signals for rearrangement (reviewed by Yancopoulos and Alt, 1986). B. PRE-BLYMPHOCYTES While pro-B lymphocytes are operationally defined as cells committed to the B lineage which have yet to rearrange their immunoglobulin genes, pre-B cells are B cell precursors in the process of rearranging their immunoglobulin genes. Early pre-B cells are generally believed to be large bone marrow pre-B cells actively rearranging their H-chain loci. Late pre-B cells are smaller cells which are either primed to rearrange, or are in the process of rearranging, their L-chain genes. The first rearrangement event that occurs in pre-B cells is a D-J rearrangement involving the H-chain locus. It has been demonstrated that D-J rearrangements may give rise to very unusual D p transcripts; these transcripts initiate upstream of the rearranged D segment and include a conserved ATG codon which is in frame with the coding exons of the p gene (Reth and Alt, 1984). Amazingly, the transcript includes, downstream of the ATG codon, a set of codons derived from the recombinational signal sequences immediately upstream of the D segment, which constitute an upstream hydrophobic leader peptide. Translation of the D p transcript leads to synthesis of the D p protein, which is translocated into the lumen of the endoplasmic reticulum (ER). It has been suggested that high-level expression of the D p protein may serve as a signal for further differentiation of pre-B lymphocytes. Preliminary studies suggest that both Dpm and Dps proteins are synthesized and that Dpm is not transported to the cell surface (S. Pillai, unpublished observations), suggesting that a signal for further differentiation may be generated from an intracellular location. It is unclear whether the generation of a Dp protein-containing cell is an obligate intermediate stage in the process of B cell differentiation, and no direct evidence exists to either confirm or refute any model which invokes the need for synthesis of a Dp protein.’ The next step in pre-B lymphocyte differentiation involves the rearrangement, again at the H-chain locus, of a V segment to the D segment that has undergone a previous rearrangement, to give rise to a D-J structure. There is a one-in-three chance of any V-to-D-J rearrangement giving rise to an in-frame rearranged H-chain immunoglobulin gene. During the joining process the addition of a small number of bases (N regions) or the I

See Note Added in Proof, p. 36.

IMMUNOGLOBULIN TRANSPORT

7

removal of a few bases contributes to junctional diversity and amplifies the creation of the immune repertoire. It is important from the viewpoint of the immune system that any given B cell clone express only a single receptor in order to maintain clonal specificity in the immune system. In any given B cell, there is the theoretical possibility of in-frame rearrangements of the H-chain gene on both maternally and paternally derived chromosomes, giving rise to two nonidentical H-chain proteins. It is now well appreciated that a productive H-chain gene rearrangement that leads to the synthesis of an intracellular p protein initiates a feedback regulation process (reviewed by Alt et al., 1986). This process is capable of shutting off H-chain gene rearrangement on the other allele and also provides a positive signal, permitting further differentiation to the stage of L-chain gene rearrangement. The feedback regulation process is of direct relevance to studies of the transport of immunoglobulins in pre-B cells. It is now clear that the signal for the feedback regulation of differentiation in pre-B cells is dependent on the synthesis of pm, not ps (Nussenzweig et al., 1987; Reth et al., 1987). At this stage of differentiation, pm is associated with the w and L surrogate immunoglobulin L chains, which are the products of the pre-B-specific A5 and vPreB genes, respectively. Important issues related to immunoglobulin transport at this stage include the localization of pm and the site from which a differentiation signal is generated by this protein. C. PRE-B-SPECIFIC SURROGATE IMMUNOGLOBULIN L CHAINS Pre-B cells have long been described as the stage of B cell ontogeny in which cells contain “free” cytoplasmic H chains. It is now well established that in pre-B cells at least a proportion of the intracellular immunoglobulin H chain is not free, but is complexed with the w and L surrogate immunoglobulin L chains. The w surrogate L chain is a 20-kDa protein that is pre-B-specific and forms disulfide-linked tetramers with the p chain (Pillai and Baltimore, 1987a; Cherayil and Pillai, 1991a). The L chain is a 15-kDa protein that is noncovalently associated with the p2-0~2complex and is also pre-B specific (Pillai and Baltimore, 1988). The w protein was not found to be associated with the D p protein in pre-B cell lines, and was not associated with p in a fibroblast cell line expressing a transfected pm gene nor in a T cell line expressing a transfected p gene. In a subset of pre-B cell lines (which we refer to as Type I1 pre-B cell lines, to distinguish them from Type I pre-B cell lines in which pm is only detectable as an intracellular protein), pm is transported to the cell surface (Findley et al., 1982; Gordon etal., 1981; Hardy et al., 1986; Hendershot and Levitt, 1984; Paige et al., 1981). In these lines, cell surface pm was found to be associated with w and L (Pillai and Baltimore, 1987a; 1988) or equivalent human

8

SHIV PILLAI

proteins (Hollis et al., 1989; Kerr et al., 1989). Indeed, in a pre-B cell line, 70213, in which pm can be induced to migrate to the cell surface in the absence of K L chain gene transcription (Paige et al., 1981), it does so in association with w and L ( S . Pillai, unpublished observations). The w chain is the product of the pre-B-specific A5 gene, and the L chain is the product of another pre-B-specific gene, the uPreB gene (Cherayil and Pillai, 1991a; Karasuyama et al., 1990; Kudo and Melchers, 1987; Sakaguchi and Melchers, 1986; Tsubata and Reth, 1990). Similar chains have more recently been described in human pre-B cell lines. The pre-B-specific human homolog of the A5 gene is the 14.1116.1 gene (Chang et al., 1986). The A5 and 14.1/16.1genes are structurally similar to conventional L-chain genes over the 3’ halves of their coding regions. The predicted structure of these genes, which respectively encode the mouse and human w surrogate L-chain proteins, includes a signal peptide followed by an amino-terminal segment which has no homology to any known protein. This is followed by an immunoglobulin J domain-like segment, and finally a domain with a structural resemblance to Ch and CK L-chain domains. A preterminal cysteine residue analogous to the preterminal cysteine in conventional L chains is disulfide linked to the free cysteine in the CHI domain. The sequence of the uPreB gene predicts a V domain-like structure preceded by a signal peptide. A likely but unproven model for the association of the p H chain with surrogate L-chain proteins is depicted in Fig. 4. It is assumed that the dvPreB protein associates with the VH domain of

FIG.4. Presumed structure of the heavy-surrogate light-chain complex.

IMMUNOGLOBULIN TRANSPORT

9

the H chain and that the carboxy-terminal half of the w/h5 protein associates with the CH1 domain, with an interchain disulfide bridge ensuring covalent association. We assume that these proteins associate with p m in pre-B cells in order to participate in the generation of a feedback signal, presumably through the six polypeptides (Pillai and Baltimore, 1988)of the pm activation complex. There are three related models for the function of the o protein in this process. The first model is the solubility/stability model, which assumes that the surrogate L chains serve a purely physical L-chain-like purpose, which is to prevent H chains from aggregating or being in an “inappropriate” conformation; an “appropriate” conformation may be necessary for movement or for engaging the proteins of the pm activation complex in order for a signal transduction event to occur. This model assumes that if, as part of the feedback process, pm needs to be transported to the cell surface or to some intracellular compartment, it cannot attain competence for transport unless ass6ciated with either a conventional L chain or surrogate L chains. The second model is the intracellular ligand/crosslinking mode. In this model, feedback regulation depends on the generation of a signal from an intracellular location, and w cannot be functionally replaced by a conventional L chain. The intracellular site of signal generation is presumed to be the ER itself or the cis-Golgi compartment. In this model, the association of surrogate L-chain proteins with p m in an intracellular location is an absolute prerequisite for both engaging and “turning on” the pm activation complex. The third model for feedback regulation is the cell-surface activation model. This model suggests the existence of a physiological differentiation stage in which, prior to rearrangement of the K locus, the pm-surrogate L-chain complex is transported to the cell surface. In such a model, an extracellular ligand may engage this pm-receptor complex and lead to the generation of a differentiation signal. In theory, such a signal, if it were to activate NFKB(Sen and Baltimore, 1986), could lead to the transcriptional activation of the K locus -and rearrangement of the K gene, leading to the next stage of differentiation. The latter two models are schematically depicted in Fig. 5. The genes for surrogate L-chain proteins are transcribed in pro-B cells prior to the onset of H-chain gene rearrangement. It is unclear whether the w/h5 protein has any function in pro-B cells. It has been suggested that w and L are coassociated in pro-B cells (Misener et al., 1990). In our studies of the w/X5 protein, we have observed a number of protein species coassociated with the w protein in a pro-B cell line (Cherayil and Pillai, 1991b). It is unclear whether any of these proteins are candidate intracellular ligands for the pw receptor which appears later in ontogeny. The major w species

10

SHIV PILLAI

I

A

I

2nd

B

FIG. 5. Models for differentiationsignal generationlfeedback regulation in pre-B lymphocytes. (A) Intracellular ligand crosslinking model. (B) Extracellular ligand activation model.

in pro-B cells is an 02 dimer. This dimer, however, is not secreted (Cherayil and Pillai , 1991b) , It appears likely that the association of p with w and L can occur only as a trimolecular complex (Karasuyama et al., 1990; Tsubata and Reth, 1990; Cherayil and Pillai, 1991a)The D p protein contains the CH1 domain of the p H chain, but lacks a V domain. The presence of the CH1 domain may have been predicted to be sufficient to permit association with the wlh5 protein; however, neither form of Dp protein is coprecipitated with w (Pillai and Baltimore, 1987),presumably because association with vPreB/L (which would require the presence of a V domain) is a necessary prerequisite for p--0 association.* TRANSITIONAL AND IMMATURE D. PRE-BTO B CELLTRANSITION: B CELLSTAGES At least three molecular events related to immunoglobulin genes and proteins characterize the pre-B to B cell transition. At this transition, cells

* See Note Added in Proof, p. 36.

IMMUNOGLOBULIN TRANSPORT

11

acquire the ability to transport membrane immunoglobulin to the cell surface, they constitutively activate transcription of the K L-chain gene, and they shut off transcription of genes encoding surrogate L-chain proteins. The transition of a pre-B cell to the antigen-responsive B stage is an antigen-independent event. The earliest B cell to emerge from the pre-B compartment in the bone marrow is probably a recently defined transitional B cell (Cherayil and Pillai, 1991a). Transitional B cells simultaneously express p--w and p-K cell surface receptors. Cell lines have been derived that represent this transitional B stage which simultaneously express the N and K genes (McGarigle et al., 1991). At the pre-B to B cell transition, the constitutive activation of NFKB and K gene transcription clearly precedes the shut-off of N gene expression. The next stage of B cell differentiation is the immature B cell stage at which cells express p-K or p-A receptors and have permanently shut off surrogate L-chain gene expression. It is presumed that at the immature B stage, cells respond negatively to ligation of their receptors by antigen and die. This is one of the two major mechanisms by which B cells are rendered tolerant to self antigens (reviewed by Goodnow et al., 1990).

E. MATUREIGM/IGD-EXPRESSING B LYMPHOCYTES The next stage of B cell differentiation is the surface immunoglobulin M/immunoglobulin D (IgM/IgD) positive mature B stage. Differentiation until this stage is antigen independent. If cells at the two previous transitional and immature B stages are exposed to antigen, they are presumed to respond negatively and may thus be eliminated. At the mature B IgM/IgD stage, cells respond positively when exposed to antigens by proliferating and undergoing further differentiation. The frequently used term “virgin B cell” covers surface immunoglobulin B cell stages prior to activation by antigen; it includes transitional, immature, and early B cells. There are three aspects to the function of membrane immunoglobulin at the mature B cell stage. Polyvalent antigens such as polysaccharide antigens, which are “T independent” (and can produce biological responses in the absence of T cell help), directly engage membrane immunoglobulin and transduce a signal which leads to B cell activation. Although, in the case of “T-dependent” protein antigens, membrane immunoglobulin may still play a signal transductional role, its major function is to internalize the cognate antigen so that the latter may be processed in an acidic endosomal compartment into peptide fragments; appropriate fragments associate with the cleft of the class I1 histocompatibility molecule and are presented on the cell surface to a cognate helper T lymphocyte. This T cell, in turn, secretes the appropriate lymphokines, such as interleukins 4 and 5, which

12

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drive further differentiation of the concerned B lymphocyte clone. From the standpoint of protein trafficking and immunoglobulin function, membrane immunoglobulin in mature B cells must therefore be part of a signaltransducing protein complex that is transported to the cell surface and must also function as an endocytic receptor. An interesting third aspect to the intracellular transport of membrane immunoglobulin at this stage of differentiation has recently emerged. The exposure of mature B cells to a cognate antigen in the absence of appropriate T cell help can lead to the down-regulation of membrane IgM, but not membrane IgD, on these cells. This is presumed to be one of the mechanisms by which clonal anergy to self antigens may be perpetuated. The actual function of membrane IgD remains unknown. IgD is not expressed at any subsequent stage of B cell ontogeny after the mature B cell stage. F. ACTIVATED AND MEMORY B LYMPHOCYTES

After mature B cells are activated by protein antigens, two unusual events involving rearranged immunoglobulin genes contribute to increased recognitional as well as functional diversity in the B lineage. Somatic mutation is a process by which rearranged immunoglobulin V segments in activated B cells are further diversified by a unique and poorly understood mutational mechanism. Somatic mutation contributes to greatly increased immune diversity at the antigen recognition level. Isotype switching, which involves a somatic deletional recombination event, leads to the generation of activated B cells with unchanged antigen specificities, but the "new" C regions, and contributes to the production of IgG, IgA, and IgE antibodies (reviewed by Rajewsky et al., 1987). Secretion of antibody, from the immunological viewpoint, should be a consequence of exposure to antigen and should not occur in early nonactivated B cells. An additional regulatory event that characterizes the activated B stage is acquisition of the ability to secrete antibody. Until and including the mature B stage, secretory IgM is synthesized but is held back intracellularly . After activation, IgM is no longer sequestered intracellularly and is secreted; for other isotypes, all of which appear in ontogeny only subsequent to activation by antigen, holdback mechanisms are irrelevant. Activated B cells may differentiate to give rise to either long-lived memory B cells or antibody-secreting plasma cells. Memory B cells may be defined as long-lived activated B cells whose life spans are measured in years instead of days. Exposure to antigen leads to proliferation and the secretion of antibody by memory B cells, which may also divide asymmetrically to give rise to plasma cells.

IMMUNOGLOBULIN TRANSPORT

13

G. PLASMACELLS:ENDSTAGEOF B LYMPHOID ONTOGENY Plasma cells are the end-stage cells of B cell ontogeny and are basically designed to be antibody-secreting factories. These cells have numerous ribosomes and extremely abundant reticular ER and Golgi compartments. Largely by posttranscriptional mechanisms, they achieve high secretoryto-membrane immunoglobulin H-chain RNA ratios; the mRNA level of secretory immunoglobulin is also regulated at the level of increased message stability (Mason el al., 1988), possibly because it is efficiently translated and is protected from degradation by virtue of association with ribosomes. These cells express no cell surface class I1 molecules and also do not express membrane immunoglobulin.

111. Intracellular Retention of Secretory Immunoglobulins From a purely teleological viewpoint, immunoglobulins should not be secreted at all stages of B cell ontogeny prior to antigen exposure. The only isotypes that are expressed by B lymphocytes prior to activation by antigen are the p and 6 isotypes. Any mechanism that prevents the secretion of immunoglobulins prior to exposure to antigen should therefore apply to these isotypes. The question of the retention of secretory IgD is a moot one; although rare IgD-producing myelomas do exist, at the mature B stage, when IgD is expressed on the cell surface along with IgM, RNAprocessing mechanisms lead to the generation of pm and ps transcripts, as well as two 6m transcripts, but no 6s transcripts. When mature B cells are activated, further differentiation leads to shut-off of the mechanisms which generate 6 transcripts. Pre-B cells are unusual from a viewpoint which has interesting implications for studying the assembly and transport of multisubunit oligomers. Immune receptors, unlike other receptors made up of more than one subunit, are synthesized in stages; the immunoglobulin H chain is synthesized prior to the L chain, and the T cell receptor p chain is synthesized before the a chain. The process of making coding region joints during immunoglobulin gene rearrangement is designed to be imprecise in order to increase junctional diversity and thus to maximize the immune repertoire. However, the efficiency of this process depends on the sequential rearrangement and expression of H-chain and L-chain genes and the stepwise selection of cells that make in-frame rearrangements. Pre-B cells, as a result, constitute natural developmental models for studying the assembly, transport, and degradation of incomplete oligomeric proteins. In bone marrow pre-B cells, alternative splicing gives rise largely to pm mRNA, and negligible amounts of ps may actually be synthesized by these

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cells in uiuo (Thorens et al., 1985). However, most pre-B cell lines synthesize roughly equivalent amounts of pm and ps, at the message as well as at the protein level. ps synthesized by pre-B cell lines is not secreted. There are two putative retention mechanisms that might serve to hold secretory immunoglobulins intracellularly . One potential mechanism applies to any immunoglobulin isotype, is not B lineage specific, and involves the association of free secretory H chains with binding protein (Bip), an abundant ER protein; this mechanism presumably involves the association of Bip with a CH1 domain. The second potential mechanism is specific for the p isotype, is developmentally regulated, and applies equally well to tetramers of ps with conventional or surrogate L chains as it does to free ps. This second mechanism presumably involves retention via a cysteine tailpiece and may not really be distinct in principle from the CH1dependent retention process. A. INTRACELLULAR RETENTION OF H CHAINAND BIP IN PRE-B AND THE SALVAGE PATHWAY CELLS:KDEL SEQUENCES A H-chain binding protein was originally described by Morrison and Scharff (1975) in a nonsecreting myeloma variant which had lost L-chain expression. This protein was rediscovered by Haas and Wabl (1983) in H-chain-only pre-B cell lines and hybridomas derived from pre-B lines. These latter workers described a doublet of proteins, which were 78 and 70 kDa in size and associated with free H chains, and referred to these proteins as the binding protein. Some of the confusion surrounding Bip was resolved when Munro and Pelham (1986) cloned the gene encoding the 78-kDa Bip and discovered that it was identical to a much-studied member of the heat-shock protein family, GRP78, which is induced by glucose starvation. The 70-kDa protein that was originally considered to be part of the Bip complex is probably a cytosolic heat-shock family protein, hsc 70. Bip/GRP78 was found to be coprecipitated with misfolded mutants of influenza hemagglutinin which were incapable of being transported to the cell surface, and the suggestion was made that Bip may play a role in identifying and sequestering misfolded or improperly assembled proteins in the ER (Gething et al., 1986). Bip/GRP78 has been demonstrated to possess a peptide-dependent ATPase activity (Flynn et al., 1989) and can be dissociated from misfolded proteins by the addition of ATP in vitro (Munro and Pelham, 1987). Bip/GRP78 has also been found to be associated transiently with assembly intermediates of some secreted proteins, and it has been suggested that the function of Bip may actually be to catalyze some step in the folding and assembly of proteins in the ER that are destined for export: It may only remain associated with incomplete

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oligomers or misfolded variants (Kassenbrock et al., 1988). Neither of these two models for the function of Bip has been established, and neither may be correct. Bip constitutes a well-studied example of a “resident” ER luminal protein which has a KDEL retention sequence and is also, like other members of this category of resident ER proteins, a calcium-binding protein. Proteins that possess a KDEL retention signal recycle back and forth between the ER and a pre-Golgi salvage compartment (Lewis et al., 1990). The KDEL receptor binds to proteins such as Bip with a high affinity in the salvage compartment; recycling to the ER occurs rapidly and in this compartment luminal proteins such as Bip have virtually no affinity for the KDEL receptor, which returns “unoccupied” to the salvage compartment. Bip-immunoglobulin H-chain complexes probably cycle back and forth in this manner in pre-B cell lines. The homolog of the Bip gene in Saccharornyces cereuesiae is the KAR2 gene. KAR2 is an essential gene in yeast, is required for nuclear fusion, and is assumed to play some important role, as yet undetermined, in the secretory process (Rose et al., 1989). KAR2 was somewhat unexpectedly found to be required for protein translocation into the ER (Vogel et al., 1990). Such a function would not have been predicted for Bip from studies of mammalian cells. Although it remains clear that Bip/GRP78/KAR2 must play some important role in cell physiology, this function remains to be determined with any certainty. The best-studied, though not necessarily the best-understood, role of Bip may be its putative ability to retain secretory immunoglobulin H chains in an intracellular location in some cell lines. This presumed role in retaining H chains intracellularly is based on the correlation of the association of Bip with nonsecreted H chains. It is equally likely that the association of Bip with H chains is a consequence of a failure of immunoglobulin secretion rather than the cause of retention. The primary basis for suggesting that Bip itself may play a role in the retention of immunoglobulins has come from studies (Bole et al., 1986; Hendershot et al., 1987; Hendershot, 1990) of a number of secreted and nonsecreted immunoglobulin mutants in a range of myeloma lines. In the absence of an L chain, wild-type immunoglobulin H-chain protein of a number of isotypes is retained intracellularly along with Bip. Mutant y immunoglobulins which lack CH 1 domains are secreted in the absence of an L chain. Mutants lacking CH2 or CH3 domains remain associated with Bip and are not secreted. Since L chains associate with the CH1 domain and since L chains displace Bip from nascent H-chain-Bip complexes, it has been proposed that Bip binds to the CH 1 domain of immunoglobulins and serves to retain unassembled H chains which are then degraded intra-

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cellularly . It is equally possible that the association of nonassembled immunoglobulin H chains with Bip is the consequence, rather than the cause, of intracellular retention. A second site for Bip association in the cysteine-containing tailpiece of the CH4 domain of ps has been suggested, in part, to explain the retention of p 2 - ~ 2complexes in early B cells. Since this cysteine-dependent mechanism could presumably be involved in the retention of ps in pre-B cells, is there any physiological role for the Bip/CH 1 domain-dependent retention of free ps? This is probably a moot question, since bone marrow pre-B cells may synthesize relatively minuscule amounts of ps mRNA to begin with. However, in a pre-B cell line which has switched to the y isotype (which lacks the cysteine-containing retention tailpiece found in p ) , y s is retained, intracellularly associated with Bip. Association of y s with a K L-chain protein in a transfected derivative of this cell line leads to the “release” of the H chain from Bip (or merely the assumption of an appropriate conformation by the tetrameric complex) and its quantitative secretion as ys2-~2 tetramers (Bachhawat and Pillai, 1991). In the next subsection, a model is suggested that unifies the cysteine tailpiece retention mechanism with the Bip holdback hypothesis. Needless to say, it remains unresolved as to whether Bip binding is a cause or a consequence of retention. B. CYSTEINE TAILPIECE-DEPENDENT RETENTION OF SECRETORY IMMUNOGLOBULINS The retention of secretory IgM is mechanistically and structurally closely linked to the processes involved in the polymerization of this molecule prior to secretion. Both ps and as H chains contain a tailpiece which contains a cysteine residue. This tailpiece plays an important role in the polymerization of these isotypes and their association with the J chain. Issues that relate to the function of this tailpiece in secretion are dealt with in greater detail in the section on immunoglobulin transport at the plasma cell stage (Section V,A). The relevance of this tailpiece to the intracellular retention of ps in the earlier stages of B cell differentiation is considered here. Numerous studies have indicated that in “virgin” splenic B cells as well as in many B cell lines that represent the early antigen-independent stages of B cell differentiation, tetramers of pm and L chain are transported to the cell surface, while ps, which also forms tetramers with an L chain, is retained intracellularly and degraded (Sibley el af., 1980; Vassali et af., 1980; Sidman, 1981; Dulis, 1983; Rubartelli et af., 1983). Three models have been suggested for the failure of IgM secretion by early B cells.

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17

1. Early B cells lack an important constituent responsible f o r the assembly of polymerized IgM, and the absence of this assembly step results in failure of secretion. Candidates for a missing or limiting low-level constituent at the B cell stage are (a) the J chain (Koshland, 1985) and (b) the IgM polymerase, a sulfhydryl oxidase (Roth and Koshland, 1981). The role of the J chain and the IgM polymerase is expanded on in the section on transport in plasma cells (Section V,A). However, the J chain is not essential for polymerization of IgM. IgM is polymerized and secreted in the absence of J chain by transfected glioma and pheochromocytoma cells (Cattaneo and Neuberger, 1987). The absence of polymerization and assembly processes in earlier B cell stages may well be tightly linked to the tailpiece-dependent retention process discussed later in this section. However, polymerization is not an absolute prerequisite for secretion; IgA is certainly secreted as monomers as well as dimers, and mutant IgM molecules that cannot form polymers are efficiently secreted (Baker et al., 1986). 2. Early B cells are intrinsically incompetent for imunoglobulin secretion because of a poorly developed ER. Although this argument has frequently been advanced, experimental evidence suggests otherwise. In a B cell line, representative of the immature B stage, transfection of a ps gene with a mutated tailpiece retention signal resulted in immunoglobulin secretion, suggesting that these cells are intrinsically competent for immunoglobulin secretion (Sitia et al., 1990). In a pre-B cell line which has switched to the y isotype (an isotype which lacks a retention signal), introduction of an L-chain gene led to efficient secretion (Bachhawat and Pillai, 1991), further indicating that early B lineage cells do not lack the machinery necessary for the secretion of secretory immunoglobulins. In the case of secretory IgM (as discussed in item l), early B cells may certainly lack some polymerizing activity, and this lack might contribute to the retention process, for tailpiece-containing immunoglobulins. 3 . The ps chain contains structural information that leads to its retention in early B lineage cells. The 19-amino-acidcysteine-containing secretory tailpiece at the carboxy terminus of the ps H chain has been demonstrated to be involved in the retention of ps in a stage-specific manner. Mutation of cys-575 (the penultimate residue) leads to the secretion of secretory IgM by B cell lines which represent early stages of differentiation and which retain and degrade wild-type ps-containing IgM molecules. Engineering this tailpiece onto a y H-chain gene converts IgG to a protein that is now retained by early B lineage cells. The current evidence suggests that in early B cells the cysteine tailpiece serves as a signal for retention of secretory IgM; this mechanism applies to

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heavy-light complexes and is therefore independent of the retention mechanism that depends on an available CH1 domain. When a deletion mutant of the ps H-chain gene lacking a CHI domain was transfected into a plasmacytoma cell line, the mutant protein was retained intracellularly and was associated with Bip. When this deletion mutant was further altered, converting the cysteine in the tailpiece at position 575 to an alanine, the resulting protein was efficiently secreted (Sitia et al., 1990). Two interpretations remain for this phenomenon. The first is that this tailpiece-dependent mechanism involves the binding of Bip to the tailpiece and hence the. retention. The alternative mechanism is that the tailpiece plays a role in retention independently of Bip, and the retained intracellular immunoglobulin remains associated with Bip. It is worth noting in this context that, although ps2-L2 tetramers and the CHI deletion mutant of ps are both retained intracellularly by means of the tailpiece mechanism, Bip association is prominent with the deletion mutant and barely detectable in the case of IgM heavy-light complexes (Sitia et al., 1990). This argues for a secondary role for Bip which remains associated with nonassembled and retained secretory immunoglobulins. In the B lineage, this retention mechanism is restricted to pre-B and early B cells. However, it is unlikely that this mechanism is B lineage specific. It appears more likely that the tailpiece-dependent retention mechanism is present in many cell types, but is abrogated in certain differentiated cells. Transfected glioma cells can secrete IgM (Cattaneo and Neuberger, 1987) and transfected fibroblasts can secrete hemimers of ps and surrogate L chains (Karasuyama et al., 1990), suggesting that Bip (which is synthesized by all cells) may not be a major player in such a retention mechanism. However, some nonlymphoid lines, including transfected Chinese hamster ovary cells and HeLa cells, retain secretory IgM (Cattaneo and Neuberger, 1987), suggesting that retention may be the phenotype of cell types which possibly lack some specialized “assembly machinery.” This putative machinery presumably leads to the masking of retention signals and is assumed to be a characteristic feature of certain specialized cells, which include activated B cells, plasma cells, and glioma and pheochromocytoma cells. When mature B cells are activated following exposure to antigen, this retention mechanism is presumably shut off and remains silent in all subsequent stages of B cell differentiation. It is possible that, as B cells differentiate, they acquire the ability to polymerize IgM and thus to mask the tailpiece retention signal. The only other isotype other than the p isotype in which a cysteine-containing tailpiece is present is a. This tailpiece in secreted IgA is believed to play an important role in the process of dimerization and association with the J chain. Since IgA is synthesized

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only by activated B cells which have undergone class switching (and have presumably shut off the tailpiece-dependent retention mechanism), this mechanism is probably of no relevance to IgA secretion. IgA can be secreted as monomers as well as dimers. In studies of an IgA-expressing B lymphoma, nonactivated lymphoma cells expressed primarily am message and virtually no as message. Activation of these cells with bacterial lipopolysaccharide led to the synthesis of secretory IgA. This IgA was not retained by these cells, arguing that the tailpiece-dependent retention mechanism for IgA is missing in activated B cells (Sitia et al., 1985). Exposure of early B lineage cells to 2-mercaptoethanol leads to immunoglobulin secretion (Alberini et al., 1990), possibly by interference with the cysteine tailpiece-dependent retention mechanism. It is possible to unify the existing knowledge of secretory immunoglobulin retention into a single framework. Two structural features of secretory immunoglobulin H chains may play a role in retention. In cells that lack L chains, an exposed CH1 domain may serve as a retention signal, particularly for the y isotype. Since retention of ps in pre-B cells could occur through the other retention signal, the tailpiece in the CH4 domain, the physiological significance of retention through the CHI domain during B cell differentiation is certainly open to question. Retention through the CHI domain as well as through the CH4 domain may depend on two alternative mechanisms. 1 . The Bip-dependent model. In this model, the CH1 domain of all isotypes as well as the CH4 tailpiece in p and Q is considered to be a binding site for Bip. Bip binding to the CH1 domain can be masked by L-chain association. The binding of Bip to the tailpiece can be masked by the process of IgM polymerization. It is conceivable that the cysteine in the CH1 domain involved in L-chain association may be part of a Bip binding site. It was suggested by Hendershot et al. (1987) that since mutants lacking this cysteine in the Q chain CH1 domain still bound Bip, this residue could not contribute to Bip binding. It is quite likely that in these mutants Bip was bound to the tailpiece and therefore the CHI cysteine might still constitute part of a Bip binding site. 2. The incomplete assembly model. This model assumes that some aspect of the free CHI domain and also of the tailpiece leads to the retention of p H chains in pre-B cells and nonpolymerized IgM in nonactivated B cells. Unpaired cysteines in the CHI domain or in the tailpiece could contribute to misfolding, thus preventing assembly and secretion, or may directly associate with retention “receptors.” Alternatively, some other structural feature of these retention domains may contribute to the holdback process. The association with Bip is considered, in this model, to be a consequence, rather than a cause, of retention.

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IV. Membrane Immunoglobulin Transport during B Cell Ontogeny

An important issue for the regulation of pre-B cell differentiation as well as the transport of immunoglobulins is the existence in uiuo of a physiological “Type 11” pre-B stage at which membrane immunoglobulin is transported to the cell surface. The existence of such a stage would suggest that a ligand may exist for the p-o receptor, which could help drive differentiation from the pre-B to the B stage. The B cell stage of lymphoid ontogeny may be subdivided into the transitional, immature, mature, activated, and memory B cell stages. In all these stages, membrane immunoglobulin functions as a signal transduction receptor and must be transported to the cell surface. In the antigenindependent immature stage, signal transduction may lead to cell death. In the later antigen-dependent stages, signal transduction through membrane immunoglobulins leads to proliferation and further differentiation. In these stages, membrane immunoglobulin must also be capable of functioning as an endocytic receptor for the internalization and processing of protein antigens. In these stages, exposure to (self) antigens in the absence of cognate T cell help may also lead to the phenomenon of receptor downregulation, which may be one of the mechanisms of perpetuating clonal anergy . A. ER DEGRADATION VERSUS CELLSURFACE TRANSPORT: FATEOF MEMBRANE IMMUNOGLOBULIN IN PRE-B CELLS

Do pre-B cells express pm on the cell surface? Is the expression of conventional K or X L chains a prerequisite for the cell surface expression of pm? What is the molecular basis for acquisition of the ability to transport pm to the cell surface at the pre-B to B cell transition? Does cell surface expression of pm precede activation of K gene expression? These closely interrelated questions are of interest from the viewpoint of understanding immunoglobulin transport and are central to understanding regulatory mechanisms involved in the pre-B to B cell transition. Most pre-B cells in the bone marrow do not express detectable amounts of pm on the cell surface. Indeed, in pre-B cultures derived from bone marrow or fetal liver, the pre-B population expressing intracellular p is clearly distinguishable from infrequently seen surface p-positive B cells. Anti-p treatment in uiuo eliminates early B cells, but leaves most pre-B cells unaffected, which is consistent with the view that most pre-B cells do not express pm on the cell surface (Burrows et al. 1978). Whether a small subpopulation of pre-B cells which express detectable surface p in associ-

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ation with surrogate L chains actually exists in the bone marrow or the fetal liver is an important unresolved issue in B cell ontogeny. h e - B cell lines in which the H-chain locus has been productively rearranged fall into two categories. The majority of these lines (which we term Type I) contain only intracellular p. In these lines, p is associated with the o and L surrogate immunoglobulin L chains and is rapidly degraded. In a subset of mouse and human pre-B cell lines (which we term Type I1 pre-B cells), pm is transported to the cell surface. In subclones of a murine lymphoma, 70213, in which most cells do not express pm on the cell surface, exposure to dextran sulfate (a B cell mitogen) leads to surface expression of p in the absence Of K L-chain synthesis (Paige et al., 1981). In Type I1 pre-B cell lines as well as in dextran sulfate-induced 70Z/3 cells, we have demonstrated that pm on the cell surface is associated with the o and L surrogate L chains. From the above studies it is clear that the expression of p on the cell surface does not depend on the synthesis of conventional K L chains, but may, nevertheless, depend on proper assembly with surrogate L-chain proteins as well as some poorly defined molecular event that may be regulated by dextran sulfate in 70213 cells. This event permits pm to evade intracellular degradation and to be transported to the cell surface. One approach to the issue of the cell surface expression of p in the pre-B and B stages of differentiation is to recognize the existence of two competing pathways for membrane immunoglobulin transport in these cells. The rapid degradation of pm in pre-B cells is part of an intracellular editing process that targets incomplete oligomeric receptors for degradation in a pre-Golgi compartment, probably the ER itself. As pre-B cells differentiate, somewhere at the late pre-B stage, they acquire the ability to “rescue” pm complexed with surrogate L chains from the intracellular ERdegradative pathway, and to transport this complex to the cell surface. Even at the B stage, after the synthesis of the K chain, both of these pathways coexist; a portion of the (presumably imcompletely or incorrectly assembled) p m in mature B cells is degraded intracellularly, and the remainder is rescued and transported to the cell surface (Dulis et al., 1982). The ER-degradative pathway for incomplete oligomers of membrane immunoglobulin in pre-B cells can be distinguished from the degradative pathway for incomplete oligomers of secretory immunoglobulin (Bachhawat and Pillai, 1991). This difference may indeed be related to the ability of nonassembled secretory immunoglobulin H chains to bind to Bip (whether or not Bip causes retention or merely associates with molecules that are incapable of movement), while nonassembled membrane immunoglobulin H chains perhaps do not associate with as efficiently with Bip. The Bip-

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associated secretory immunoglobulin may shuttle back and forth between the ER and the salvage compartment prior to degradation. Alternatively, transmembrane hydrophilic topogenic sequences may determine the rapid degradation of nonassembled membrane immunoglobulins. The possible role of such a topogenic sequence is described in the next subsection. In pre-B cells, pm that is either free or associated with w and L is rapidly degraded. It is unclear whether p chains that are associated with surrogate L chains are less susceptible to degradation than are free p chains. However, the levels of expression of the genes for surrogate L chains (the A5 and uPreB genes) are similar in both Type I and Type I1 pre-B cell lines. Similarly, the levels of expression of two B lineage genes, the mb-I and B29 genes, whose products play a role in the surface expression of pm in B cells are also comparable to mature B cell levels in both Type I and Type I1 pre-B cell lines. What regulates the ability to transport pm to the cell surface at the pre-B to B cell transition? Issues such as the role of pmassociated proteins, the assembly process, and the hydrophobicity of pm are discussed in detail in Section IV,B. B. TRANSPORT OF MEMBRANE IGM: ASSOCIATED PROTEINS AND THE ROLEOF HYDROPHOBICITY As discussed in Section IV,A, membrane immunoglobulin H chains have two distinct fates after synthesis. One constitutive “pathway” for this protein involves its intracellular degradation; incompletely assembled or misfolded mmebrane immunoglobulins are probably being constantly directed into this degradatory pathway. The site of degradation is a preGolgi compartment which may the ER itself. The second fate of membrane immunoglobulin is to be rescued from the intracellular degradative pathway, presumably by virtue of being properly and completely assembled. What constitutes complete and proper assembly of membrane immunoglobulin? Since intriguing correlations have been made between the acquisition of hydrophobicity and the cell surface transport of IgM, is there an underlying theme, possibly of heuristic value, which connects assembly, hydrophobicity, and cell surface transport? It has long been appreciated that the mere association of pm and K is insufficient for the surface expression of p. Indeed, from studies of Type I1 pre-B cells which express p on the cell surface in association with surrogate L chains, it has been clear that expression of the K L chain was not the key event leading to the surface expression of p. In plasma cells which secrete ps in association with conventional L chains, pm was associated with K , but was not transported to the cell surface (Sitia et al., 1987; Hombach et al., 1988a). Indeed, in a pre-B cell line expressing the y iso-

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type and a transfected K L-chain gene, ys2-~2tetramers were secreted, while ym2-~2 tetramers remained intracellular (Bachhawat and Pillai, 1991). All these results suggested that an additional posttranslational event or protein may be necessary for the surface transport of membrane immunoglobulin. In studies of the biosynthesis and fate of pm in pre-B cells and in B cells, we demonstrated that, in B cells, three distinct forms of pm may be identified (PiUai and Baltimore, 1987b). The initially synthesized pm protein is relatively hydrophilic (and partitions into the aqueous phase upon fractionation with Triton X-114). This relative hydrophilicity is perhaps not too surprising when one considers that the transmembrane region of pm contains nine serine and threonine residues. The hydrophilic form is referred to as the pml form. After a short while, this protein is converted to a relatively hydrophobic form, referred to as the pm2 form. In B cells, the pm2 form acquires endo H resistance prior to conversion to a terminally glycosylated slower migrating pm3 form, which is transported to the cell surface. Since the acquisition of a relatively long-lived hydrophobic form of pm correlates with the transport of this protein to the cell surface in B cells, it seemed likely that understanding the basis for the acquisition of hydrophobicity might yield information relevant to the mechanism by which pm is transported to the cell surface. The acquisition of relative hydrophobicity by pm postsynthetically is probably relevant to the conformation of the fully assembled membrane IgM molecule that is recognized for transport, and is not in any way a requirement for proper anchoring in the ER membrane (Pillai and Baltimore, 1987b). The relevance of the relative hydrophobicity of the pm molecule to the process of cell surface transport was dramatically highlighted in a study on the transport of wild-type and mutant IgM proteins in nonlymphoid cells (Williams et al., 1990). In non-B cells, transfection of a gene encoding the wild-type membrane p H-chain protein and a A L-chain gene leads to the formation of intracellular membrane IgM tetramers which are not transported to the cell surface. However, when a hydrophilic stretch of five amino acids in the anchor region of the pm gene was replaced by nucleotides encoding hydrophobic amino acids, transfection experiments on fibroblasts revealed that this relatively hydrophobic pm protein could be transported to the cell surface in association with L-chain. From these studies, it seems likely that, whatever the requirements for surface transport of IgM entail, in terms of either associated proteins or posttranslational modifications or both, the net effect of these assembly-related events may well be mimicked by the artificially acquired hydrophobicity of the pm protein. The hydrophilic TTAST stretch may be viewed as a topogenic sequence that targets pm for retention, and posttranslational

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processes such as assembly with other hydrophobic proteins may “mask” this region and permit transport to the cell surface. In the past few years, a number of proteins have been described in pre-B and B cells that are associated with membrane immunoglobulin. On the surface of Type I1 pre-B cells, p m is known to be associated with the o and L surrogate L-chain proteins. These surrogate L chains have been described in considerable detail in Section IIC. A functional role of these proteins in permitting the transport of pm to the cell surface has been suggested in transfection experiments. Transfection of the A5 and vPreB genes along with a modified p m gene (incorporating transmembrane and cytoplasmic tail structures from a class I gene) led to the expression of p and surrogate L chains on the surface of a recipient myeloma cell line (Tsubata and Reth, 1990). In addition to w and L , up to six other polypeptides have been reported to associated with p m in Type I1 pre-B cell lines (Pillai and Baltimore, 1988; Takemori et al., 1990). Two of these proteins may correspond to the mb-l/Iga and B29/Igp proteins that have been studied in some detail in B cells and are described here. The other four have been characterized in a limited fashion, and their role in the transport of pm remains to be established. The use of digitonin as a lysis buffer and two-dimensionalnonreducing/ reducing gel systems for analysis has led to the characterization of two proteins associated with membrane IgM in B cells (Hombach et al., 1988b, 1990a).These proteins are believed to form a disulfide-linked heterodimer that is associated with membrane IgM; the exact stoichiometry of this complex and the proportion that is actually disulfide bonded remain to be determined with certainty. The first of these proteins, originally described as B34, is now known as IgM(a). This protein is a 34-kDa protein and has been confirmed to be the product of the mb-Z gene (Hombach et al., 1990a; Sakaguchi et af., 1988). The ability of subclones of a myeloma line to transport p m to the cell surface was correlated directly with the presence of this protein. Introduction of the mb-1 gene into a cell line that initially lacked the ability to transport IgM to the cell surface led to the establishment of a transfectant cell line which expressed IgM(a)protein, and which was now competent to transport pm to the cell surface. A second protein that was found to be associated with membrane IgM in B cells and which is at least in part disulfide linked to IgM(a) is a 39-kDa protein termed Ig(p). This protein, unlike IgM(a) (which is specifically associated with the p isotype), is presumed to be associated with membrane immunoglobulin of all isotypes. Ig(p) has recently been demonstrated to be the product of the B lineage-specific B29 gene (Hombach et al., 1990b; Hermanson et al., 1988).Overexpressionof the B29 gene in a myeloma line led to an increase in the surface expression of IgM. Both mb-1 and B29 are expressed from the earliest pro-B stage of

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ontogeny and by all surface immunoglobulin-positive lymphocytes. Structurally, the cDNA sequences of both these genes suggest that they encode transmembrane glycoproteins. Both genes encode signal peptides, extracellular domains with N-glycosylation sites, transmembrane anchor regions, and cytoplasmic tails which bear some homology to one another as well as to the cytoplasmic tails of the T cell receptor-related CD3 y chain, suggesting that these proteins may be involved in signal transduction through membrane IgM. Indeed, both these proteins have been demonstrated to be N-glycosylated and to be phosphorylated on tyrosine residues in response to crosslinking of membrane immunoglobulin (Campbell and Cambier, 1990). A separate protein associated with membrane IgD expressed in cell lines which have lost mb-1 and the ability to transport surface IgM has been described. This 35-kDa protein is presumed to be the 6 isotype-specific equivalent of mb-1 (Wienands et al., 1990). This protein is structurally related to the mb-1 product, but must be predicted to be the product of a distinct gene (since it has been identified in clones that lack mb-1 transcripts). The probable existence of a y isotype-specific “mb-1”-like protein has been postulated based on the inability of ym2-~2 tetramers to be transported to the cell surface in a transfected pre-B cell line (Bachhawat and Pillai, 1991). The acquisition of the ability to transport membrane immunoglobulins of various isotypes during development and its correlation with isotype-specific associated proteins remains a poorly explored avenue. Many issues remain to be resolved regarding the role of the products of the mb-1 and B29 genes in transporting membrane IgM to the cell surface. Although it is tempting to draw analogies with the role of the CD3 complex proteins in the transport of the T cell receptor to the cell surface, certain differences need to be addressed. The product of the mb-2 gene (unlike CD3 chains) can apparently be expressed on the surface of some cells in the absence of membrane IgM. Both mb-1 and B29 are expressed at levels comparable to those seen in B cells in Type I pre-B cell lines that express only intracellular p. It is likely that other IgM-associated proteins may exist in the B cell stage that may play a role in transporting membrane IgM to the cell surface; these proteins remain to be identified. A “minimal” model for the structure of the B cell receptor is provided in Fig. 6. An interesting suggestion has been made regarding the possible role of assembly of IgM with mb-1 (this suggestion could apply equally well to B29 or any other IgM-associated protein) in assisting transport to the cell surface. Since, in nonlymphoid cells, replacement of five hydrophilic amino acids in the transmembrane region of the H chain with hydrophoSee Note Added in Proof, p. 36.

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gatively charged loop

MEMBRANE

K

K

FIG. 6. A “minimal” model for the B cell receptor.

bic residues is sufficient to make intracellular IgM competent for surface transport, it was suggested that mb-1 may, by associating with the H chain, mask the hydrophilic stretches in the transmembrane region, and the ensuing hydrophobicity is sufficient for ensuring transport to the cell surface (Williams et al., 1990). The possibility that a posttranslational modification such as the myristylation of IgM may play a role in the acquisition of hydrophobicity in the process of assembly and surface transport remains to be excluded (Pillai and Baltimore, 1987b). From the studies of membrane immunoglobulin discussed here as well as from interesting studies of the degradation and transport of the T cell receptor-CD3 complex (Bonifacino et al., 1990), an interesting concept has emerged that may apply generally to the assembly of transmembrane heterooligomers. Hydrophilic stretches in the transmembrane segments of individual chains of multisubunit membrane proteins are probably involved in the recognition of partners and in the assembly process. The process of assembly leads to the masking of the hydrophilic transmembrane stretches and prevents rapid degradation. The process may be regulated by the need to attain a threshold level of hydrophobicity that permits transport to proceed; alternatively, being below this threshold, hydropho-

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bicity may serve as a signal for ER retention or degradation, thus abrogating the transport of incompletely assembled oligomers. The “packing” process could conceivably be catalyzed by posttranslational processes such as acylation (Pillai and Baltimore, 1987b). There is evidence (B. J. Cherayil and S. Pillai, unpublished observations) suggesting that the ratelimiting step at the pre-B to B transition which drives assembly and transport may indeed be the efficiency of the process of covalent heavy-light assembly, which may be a prerequisite for engaging transmembrane masking proteins such as mb-1 and B29. C. MEMBRANE IMMUNOGLOBULIN RECYCLING, ENDOCYTOSIS, AND DOWN-REGULATION Apart from its ability to be transported to the cell surface in B cells, the membrane immunoglobulin molecule or an associated protein must carry structural information permitting endocytosis, recycling, and receptor down-regulation functions. When the antigen receptor on B cells encounters a cognate protein antigen, the antigen is rapidly internalized into an endocytic compartment. In order for antigen presentation to occur, the contents of this compartment must be accessible to processing proteases and must intersect with vesicles containing class I1 molecules. The internalized protein is then cleaved into peptides, and the appropriate peptide then associates with the antigen-binding groove on the class I1 molecule and is presented on the surface of the cell. Membrane immunoglobulin is then presumably recycled to the cell surface. Ligation of membrane immunoglobulin by a multivalent antigen (as mimicked by antiimmunoglobulin crosslinking) leads to internalization by nonselective endocytosis (Guagliardi et al., 1990). Monovalent protein antigens are internalized into clathrin-coated vesicles (Watts et al., 1989). However, ligands taken up by selective or nonselective endocytosis are known to be delivered to the same endosomes (Tran et al., 1987). The transmembrane region and the cytoplasmic tail of each membrane immunoglobulin H-chain isotype are highly conserved across species. The function of membrane immunoglobulin in terms of signal transduction as well as for the endocytic functions that are essential for antigen presentation is presumed to be mediated in part via the proteins that are associated with pm (including the products of the mb-1 and B29 genes). The cytoplasmic tail of pm contains only three amino acids; the mb-1 and B29 gene products have considerably longer cytoplasmic tails which contain conserved cytoplasmic residues known to be phosphorylated in response to activation. Presumably, ligation of membrane immunoglobulin leads to the appro-

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priate conformational changes/phosphorylation events that regulate the generation of a differentiation signal, as well as the internalization and recycling of the receptor itself. Attempts have been made to dissociate the signal transduction and antigen presentation functions of membrane immunoglobulin by making mutations of the transmembrane region and cytoplasmic tail regions of the pm gene, followed by transfection and functional analysis (Webb et al., 1989; Shaw et al., 1990). Replacement of the transmembrane region of pm with a class I1 transmembrane anchor, not surprisingly, abolished the ability of this protein to transduce a signal. Deletion of the KVK cytoplasmic tail severely compromised both signal transduction and antigen presentation functions. The hydroxyl group of the tyrosine residue (position 587) and the adjacent serine residue (position 588) were demonstrated to be of critical importance in signal transduction (Shaw et al., 1990). Interestingly, conversion of Tyr-587 alone to a phenylalanine resulted in a molecule that was able to signal, but which lacks the ability to present antigen. This dissociation of signal transduction from antigen presentation suggests that different protein contacts are required for these two processes and that different pm-associated proteins may help mediate these functions. In Fig. 7, a schematic view of the last 41 amino acids of the pm protein is provided, indicating residues which, based on the analysis of mutations in this region, are known to be of functional importance. Closely related to the functions of membrane immunoglobulin just discussed is the phenomenon of receptor down-regulation, which may be an important mechanism for the generation of clonal anergy. In “double” transgenic mice expressing hen egg white lysozyme as well as the rearranged immunoglobulin genes which recognize lysozyme, a feature of anergic B lymphocytes was the expression of relatively high antigenspecific IgD levels in conjunction with low surface IgM levels (Goodnow et al., 1990). A model for anergy in these cells invokes the specific downregulation of surface IgM, possibly as a result of exposure to monomeric self antigen in the absence of cognate T cell help. Since different proteins have been found to be associated with IgM and IgD, selective posttranslational changes in one isotype-specific protein may be invoked to explain such a specific down-regulation event. It is indeed conceivable that even in memory B cells (which may express other isotypes, including IgG, IgE, and IgA) similar down-regulation mechanisms may play a role in the maintenance of anergy . The role of isot ype-specific membrane immunoglobulinassociated proteins and of specific transmembrane residues on the immunoglobulin molecule and in these processes may be predicted, but remain to be established.

IMMUNOGLOBULIN TRANSPORT

29

E

(-)

E (-) V

N A E

G

E

(-1

(-1 (4

F

EXTRACELLULAR

(-) E N

L W *Fl RetentioniDegradation signal

LFL

L

MEMBRANE

SL

s Y7587) T~

(+I

K (595) (596) (+) K (597)

v

T

(588) INTRACELLULAR

FIG. 7. Membrane exon-encoded residues of the pm protein depicting residues relevant to function and transport. For details, see text.

D. MEMBRANE IMMUNOGLOBULIN RETENTIONIN PLASMA CELLS Plasma cells presumably do not need to respond to antigen and need not express membrane immunoglobulin on the cell surface. This issue has not, however, been established with certainty, and few studies have been performed using actual plasma cells. In many myeloma lines, membrane immunoglobulin is retained intracellularly (Hombach et al., 1988a; Sitia et al., 1987). In some lines, this loss of expression can be explained by the loss of mb-1 expression. In situ hybridization of mb-1 and B29 genes or immunohistochemical studies using antibodies for these proteins must be performed on tissue sections to confirm whether these genes are actually shut off in plasma cells in the course of B cell development. V. Immunoglobulin Secretion in Plasma Cells

Although numerous studies have been published on the secretion of immunoglobulins by plasma cells, many unanswered questions remain regarding the details of this process. In brief, no vital role in the process of

30

SHIV PILLAI

transport itself can be ascribed to posttranslational modifications of immunoglobulin, to the process of assembly into higher oligomers (for IgM and IgA), or to the J chain. An absolute requirement does exist for association with an L chain and for the heavy-light complex to be “conformationally appropriate” for transport. OF A. ROLEOF THE J CHAINAND ASSEMBLY POLYMERIC IMMUNOGLOBULINS

Prior to the establishment of the tailpiece-dependent retention mechanism of secretory IgM in early B cells, a popular model for IgM secretion invoked the need for assembly of J chain-containing polymeric IgM prior to secretion. The immunoglobulin J chain (Koshland, 1985) is a 15-kDa protein associated with the cysteine-containing tailpiece in IgM pentamers and in IgA dimers. The J chain gene is transcriptionally activated in the course of antigen- and lymphokine-induced activation of B lymphocytes. The J chain is essential neither for the oligomerization of IgM monomer units nor for IgM secretion. In a transfected glioma cell line, IgM assembles to form pentamers and hexamers in the absence of J chain and is secreted (Cattaneo and Neuberger, 1987). Indeed, assembly of IgM and IgA into polymeric forms is not a prerequisite for secretion. Monomeric IgA is secreted efficiently and so is a mutant IgM that is incapable of forming pentamers (Baker et al., 1986). The probable role of the J chain lies in the recognition of assembled immunoglobulins by the polyimmunoglobulin receptor prior to transcytosis. The issue of IgM assembly has been reviewed in depth (Davis and Shulman, 1989). AND SECRETION B. POSTTRANSLATIONAL MODIFICATIONS

Numerous posttranslational modifications of immunoglobulins ranging from N-linked and 0-linked glycosylation to tyrosine sulfation have been examined from the viewpoint of secretion (reviewed by Wall and Kuehl, 1983). No known posttranslational modification is essential for the secretory process. C. ROLEOF L CHAINSAND “APPROPRIATE” CONFORMATION FOR SECRETION Immunoglobulin L chains play a crucial role in immunoglobulin secretion. Indeed, it is likely that a small conserved region on the VL domain may serve as a “recognition patch” for the secretory machinery. Studies of the role of L chain in immunoglobulin secretion support, albeit in a

IMMUNOGLOBULIN TRANSPORT

31

limited way, the notion that secretion is not merely a “bulk flow” process for nonretained nonanchored proteins that enter the ER. H-chain proteins in the absence of L chains are retained intracellularly in association with Bip. In an H-chain-only myeloma cell line (Pepe el al., 1986), as well as in a ys-containing pre-B cell line (Bachhawat and Pillai, 1991), introduction of an L-chain gene leads to efficient secretion of fully assembled immunoglobulin. L-chain proteins in the absence of H chains are secreted. A notion that is currently gaining ground is that L chains may possess a steric determinant that is crucial for the secretion of free L chains as well as heavy-light tetramers. L chains with point mutations have been identified that are not secreted (Mosmann and Williamson, 1980; Wu et al., 1983; Nakaki et al., 1989; Dul and Argon, 1990). Nonsecreted L-chain proteins have also been found to be associated intracellularly with Bip. Mutations in both the VL and CL domains have been identified that prevent secretion. Dul and Argon (1990) tested the hypothesis that a group of conserved residues (residues 57-65) on L-chain proteins may form part of a structural patch that is recognized by the intracellular secretory machinery. Conversion of a phenylalanine at position 62 to a serine resulted in a nonsecreted L chain. This mutant L chain bound Bip. In the presence of H-chain protein, however, this L chain was assembled into a functional antigenbinding antibody. This antibody molecule did not bind Bip and was recognized by a panel of monoclonal and polyclonal antibodies against wild-type L chains, indicating that no gross conformational alteration had occurred as a result of the point mutation. The assembled and functional antibodies containing a serine residue at position 62, however, were not secreted. These data suggest that structural recognition of conserved motifs may play a role in the progress of the antibody molecule along the secretory pathway. Figure 8 outlines retention and transport signals that influence immunoglobulin secretion. M. Summary: Choices between RetentiodDegradation and Transport of Immunoglobulins Are Dictated by Function

In pre-B lymphocytes, secretory immunoglobulin has no function. At this stage, ps is retained intracellularly. Two retention signals have been identified, one in the CH1 domain and the other in the CH4 cysteine tailpiece. The retention mechanism may involve association with Bip or possibly with an ER protein or proteins that sequester luminal proteins that contain free cysteines. At this stage, pm is associated with the o and L proteins and is degraded intracellularly. The p 2 - ~ 2 - ~complex 2 (in associ-

32

SHIV PILLAI chain

Recognition patch on L chain for secretion

-

Putative Bip binding sites or retention signals

Tailpiece

Ht

/

FIG. 8. Schematic view of retention and transport signals on secretory immunoglobulins. The domain structure of the heavy chain depicted is typical of hs. L chain, light chain; H chain, heavy chain; V, variable region; VL, variable region of the light chain; CL, constant region of the light chain; SH, sulthydryl.

ation with other proteins) is presumed to be involved in generating a signal for further differentiation. Intracellular degradation of pm in early (Type I) pre-B cells probably involves recognition of a hydrophilic signal in the transmembrane region of pm. Late in the pre-B stage in Type I1 pre-B cells, pm in association with surrogate L chains is transported to the cell surface. Cell surface transport probably depends on efficient covalent pm-o assembly and also the posttranslational acquisition of hydrophobicity. The mb-1/IgMa and B29/Igp proteins may serve to mask the hydrophilic transmembrane residues in pm which target it for ER degradation. In nonactivatedhirgin B cells, secretory ps2-~2tetramers are retained intracellularly by a mechanism involving the cysteine tailpiece on ps and association with Bip. Bip does not associate with tetrameric membrane immunoglobulins (Sitia et al., 1990). Membrane immunoglobulin is transported to the cell surface in association with IgM(a) (which is specific for

IMMUNOGLOBULIN TRANSPORT

33

the p isotype) and Ig(p), which presumably associates with membrane immunoglobulins of all isotypes. At this stage, membrane immunoglobulin functions as the antigen receptor which is involved both in signal transduction and endocytosis (for antigen presentation). Mutations in the transmembrane domain can dissociate signal transduction from endocytosis and suggest that the endocytic pathway can be regulated by pm-associated proteins. Receptor down-regulation may serve to sustain a state of clonal anergy . After B cells are activated by antigen, class switching may occur. Isotype-specific associated proteins may play a role in the surface transport of various membrane immunoglobulin classes. Assembly processes help ps and as to bypass retention mechanisms. The y and E isotypes lack a cysteine tailpiece and are secreted in association with L chains. At this stage, polymerization masks the cysteine tailpiece of ps and a s and retention is abrogated. While considering transport along the secretory pathway, two broad mechanisms have been proposed. One model suggests that, after proteins are translocated into the ER, many categories of retention and transport signals are recognized by receptors which direct vesicles to appropriate locations. Mannose 6-phosphate is a tag for lysosomal transport, and the KDEL signal ensures that ER luminal proteins do not go beyond the salvage compartment. In such a model, proteins destined for the cell surface or for secretion have specific steric features recognized by receptors which guide vesicular transport. The second model is the bulk flow model (Rothman, 1987), which suggests that once a protein enters the ER it will be transported to the cell surface by “bulk flow,” unless retention signals or lysosomal transport signals subvert this process. This model suggests that no receptors are necessary to direct vesicular transport to the plasma membrane. A separate concept particularly relevant to immunoglobulin transport and which is compatible with both models above is the need for oligomerization and assembly as a prerequisite for cell surface transport (Kreis and Lodish, 1986). An “exposed” interface prior to assembly could function as a retention signal which requires masking by the process of assembly. Alternatively, the fully assembled complex may provide the correct stereochemical configuration for further transport. It remains unclear whether transport of membrane and secretory immunoglobulins merely reflects the abrogation of retentioddegradation mechanisms or actually depends in any way on specific steric recognition of domains on immunoglobulin molecules for movement to occur. The recent demonstration of a patch on L-chain molecules which is essential for immunoglobulin movement suggests a possible role for steric recognition events, as opposed to bulk flow, in the absence of retention.

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SHIV PILLAI

ACKNOWLEDGMENTS I thank David Baltimore for introducing me to pre-B lymphocytes, and Marian Koshland, Bobby Cherayil, Anand Bachhawat, and Sudhir Krishna for many invaluable discussions. I also thank Ravi Iyer for his help with the figures and Michael Reth for communicatingresults prior to publication. This work was supported by National Institutes of Health grant A1 27835 and by the Arthritis Foundation.

REFERENCES Alberini, C. M., Bet, P., Milstein, C., and Sitia, R. (1990). Nature (London)347,485-488. Alt, F. W., Blackwell, T. K., Depinho, R. A., Reth, M., and Yancopoulos, G. D. (1986). Immunol. Rev. 89,5-63. Bachhawat, A-.K., and Pillai, S. (1991). J . Cell Biol. (in press). Baker, M. D., Wu, G. E., Toone, W. M., Murialdo, H., Davis, A. C., and Shulman, M. J. (1986). J . Immunol. 137, 1724-1728. Bole, D. G., Hendershot, L. M., and Kearney, J. F. (1986). J . Cell Biol. 102, 1558-1566. Bonifacino, J. S., Cosson, P., and Klausner, R. D. (1990). Cell 63,503-513. Burrows, P. D., Kearney, J. F., Lawton, A. R., and Cooper, M. D. (1978).J. Immunol. 120, 1526-153 I . Campbell, K. S., and Cambier, J. C. (1990). EMBOJ. 9,441-448. Cattaneo, A., and Neuberger, M. S. (1987). EMBO J . 6,2753-2758. Chang, H., Dmitrovsky, E., Hieter, P. A., Mitchell, K., Leder, P., Turoczi, L., Kirsch, I. R., and Hollis, G. F. (1986). J. Exp. Med. 163,425-435. Cherayil, B. J., and Pillai, S. (1991a). J . Exp. Med. 173, 111-116. Cherayil, B. J., and Pillai, S. (1991b). Manuscript in preparation. Davis, A. C., and Shulman, M. J. (1989). Immunol. Today 10, 118-128. Dul, J. L.,and Argon, Y. (1990). Proc. Natl. Acad. Sci. U.S.A. 87,8135-8139. Dulis, B. H. (1983). J. Biol. Chem. 258,2181-2187. Dulis, B. H., Kloppel, T. M., Grey, H. M., and Kubo, R. T. (1982). J. Biol. Chem. 257 4369-4374. Findley, H. W.,Cooper, M. D., Kim, J. H., Alvarado, C., and Ragab, A. H. (1982). Blood 60, 1305- 1309. Flynn, G. C., Chappell, T. G., and Rothman, J. E. (1989). Science 245,385-390. Gething, M.-J., McCammon, K., and Sambrook, J. (1986). Cell 46,939-950. Goodnow, C. G., Adelstein, S., and Basten, A. (1990). Science 248, 1373-1379. Gordon, J., Hamblin, T. J., Smith, J. L., Stevenson, F. K., and Stevenson, G. T. (1981). Blood 58,552-556. Gu,H., Kitamura, D., and Rajewsky, K. (1991). Cell 65,47-53. Guagliardi, L. E., Koppelman, B., Blum, J. S., Marks, M. S., Cresswell, P., and Brodsky, F. M . (1990). Nature (London) 343, 133-139. Haas, I. G., and Wabl, M. (1983). Nature (London)306,387-389. Hardy, R. R., Dangl, J. R., Hayakawa, K., Jager, G., Herzenberg, L. A., and Herzenberg, L. A. (1986). Proc. Natl. Acad. Sci. U.S.A. 83, 1438-1442. Hendershot, L. M. (1990). J. Cell Biol. 111,829-837. Hendershot, L., and Levitt, D. (1984). J. Immunol. 132,502-509. Hendershot, L. M., Bole, D., Kohler, G., and Kearney, J. F. (1987). J . Cell Biol. 104, I61-767. Hermanson, G., Eisenberg, D., Kincade, P., and Wall, R. (1988). Proc. Natl. Acad. Sci. U.S.A. 85,6890-6894.

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Hollis, G. F., Evans, R. F., Stafford-Hollis, J. M.,Korsmeyer, S. J., and McKearn, J. P. (1989). Proc. Natl. Acad. Sci. U . S . A . 86,5552-5556. Hombach, J., Sablitzky, F., Rajewsky, K., and Reth, M. (1988a). J. Exp. Med. 167,652-657. Hombach, J., Leclercq, L., Radbruch, A., Rajewsky, K., and Reth, M. (1988b).EMBO J . 7, 345 1-3456. Hombach, J., Tsubata, T., Leclercq, L., Stappert, H., and Reth, M. (1990a). Nature (London) 343,760-762. Hombach, J., Lottspeich, F., and Reth, M. (1990b). Eur. J. Immunol. 20,2795-2799. Karasuyama, H., Kudo, A., and Melchers, F. (1990). J . Exp. Med. 172, %9-972. Kassenbrock, C. K., Garcia, P. D., Walter, P., and Kelley, R. B. (1988). Nature (London) 333,90-93. Kerr, W. G . , Cooper, M. D., Feng, L., Burrows, P. D., and Hendershot, L. M. (1989). Int. Immunol. 4,355-361, Koshland, M. E. (1985). Annu. Rev. Immunol. 3,425-452. Kreis, T. E., and Lodish, H. F. (1986). Cell 46,929-937. Kudo, A., and Melchers, F.(1987). EMBO J . 6,2267-2272. Lewis, M. J., Sweet, D. J., and Pelham, H. R. B. (1990). CeN61, 1359-1363. Mason, J. O., Williams, G., and Neuberger, M. S. (1988). Genes Deu. 2, 1003-1011. McGarigle, M., Krishna, S., and Pillai, S. (1991). Manuscript in preparation. Misener, V., Jongstra-Bilen, J., Young, A. J., Atkinson, M. J., Wu, G. E., and Jongstra, J. (1990). J . Immunol. 145,905-909. Morrison, S. L., and Scharff, M.D. (1975). J . Immunol. 114,655-659. Mosmann, T. R., and Williamson, A. R. (1980). Cell 20,283-292. Munro, S . , and Pelham, H. R. B. (1986). Cell 46,291-300. Munro, S . , and Pelham, H. R. B. (1987). Cell 48,899-907. Nakaki, T.,Deans, R. J., and Lee, A. S. (1989). Mol. Cell Biol. 9,2233-2238. Nussenzweig, M.C., Shaw, A. C., Sinn, E., Danner, D. B., Holmes, K. L., Morse, H. C., and Leder, P. (1987). Science 236,816-819. Oettinger, M.A., Schatz, D. G., Gorka, G., and Baltimore, D. (1990). Science 248, 15171523. Paige, C. J., Kincade, P. W., and Ralph, P. (1981). Nature (London)292,631-633. Pepe, V. H., Sonenshein, G. E., Yoshimura, M. I., and Shulman, M. J. (1986). J. Immunol. l37,2367-2372. Pillai, S., and Baltimore, D. (1987a). Nature (London) 329, 172-174. P h i , S., and Baltimore, D. (1987b). Proc. Natl. Acad. Sci. U.S.A. 84,7654-7658. Pillai, S., and Baltimore, D. (1988). Curr. Top. Microbiol. Immunol. 137, 136-139. Rajewsky, K., Forster, I., and Cumano, A. (1987). Science 238, 1088-1093. Reth, M. G., and Alt, F. W. (1984). Nature (London) 312,418-423. Reth, M. G., Petrac, E., Wiese, P. Lobel, L., and Alt, F. W. (1987). EMBO J . 4,361-366. Reth, M., Hombach, J., Wienands, J., Campbell, K. S., Chien, N., andcambier, J. C. (1991). Immunol. Today 12, 196-201. Rose, M. D., Misra, L. M., and Vogel, J. P. (1989). Cell 57, 1211-1221. Roth, R. A., and Koshland, M. E. (1981a). Biochemistry 20,6594-6599. Rothman, J. E. (1987). Cell 50,521-522. Rubartelli, A., Sitia, R., Zicca, A., Grossi, C. E., and Ferrarini, M. (1983). Blood 62, 495-504. Sakaguchi, N., and Melchers, F. (1986). Nature (London) 324,579-582. Sakaguchi, N., Kawashimura, S., Kimoto, M., Thalmann, P., and Melchers, F. (1988). EMBO J . 7,3457-3464. Schatz, D. G., Oettinger, M. A., and Baltimore, D. (1989). Cell 59, 1035-1048.

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Sen, R., and Baltimore, D. (1986). Cell 47,921-928. Shaw, A. C., Mitchell, R. N., Weaver, Y.K., Campos-Torres, J., Abbas, A. K., and Leder, P. (1990). Cell 63,381-392. Sibley, C. H., Ewald, S. J., Kehry, M. R., Douglas, R. H., Raschke, W. C., and Hood, L. E. (1980). J . fmmunol. 125,2097-2105. Sidman, C. (1981). Cell 23,379-389. Sitia, R., Rubartelli, A., Kikutani, H., Hammerling, U., and Stavnezer, J. (1985). J. fmmunol. 135,2859-2864. Sitia, R., Neuberger, M. S., and Milstein, C. (1987). EMBO J . 6, 3969-3977. Sitia, R. Neuberger, M., Alberini, C., Bet, P., Fra, A., Valetti, C., Williams, G., and Milstein, C. (1990). Cell 60,681-690. Takemori, T., Mizuguchi, I. M., Miyazoe, I., Nakanishi, M., Shigemoto, K., Kimoto, H., Shirsawa, T., Maruyama, N., and Taniguchi, M. (1990). EMBO J . 9,2493-2500. Thorens, B., Schulz, M.-F., and Vassali, P. (1985). EMBO J . 4, 361-368. Tran, D., Carpentier, J. L., Sawano, F., Gorden, P., and Orci, L. (1987). Proc. Natl. Acad. Sci. U.S.A. 84,7957-7961. Tsubata, T., and Reth, M. (1990). J . Exp. Med. 172,973-976. Tsubata, T., Tsubata, R., and Reth, M. (1991). Eur. J . fmmunol.21, 1359-1363. Vassali, P., Tartakoff, A., Pink, J. R. L., and Jaton, J. C. (1980). J. Biol. Chem. 255, 11822-11827. Vogel, J. P., Misra, L. M., and Rose, M. D. (1990). J . Cell Biol. 110, 1885-1895. Wall, R., and Kuehl, M. (1983). Annu. Rev. fmmunol. 1,393-422. Watts, C., West, M. A., Reid, P. A., and Davidson, H. W. (1989). Coldspring HarborSymp. Quant. Biol. 54,345-352. Webb, C. F., Nakai, C., and Tucker, P. W. (1989). Proc. Natl. Acad. Sci. U . S . A . 86, 1977- 198 1. Wienands, J., Hombach, J., Radbruch, A., Riesterer, C., and Reth, M. (1990). EMBO J. 9, 449-455. Williams, G. T., Venkitaraman, A. R., Gilmore, D. J., and Neuberger, M. S. (1990). J . Exp. Med. 171,947-952. Wu, G. E., Hozumi, N., and Murialdo, H. (1983). Cell 33,77-83. Yancopoulos, G. D., and Alt, F. W. (1986). Annu. Rev. Immunol. 4,339-368. NOTEADDEDI N PROOF.Since the submission of this review the work of Rajewsky and colleagues (Gu er al., 1991) suggests that the D p protein serves as a negative signal, eliminating early pre-B cells in which a particular reading frame has been generated by D-J rearrangement at the H-chain locus. Some evidence has also been obtained for low levels of D p protein on the surface of pre-B cells in association with surrogate light chains (Tsubata et al., 1991). Evidence has also been obtained indicating that alternatively glycosylated forms of the mb-1 protein associate with IgM and IgD, indicating that only a single Ig(a) protein may exist (unpublished observations cited in Reth et al., 1991).

INTERNATIONAL REVIEW OF CYTOLOGY, VOL. 130

The Cytoskeletal System of Nucleated Erythrocytes WILLIAMD. COHEN Department of Biological Sciences, Hunter College of CUNY, New York, New York 10021; and The Marine Biological Laboratory, Woods Hole, Massachusetts 02543

I. Introduction

The blood of all vertebrates other than mammals contains nucleated erythrocytes throughout life, with rare exception. Indeed, this is one of the simple universal characteristics distinguishing mammals from nonmammals. Thus, the erythrocytes of fish, amphibians, reptiles, and birds are typically flattened ellipsoids, with biconvexity produced by a bulging nucleus (Ramon-Cajal, 1933; Andrew, 1965; Rowley and Ratcliffe, 1988) (Fig. 1). In contrast, essentially all erythrocytes in the blood of adult monotremes, marsupials, and placental mammals are anucleate, and they assume the familiar flattened biconcave discoid shape under static (nonflow) conditions (Briggs, 1936; Ralston, 1985). However, during early embryonic development, even mammalian blood is populated by nucleated erythrocytes (Dantschakoff, 1908; Maximow, 1909; Yadav, 1972; Block, 1964). These constitute the “primitive generation” that originates in the so-called “blood islands” of the yolk sac, in common with the first generation of erythrocytes in all other vertebrates. In addition, various invertebrates, including blood clams (Mollusca), certain sea cucumbers (Echinodermata), and marine worms or wormlike species representing several phyla (Annelida, Sipuncula, and Priapulida), also package respiratory proteins within nucleated erythrocytes (Cohen and Nemhauser, 1985). Beyond interest in erythrocytes per se, there are good reasons for studying the cytoskeletal system of nucleated erythrocytes. First, these are cells specialized with respect to the generation and maintenance of a particular shape, and in their mechanical responses to deformation. As such, they are likely to have a highly differentiated cytoskeletal system displaying few extraneous properties. Second, they are readily available in quantity and in pure populations with only minor variation of cell shape or size for a given species, the one major variable being age. Third, their cytoskeletal system is highly compartmentalized compared to other cell types, simplifying interpretation of data and permitting at least some de31

Copyright 6 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

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WILLIAM D. COHEN

FIG. 1. Erythrocytes of the frog Runa pipiens. (a) Edge and face views; (b) oblique view illustrating nuclear bulge. Scanning electron microscopy of glutaraldehyde-fixed critical point-dried cells.

gree of cytoskeletal fractionation. Fourth, because erythrocytes exist naturally as individual cells in a fluid tissue, their external morphology and internal structure are maintained independently of contact with other cells, and their cytoskeletal system can be considered functionally complete. Fifth, their distinctive universal shape facilitates an experimental approach to questions about the relationship between cellular morphogenesis and biogenesis of the cytoskeleton during cell differentiation. For these reasons, the past decade has seen the exploitation of nucleated erythrocytes as a model system for a variety of studies on cytoskeletal structure, function, and biogenesis. Such work represents a natural step toward analysis of a cytoskeletal system at the next level of complexity beyond that of the mammalian erythrocyte, that is, one with greater resemblance to that of eukaryotic cells in general. In this status report on our understanding of the nucleated erythrocyte cytoskeletal system, coverage is not exhaustive. The focus is on cytoskeleton formation and function, particularly that of the marginal band (MB) of microtubules (MTs). Although much of this chapter is concerned with cytological, ultrastructural, and molecular observations on nonmammalian erythrocytes, recent comparative work on the cytoskeleton of primitive nucleated erythrocytes in developing mammals is also discussed. In pointing out areas of ignorance or disagreement throughout, I have taken the liberty of speculating occasionally in the hope that this will stimulate further investigation.

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11. Nucleated Erythrocytes: A Phylogenetic and Physiological Portrait

In the gallery of cells presented diagrammatically in Fig. 2, nucleated erythrocytes of five nonmammalian vertebrate classes are represented: cartilaginous fish, bony fish, amphibians, reptiles, and birds (Fig. 2, cells a-g). Although cell size is fairly uniform for a given species, among different species there is considerable size variation (Ram6n-Caja1, 1933; Andrew, 1965; Goniakowska-Witalidska and Witalitiski, 1976). The Amphibia typically have relatively large erythrocytes (Fig. 2, cells a-c), those of the giant salamanderAmphiuma tipping the vertebrate scale with a long axis of 70 pm. At the other extreme are erythrocytes of most birds and bony fish (Fig. 2, cells f and g), usually 15 p m or less. In an intermediate range are the erythrocytes of cartilaginous fish (Elasmobranchs)and reptiles, the latter typically somewhat smaller (Fig. 2, cells d versus e). Despite these considerable size differences, the flattened ellipsoidal biconvex shape of nucleated nonmammalian vertebrate erythrocytes is essentially universal. Similar morphology occurs in mammalian primitivegeneration nucleated erythrocytes as well (Fig. 2, cell h), with the exception that many cells have reduced ellipticity or even discoidal profiles, as

-

a b C d e f 9 FIG. 2. Size and morphology of vertebrate erythrocytes. Cells a-g, nonmammals; cells

h-j, mammals. (a) Amphiuma tridactylum (giant salamander); (b) Notophthalmus uiridescens (Eastern newt or salamander); (c) Rana pipiens (leopard frog; face and edge view); (d) Mustelus canis (smooth dopfish); (e) Anolis carolinensis (anole, a lizard); (f) Carassius auratus (goldfish); (9) Callus domesticus (chicken); (h) Monodelphis domestica (gray shorttailed opossum; primitive erythrocyte of neonate); (i) Camelus dromedarius (camel, adult; face and edge view); (i)Homo sapiens (human, adult; face and edge view).

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WILLIAM D.COHEN

observed in marsupial neonates (Cohen et al., 1990). In mammals of the camel family (e.g., camel, guanaco, and llama), circulating definitive erythrocytes are anucleate, but they are otherwise unusual among mammals, being elliptical even under nonflow conditions and flattened without biconcavity (Cohen, 1976; Cohen and Terwilliger, 1979) (Fig. 2, cell i). In all other mammals, typical mature definitive erythrocytes are biconcave disks when not in flow, as shown in face and edge views of a human erythrocyte (Fig. 2, cellj). The fundamental morphology of nucleated erythrocytes does not depend on the presence of the nucleus itself. In certain species representing several genera of salamanders (Emmel, 1924; Villolobos et al., 1988), >80% of the circulating erythrocytes are anucleate, yet most are flattened and ellipsoidal (Cohen, 1982). In addition, flattened elliptical shape is observed in other types of nucleated blood cells, such as nonmammalian vertebrate thrombocytes (Behnke, 1970a, b; Fawcett and Witebsky, 1964) and invertebrate clotting cells (Cohen and Nemhauser, 1985). Therefore, this shape is correlated with a common environment, rather than a common cargo or function. The real world of mature nucleated erythrocytes is not the microscope slide or the test tube in which we usually examine them, but rather a dynamic flowing tissue in which they are continuously subjected to pressure, deformation, and mechanical and osmotic testing. Their properties as observed under static experimental conditions can be misleading, a point pursued in Sections III,E and IV,A. What, then, is the functional significance of flattened ellipsoidal shape in nucleated erythrocytes? Flattening presumably enhances respiratory gas exchange by reducing diffusion distances, and, perhaps even more importantly, it orients cells in flow in such a way as to reduce overall blood viscosity (Chien, 1975; Fischer, 1978). Ellipticity reduces effective cell diameter for passage through narrow capillaries (Bloch, 1962), and it also appears to be important for cell orientation during blood flow, though its precise contribution is less clear (Cohen, 1978a; Fischer, 1978). Differences between nucleated erythrocytes of nonmammalian vertebrates and the nonnucleated definitive erythrocytes of mammals extend well beyond morphology. Typical adult mammalian erythrocytes are highly adaptable to physiological or experimental flow conditions, under which they rapidly assume shapes such as umbrellas and flattened ellipsoids. Although the nucleated erythrocytes of nonmammals are able to twist and bend as they negotiate turns of small radii, and to regain normal (i.e., equilibrium) shape rapidly, they exhibit much lower deformability than mammalian definitive erythrocytes during capillary flow (Usami et al., 1970; Gaehtgens et al., 1981a,b). In addition, under experimental conditions in uitro, mammalian erythrocytes align and elongate into flat-

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tened ellipsoids in the flow direction without tumbling, whereas nonmammalian vertebrate erythrocytes assume more random positions and flip or tumble (Fischer, 1978; Gaehtgens et al., 1981a). The latter behavior is also exhibited by mammalian erythrocytes that have been made more rigid by glutaraldehyde fixation (Chien, 1975; Fischer, 1978). In mammalian definitive erythrocytes, the membrane exhibits “tank-treading”; that is, any point on the membrane will translocate all the way around the cell under flow conditions (Fischer and Schmidt-Schonbein, 1977; Fischer, 1978; Fischer et al., 1978). The general effect of membrane tank-treading is increased stability of cell orientation in flow and further lowering of blood viscosity, reducing the circulatory work requirement. Neither membrane tank-treading nor its physiological effects have been observed in nucleated erythrocytes of nonmammalian vertebrates, suggesting that tank-treading represents an evolutionary advance in erythrocyte design. Definitive mammalian erythrocytes also readily crenate, forming numerous surface protrusions, whereas those of nonmammals do not. In summary, definitive anucleate mammalian erythrocytes are morphologically much more responsive and behave like “fluid droplets,” while nucleated nonmammalian erythrocytes more closely resemble “solid bodies” (Fischer, 1978; Fischer er al., 1978). The basis for these physiological differences is to be found in large part in differences between the cytoskeletal systems of these two erythrocyte types. That of nucleated erythrocytes consists of the following major components: the MB of MTs; the cell surface-associated cytoskeletal network, or membrane skeleton (MS); and intermediate filaments (IFs) of the vimentin class. These components, interacting as a system, are believed to be responsible for the generation and maintenance of nucleated erythrocyte morphology and for mechanical properties critical to cell function. Of the three, only the MS has a counterpart in typical definitive mammalian erythrocytes. The basic features of the MB/MS system are illustrated in Fig. 3, using the dogfish erythrocyte as a typical example. A somewhat oversimplified diagram of the cytoskeletal system of mature nucleated erythrocytes is presented in Fig. 4. The MB is completely enclosed within the MS and tightly apposed to it as a supporting frame (Fig. 4a). The nucleus is suspended between MS networks lining the inner surface of the plasma membrane on opposite sides of the cell, producing a bulge in edge view (Fig. 4b). For simplicity, the diagram omits IFs, which traverse the region between nucleus and MS. In categorizing erythrocyte behavior as fluid versus solid, it is important to note that the fundamental distinction is not equivalent to primitive versus definitive, mammalian versus nonmammalian, or even nucleated versus nonnucleated. With respect to structure and behavior, primitive

FIG. 3. The cytoskeletal system of dogfish erythrocytes (Mustelus canis), typical of nearly all nonmammalian vertebrates. (a) Erythrocyte cytoskeleton produced by Triton X-100 lysis of cells under microtubule (MT)-stabilizing conditions; uranyl acetate-stained whole-mount, transmission electron microscopy (TEM). The marginal band (MB) and the nucleus (N)are densely stained. The membrane skeleton (MS) appears as a more lightly stained network bordered by the MB. (b) Thin cross section through MB in simultaneously lysed and fixed cell. MTs constituting the MB are enclosed with the MS at the cell extremity. Remnants of the membrane bilayer and hemoglobin (fuzzy clumps) are present due to the preparation method used. (c) MB isolated by detergent-based MS dissolution (whole-mount, TEM). (d) Lane I: SDS-PAGE pattern of whole cytoskeletons, showing spectrin-region (S) and tubulin-region (T) polypeptides, plus some actin (A, faint) and a few other components; lane 2: isolated MBs, at comparable tubulin-region loading, exhibiting the same tubulin pattern as in whole cytoskeleton and only minor amounts of other components. (b) From Cohen et al. (1982a); reproduced from the Journal of Cell Biology, 1982, 93, 828-838, by copyright permission of the Rockefeller University Press. (c and d) From Sanchez et al. (1990); reproduced from the European Journal ofcell Biology, 1990,52,349-358, by permission of Wissenschaftliche Verlasgesellschaft MBH.

CYTOSKELETAL SYSTEM OF NUCLEATED ERYTHROCYTES

a

43

b

FIG.4. Model of the nucleated erythrocyte cytoskeletal system, diagrammatically simplified. (a) Face view, showing the marginal band (MB) completely enclosed within the membrane skeleton (MS), and in contact with it. (b) Edge view, showing biconvexity due to the presence of the nucleus. Intermediate filaments, believed to connect the inner cell surface to the nucleus or to the opposing inner cell surface, are omitted for simplicity. Modified from Cohen and Nemhauser (1985), “Marginal bands and the cytoskeleton in blood cells of marine invertebrates,” in Blood Cells of Marine Invertebrates: Experimental Systems in Cell Biology and Comparative Physiology (W. D. Cohen, ed.), pp. 1-49, with permission of WileyLiss Div. of John Wiley & Sons, Inc.

and definitive erythrocytes of nonmammals are not significantly different, and the same can be said for primitive erythrocytes of mammals versus nonmammals, and for the anucleate erythrocytes of certain nonmammalian vertebrates versus nucleated ones, as documented in subsequent sections. Rather, the real distinction is between erythrocytes in which MBs, interacting with other cytoskeletal elements, play a role in the determination of erythrocyte morphology and mechanicalhheological properties (solid body behavior) and erythrocytes in which MBs have no such role (fluid droplets) (Cohen, 1978a; Fischer, 1978). 111. The Marginal Band of Mature Erythrocytes

All of the mature nucleated erythrocytes of nonmammalian vertebrates (Fig. 2, cells a-g) contain MBs. The MB is principally a hooplike continuous MT bundle located close to the plasma membrane in the plane of flattening. Few, if any, MTs are found elsewhere in the cells; thus, the MB is one of the simpler MT systems in terms of organization and spatial

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distribution. Therein lies its fundamental attraction as the subject of a variety of experimental studies in recent years. For erythrocytes of nonmammalian vertebrates, MB thickness in the light microscope and MT number per electron microscopic (EM) cross section are positively correlated with cell size (Small and Davies, 1972; Goniakowska-Witalinska and Witalinski, 1976; Cohen, 1978b). Thus, the Amphiuma erythrocyte MB (Fig. 2, cell a) is about 1pm thick and contains hundreds of MTs per cross section (Small and Davies, 1972), whereas the corresponding values for goldfish (Fig. 2, cell f ) are -0.1 pm with eight to 10 MTs (Weinreb and Weinreb, 1965). For diverse species representing most vertebrate classes, the relationship between cell long axis ( L ) and MT number (n) is roughly logarithmic; log n = 0.05L + 0.3, with greater deviations occurring in the smaller cells (derived from the data of Goniakowska-Witalinska and Witalinski, 1976). Species can thus be selected so as to provide MBs of size and thickness appropriate to a particular experimental problem. MBs are also present in mammalian blood platelets (Behnke, 1970b), nonmammalian vertebrate thrombocytes (Fawcett and Witebsky, 1964), and invertebrate nucleated erythrocytes and clotting cells, representing a wide phylogenetic range (Cohen and Nemhauser, 1985). Although these MBs are of considerable interest from a comparative standpoint, the discussion here focuses principally on MBs of vertebrate erythrocytes.

A. STRUCTURE In their original paper on the ultrastructural features of erythrocyte MBs, Fawcett and Witebsky (1964) raised fundamental questions regarding their construction. Remarkably, these questions have not yet been answered unequivocally for a single species. For purposes of discussion, it is helpful to set forth several hypothetical models (Fig. 5). A closed-hoop model (Fig. 5, model I) would seem least likely for any MB, although not totally absurd. MT annealing (Rothwell et al., 1986) allows for the possibility that a +MT end might grow around the cell perimeter and join onto its own -end, but such a rendezvous is certainly a complex scenario. In fact, closed MT hoops have never been seen separating from others during MB isolation experiments, and the model I prediction that MBs will always exhibit the same MT number at opposite sides in cross section is not supported by observation. Moreover, in at least some erythrocyte cytoskeletons, individual MTs appear to traverse the circumference more than one complete turn (Miller and Solomon, 1984; Cohen and Nemhauser, 1985). The overlapping MT segment model (Fig. 5, model 11) is also highly unlikely. Individual MTs, when traceable within erythrocyte cytoskel-

CYTOSKELETALSYSTEMOFNUCLEATEDERYTHROCYTES

I. CLOSED

11.OVERLAPPING SEGMENTS

HOOPS

3

P

V

4-

-4

A

4

111.SINGLE

VERY LONG MICROTUBULE

45

3-

A

4 SMALL NO. LONG MICROTUBULES OF SAME OR MIXED POLARITY

FIG. 5. Possible models of marginal band (MB) construction. Models I and I1 are highly unlikely for any MB; model I11 may be correct for MBs of some species; model IV is probably correct for MBs of most species. Numbers adjacent to arrowheads indicate the numbers of microtubules that would be observed in cross section through the MB at various points.

etons or isolated MBs, appear to be very long, and few MT ends are normally visible. In addition, kinetic data indicate that, for reassembling chicken erythrocyte MBs at least, there are probably only one to three free growing MT ends present (Miller and Solomon, 1984), and chicken erythrocyte MT protein characteristicallyforms very long MTs in vitro (Murphy and Wallis, 1983b).

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The literature thus supports models in which MBs are constructed of only one or a few extremely long MTs. For relatively thin MBs of small erythrocytes (e.g., birds and bony fish), the single coiled MT (Fig. 5 , model 111)may well apply. It may also be correct for the mammalian platelet MB, although this has not yet been demonstrated directly (Nachmias et al., 1977; Nachmias, 1980; White et al., 1986). For an elliptical erythrocyte in which L = only 10 pm, a single MT with but 10 windings would be almost 300 pm long! This model predicts a maximum difference of only one MT at opposite sides in a given MB cross section, regardless of the number of windings. Although not yet studied systematically, a difference of 1 is frequently observed in thin MBs such as those of goldfish, in which low MT number makes accurate counting feasible (W. D. Cohen, unpublished observations). However, the single-MT model cannot apply universally because, depending on the species, some (turtle and bullfrog) or all (mudpuppy) MBs have been found to contain MTs of mixed polarity, as determined using the tubulin “hook” method (Euteneuer et al., 1985). For these cells, at least, MBs are believed to consist of a relatively small number of very long MTs of opposite polarities (Fig. 5 , model IV). For two multiply wound MTs, model IV predicts a maximum difference of 2 at opposite MB sides regardless of the number of windings of either MT, with each additional multiply wound MT increasing the maximum difference by 1 . This is as yet untested. The polarity studies by Euteneuer et al. (1985) also revealed (1) a mirror-image relationship between MT polarity patterns in opposite-side MB cross sections, (2) relative constancy of MT number and polarity pattern throughout MB length (i.e., no MT overlap zone), and (3) variation in MT polarity patterns in different MBs of an individual animal and species. Features 1 and 2 are consistent with a model proposed originally for experimentally induced reassembly of the blood clam erythrocyte MB (Nemhauser et al., 1983) (Fig. 9, discussed in Section 111,D). Feature 3 implies a degree of permissible variability in the MB biogenetic mechanism. It is interesting that the percentage of cells having mixed MT polarity was highest in the largest cells studied (100% in the mudpuppy), lowest in the smallest ones (14%in the turtle), and intermediate in those of intermediate size (43% in the bullfrog) (Euteneuer et al., 1985). Such a correlation could be tested using much smaller erythrocytes of birds or bony fish, having very thin MBs. Direct determination of the actual number of MTs composing a given MB is quite difficult in practice. The possibility of artifact due to MT breakage during MB isolation or to poor in situ fixation cannot be eliminated with certainty. In addition, MB curvature precludes complete threedimensional reconstruction by serial sectioning, although up to 60% of MB

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length has been examined using a tilt stage (Euteneuer et al., 1985). The best current approach to this problem may be high-resolution videoenhanced contrast microscopy of relatively thin MBs undergoing experimentally induced disorganization in real time, combined with negative staining to verify that the MTs osbserved by video are truly single. With respect to three-dimensional arrangement, MBs do not consist of MTs packed uniformly into a tight cylindrical shape, but rather more asymmetrical ribbonlike arrays (Behnke, 1970a,b; Small and Davies, 1972; Cohen et al., 1982a). Negatively stained isolated MBs often look ribbonlike when flattened onto the substrate, and in scanning electron micrographs they appear as thick ribbons (Bertolini and Monaco, 1974; Cohen, 1978b; Sanchez et al., 1990). Electron microscopy of MBs in situ or after isolation frequently reveals inter-MT cross-bridges (e.g., Cohen et al., 1982a; Centonze et al., 1984), but their number and spatial distribution are unknown. In very large erythrocytes of amphibians (e.g., Amphiurna) the MB is more highly flattened and ribbonlike at the two ends of the elliptical cell than elsewhere. This was noted by Meves (191l), and can be seen immediately upon cell lysis under MT-stabilizing conditions (Cohen, 1978b). It is possible that such flattening permits the MB to negotiate a smaller radius of curvature at these extremities, thereby allowing large cells with otherwise thick MBs to attain elliptical, as opposed to discoidal. cell shape. Regardless of functional significance, the question remains open as to how this structural differentiation is achieved. B. MECHANICAL PROPERTIES The MB is flexible both in situ and after isolation; that is, it can bend and twist without breaking. This was well documented by Meves (191l), and Fawcett and Witebsky (1964) also noted that MBs could form and sustain loops in situ. Goniakowska-Witalinska (1974) observed figure-eight MB twisting in situ in amphibian erythrocytes swollen by exposure to hypoosmotic media, in which MBs appeared to be separated by a considerable distance from the plasma membrane except at the two ends of the cellular ellipse. MBs in erythrocyte cytoskeletons prepared by detergent lysis also typically twist into figure-eight forms (Bertolini and Monaco, 1974;Cohen, 1978b)(Fig. 6 a +. b), but here, twisting appears to be an accomodation to reduction in MS surface area, with the MB confined within. When the MS is removed from cytoskeletons containing figure-eight MBs, using high salt, proteases, or detergents (Cohen, 1982; Cohen and Ginsburg, 1986; Sanchez et al., 1990), the isolated MBs typically reassume a more planar configuration (Fig. 6c). Therefore, MS shrinkage or contraction probably

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-

(el tangential stripping

cell lysis

(a] cytoskeleton in situ

MS

Giz?

-

(b] f i g i r e 8 twisting

(cl isolated MB circularirat ion

FIG. 6. Mechanical behavior of the cytoskeleton and isolated marginal band (MB) in uitro. The nucleus is omitted for simplicity; for a detailed explanation, see text.

imposes forces on the MB sufficient to cause twisting (see Section V,D and Fig. 15). In a figure-eight twist, there are two possible mirror-image configurations: right-handed or left-handed. Counts in several amphibians have shown that the MB twist direction in figure-eight cytoskeletons is nonrandom, strongly favoring right-handedness (Cohen, 1978b). The reason for this is unknown, but MB construction by unequal numbers of MTs of opposite polarity (Euteneuer et al., 1985) could be a factor. Additional information on MB mechanics has been obtained by studying MB structure during isolation and subsequent experimentally induced disorganization (Fig. 6c-e). MBs in intact nucleated erythrocyte cytoskeletons typically retain the ellipticity of living cells after cell lysis (Fig. 6a). However, they become much more circular when released from the cytoskeleton by MS dissolution, regardless of the particular agent or conditions utilized (Bertolini and Monaco, 1974; Cohen, 1982; Cohen and Ginsburg, 1986; Sanchez et al., 1990)(Fig. 6c). Isolated MBs remain intact even during micromanipulation (Cohen, 1978b; Waugh et al., 1986; Waugh and Erwin, 1989),their integrity presumably attributable to the preservation of inter-MT cross-bridges. Isolated MBs transected accidentally, or experimentally by means of microneedles, typically open outward so as to become more linear (Fig. 6d). Continued proteolysis of MBs after proteolytic isolation from cytoskeletons frequently results in the “stripping” of MTs or MT bundles from the MB, with linearization tangential to the curved MB surface (Fig. 6e). Mechanical properties of isolated MBs have been studied by means of micromanipulation on hooks, accompanied by measurements using calibrated glass fibers (Waugh et al., 1986; Waugh and Erwin, 1989). MBs were sufficiently flexible to return to their original shape after extension

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into a highly elongated oval, maintaining essentially constant circumference during deformation and recovery (Fig. 6f). For MBs of the newt Notophthalmus uiridescens isolated using proteases or high KCl, average flexural rigidity (indicating resistance to bending) was -9 x lo-’’ dyn-cm’, and average extensional rigidity (resistance to increase in length produced by axial forces) was 0.017 dyn. Waugh and co-workers concluded that MBs are essentially inextensible compared with the erythrocyte membrane, implying maintenance of constant MB circumference during erythrocyte deformation. In addition, the flexural rigidity of isolated MBs was several thousand times greater than that calculated for the membrane, indicating that MBs stabilize the overlying cell surface against indentation. Cytoskeletal behavior, as summarized in Fig. 6, is consistent with a mechanical model in which the intact MB is stressed due to curvature [originally described as bending strain (Cohen, 1978b; Joseph-Silverstein and Cohen, 1984, 198S)l. In other words, the curved MTs behave as if preferring to be straight. Within the complete system, the MB is assumed to be deformed into an ellipse through forces applied by other cytoskeletal elements (Cohen, 1978b; Waugh and Erwin, 1989) (Fig. 6a). Release from the cytoskeleton during isolation would produce MB circularization due to the spontaneous equalization (i.e., redistribution) of strain (Fig. 6a-c), and loss of continuity by transection or proteolytic stripping would result in linearization as bending strain is relieved (Fig. 6d and e). The mechanical characteristics of the MB in mature nucleated erythrocytes thus indicate that it is a strained flexible structural frame, that is, a relatively passive and stable skeletal structure providing for the attachment and support of other structural elements. This is considered further in Section II1,E. C. MOLECULAR COMPONENTS 1. Tubulin

MBs consist principally of MTs, and thus tubulin is their major molecular component. The properties of nucleated erythrocyte tubulin have been studied by Murphy and co-workers, using chicken erythrocytes (Murphy and Wallis, 1983a,b, 1985; Murphy et al., 1987; Rothwell et al., 1986). Tubulin was found to constitute 1% of total cell protein, with less than one-half in the MB. Erythrocyte tubulin assembled with greater efficiency and lower nucleation rate than did brain tubulin, producing much longer MTs under similar conditions. It was proposed that the assembly of long MTs typical of MBs might be regulated by a tubulin oligomer pool, limiting the nucleation rate and accounting for at least some non-MB tubulin in the cell (Murphy and Wallis, 1985).

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The characteristics of chicken erythrocyte tubulin in uitro may derive, in part, from its unusually divergent p subunit (Murphy and Wallis, 1983a; Murphy et al., 1987). This isotype, cp6, is the product of a gene expressed only in developing erythrocytes and thrombocytes in chick bone marrow, i.e., cells assembling MBs (Murphy et al., 1986). It is one of six known chicken p-tubulin isotypes, constituting 95% of the p-tubulin in mature chicken erythrocytes, cp3 making up the remaining 5% (Joshi et al., 1987). Another p-tubulin isotype, MPl , identified in the mouse, is proposed to be involved in MB formation in mammalian primitive erythrocytes and platelets. It differs from cp6 in 18% of amino acid residues, but also shares with it unique amino acid similarities at many positions throughout its structure (Wang et al., 1986; Murphy et al., 1987). Since Mpl was identified using a mammalian bone marrow library, and since mammalian bone marrow erythroblasts do not contain MBs (Repasky and Eckert, 1981, vs. Grasso, 1966), it is possible that Mpl is platelet specific and that there exists another p-tubulin unique to mammalian primitive erythrocyte MBs. It is important to note, however, that the broader question as to whether MB-specific p-tubulins occur universally has not yet been addressed systematically. Data on MB tubulins in other species are more limited. After disassembly of dogfish erythrocyte MBs in living cells at low temperature, four major polypeptides increased in the cytosol (Cohen et al., 1982a),all in the tubulin region of sodium dodecyl sulfate (SDS) gels. Isolated dogfish erythrocyte MBs also yield these four polypeptides, with the same stoichiometry as in the whole cytoskeleton (Sanchez et al., 1990),and preliminary Western blotting indicates that all are tubulins (Sanchez and Cohen, 1990 unpublished observations). MT protein, obtained from dogfish erythrocyte MBs either by low-temperature extraction of whole cytoskeletons or by isolated MB disassembly, reassembles readily into MTs in uitro (Cohen et al., 1982b). 2 . Other MB-Associated Proteins The term “MB-associated proteins” as used here includes those indirectly associated with the MB as well as MT-associated proteins (MAPS) having MT binding sites. Possible functions for the latter include nucleation and stabilization of MTs during MB assembly, cross-bridging of MTs into bundles, and linking of the MB to the MS and the plasma membrane. In erythrocytes of the amphibian Bufo marinus (marine toad), the MB contains a protein antigenically similar to brain MAP 2, and gold-labeled anti-MAP 2 binds to MB cross-bridges in this species (Sloboda and Dickersin, 1980; Centonze et al., 1984, 1985). In addition, a protein of M , 280,000 obtained from the cytoskeleton cross-reacts with MAP 2 antibody and can cause bundling of reassembled brain MTs (Centonze and Sloboda, 1986).

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Recently, Feick et al. (1991; see also the review by Wiche et al., 1991) have isolated syncolin, a high-molecular-weight MT-bundling protein from chicken erythrocytes that is probably the same protein studied in Sloboda’s laboratory. The starting material consisted of taxol-polymerized MTs obtained from extracts of sonicated cells, from which syncolin was released at low temperature. Syncolin comigrated with MAP 2 in SDSpolyacrylamide gel electrophoresis (SDS-PAGE) (M, 280,000) and also showed cross-reactivity with anti-MAP 2. However, it was quite different in structure, exhibiting heat lability and assembling into 13-nm spheroids of 990,000 average molecular weight, rather than being fibrous. Two observations suggested to the authors that syncolin was involved specifically in MT-MT interactions, as opposed to functioning as either a spatial determinant or a MT-MWmembrane link: (1) syncolin remained diffusely associated with the cytoskeleton after low-temperature MB disassembly, but reassociated with the MB after temperature-induced MB reassembly, and (2) in mature erythrocytes, syncolin showed immunofluorescent colocalization only with MBs, even when MBs were highly twisted. These observations do not constitute a compelling argument for MT-MT bridging, however, since the interface between MB and MS would likely be intact even in cytoskeletons containing highly twisted MBs. It would thus be helpful to determine syncolin distribution in intact chicken erythrocyte cytoskeletons versus isolated MBs. Other studies have not demonstrated the presence of high-molecularweight MAP 2- or syncolin-like MAPs, or have yielded equivocal results. Temperature cycling experiments revealed only two putative MAPs of M , -80,000 and 100,000 that coassemble with an invertebrate (blood clam) erythrocyte MB in living cells (Joseph-Silverstein and Cohen, 1986). In Xenopus erythrocytes, cytoskeletal polypeptides of appropriate molecular weight did not cross-react with antibodies to mammalian or avian MAP 2 (Gambino et al., 1985). MBs isolated from amphibian (Triturus cristatus) erythrocytes were reported to contain numerous non-tubulin components, including actin, myosin, M , 90,000 glycoprotein, and other highmolecular-weight proteins (Monaco et al., 1982), but many of these appear to be contaminants resulting from incomplete MS removal (Cohen and Ginsburg, 1986). Neither myosin nor high-molecular-weight MAPs have been found in MBs isolated from dogfish erythrocytes, although minor amounts of spectrin and actin have been observed (Sanchez et al., 1990). Tau protein, but not MAP 2, has been found in MT protein preparations purified from chicken erythrocytes by temperature cycling, and the MB of chicken erythrocyte cytoskeletons binds anti-tau in situ (Murphy and Wallis, 1985). Although a high-molecular-weight component possibly corresponding to syncolin is present in whole dogfish erythrocyte cytoskeletons, MBs isolated from these cytoskeletons by a recently developed

-

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WILLIAM D.COHEN

detergent-based method have been found to contain tau proteins, but not high-molecular-weight MAPs (Sanchez et al., 1990; Sanchez and Cohen, 1990). The presence of tau in MBs may reflect a role in MT nucleation (Murphy and Wallis, 1985), MT-MT cross-bridging, or both. Considerable information on tau properties is now available, derived in part from studies related to its presence in the paired helical filaments of Alzheimer’s disease (Ihara etal., 1986; Grundke-Iqbal et al., 1986). Like MAP 2, tau proteins have an MT-binding domain consisting of three 19-amino-acid repeats near the carboxy-terminal end (Lee et al., 1988). The long amino-terminal remainder of the molecule, thought to project away from the MT, probably does not have an MT crosslinking function. Rather, the shorter carboxyterminal portion beyond the repeats (Lewis et al., 1989), or possibly including the last repeat (Lewis and Cowan, 1990), appears able to bind to the same region of another tau (or MAP 2) molecule. Thus, binding between two tau molecules on adjacent MTs would create an inter-MT cross-bridge. The ability of tau to induce MT bundling in living cells has been demonstrated elegantly by engineering the expression of tau or tau variants in cultured cells which normally do not produce it (Kanai et al., 1989; Lewis et al., 1989). One appeal of a tau-tau cross-bridge is that MB MTs would require only one type of MAP binding site, rather than a different one for each end of a more specialized bridge protein. In addition, if the same type of bridge were made with a membrane/MS-bound form of tau, MTs could link both to the inner cell surface and to other MTs without having to bind MAPs selectively. Such a mechanism is speculative, of course, and a membrane-bound form of tau has not yet been reported. However, polymerized actin has been localized to the plane of the mature erythrocyte MB (Kim et al., 1987), and binding of tau protein to actin has been demonstrated in uitro (Selden and Pollard, 1983). Studies in two distantly related species suggest that tau is a common component in vertebrate MBs (Murphy and Wallis, 1985; Sanchez et al., 1990). As shown in curved paracrystals, tau has elasticity that decreases with increased phosphorylation [length and stiffness increase (Lichtenberg ef al., 1988; Lee, 1990)l. Such elasticity could be an important factor in MB mechanical properties. MTs bridged by tau carboxy terminal regions would be very closely spaced (Lewis ef al., 1989), as usually observed in situ and in isolated MBs. However, relatively weak tau-tau binding might also permit temporary separation of the MTs making up the MB in regions of extensive cell deformation, with MTs “zipping” back together upon removal of the deforming forces. This could explain why MB MTs in situ are sometimes observed to be separated from each other by a considerable distance, whereas in isolated MBs they are usually tightly bundled.

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Actin and ezrin are two additional proteins that appear to be MB associated. Use of rhodamine-labeled phalloidin on mature chicken erythrocytes has revealed polymerized actin with a distribution similar to that of MB tubulin (Kim et a f . , 1987). It is not yet clear whether any of this actin composes the MB itself, or whether it is principally associated with the MS in the plane of flattening. Another protein localized to the MB plane via monoclonal antibody binding has at least some properties identical to those of ezrin (Birgbauer and Solomon, 1989), a M , -80,000 component isolated previously from intestinal brush border (Bretscher, 1983). Localization of erythrocyte ezrin in the plane of flattening during MB biogenesis follows, rather than precedes, that of the MB MTs, and thus it is not a likely candidate for the spatial control of MB formation in erythroblasts (Birgbauer and Solomon, 1989). However, it might well be involved in the control of MB reassembly after experimentally induced disassembly. INDUCED MB REASSEMBLY IN MATURE CELLS D. EXPERIMENTALLY The basic sequence of morphological stages during the differentiation of nucleated erythrocytes, and the accompanying changes in cytoskeletal properties, are summarized in Fig. 7. The focus in this section is on the mature erythrocyte MB (Fig. 7, cell 4), with earlier stages of the sequence discussed in Section IV. Colchicine or related MT inhibitors applied at physiological temperature appear to have little or no effect on MB MTs or the shape of mature cells, regardless of species (Fig. 7, cell 4-4a) (Behnke, 1970a,b; Barrett and Dawson, 1974; Cohen et al., 1982a; Gambino et af., 1985; Kim et al., 1987). In some species (e.g., frogs), exposure to low temperature (0-4°C) also has no effect, whereas in others (e.g., chicken, dogfish, and blood clam) such exposure induces MB disassembly (cell 4+4b) (Behnke, 1970a,b; Cohen et al., 1982a; Nemhauser et al., 1983). Despite lowtemperature MB disassembly, flattened elliptical cell morphology is retained (Fig. 7, cell 4b). Return of such cells to physiological temperature appropriate to the species results in MB reassembly, usually in 1-2 hours (Fig. 7, cell 4b+4c). Although colchicine and related inhibitors do not induce MB disassembly, they block reassembly at physiological temperature after low-temperature MB disassembly (Fig. 7, cell 4b+4d) (Cohen et al., 1982a; Nemhauser et af., 1983). In the case of mature blood clam erythrocytes, a pair of centrioles is physically associated with the MB in every mature cell (Cohen and Nemhauser, 1980) (Fig. 8). These centrioles form part of an MT organizing center (MTOC) that participates in temperature-induced MB reassembly (Nemhauser et af., 1983). MT growth initiates at this MTOC, and individual MTs or thin MT bundles elongate so as to contact the MS within

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KEY: +T=IOWtemp. (0-4Oc) 4 T =physiol. temp COI=colehiclne

CyB= cylochalasin B CaI=calcium ionophore c> =sphere, no MB Q=flattened disc +MB flattened ellipse+ MB

o=

blood clam)

o=

flattened ellipse, noMB

FIG. 7. Properties of the cytoskeleton during morphogenesis and maturationof nucleated erythrocytes. The natural pathway from erythroblast to mature erythrocyte is presented in steps 1-4 (heavy arrows). All other steps (2a-2d; 4a-4e) represent experimental manipulations. See text for a detailed explanation.

-15-20 minutes. Inter-MT cross-bridging occurs after a significant number of MTs are already at the periphery in the plane of flattening. In the sequence illustrated in Fig. 9, it is proposed that MTs grow in opposite directions around the cell periphery (Fig. 9a) and cross each other distally (Fig. 9b), eventually closing the MB proximal to the MTOC (Fig. 9c) and continuing on in their respective directions. This would produce MBs of mixed MT polarity, corresponding to model IV (Fig. 5, Section 111,A). In contrast, centriole-containing MTOCs are not observed during temperature-induced MB reassembly in mature chicken erythrocytes (Miller and Solomon, 1984). Here, MT growth appears to be largely localized to the periphery, and MT numbers as observed in whole mounts are consistent with model I11 (Fig. 5 ) . With the exception of skate erythrocytes (Cohen, 1986), MB-associated centrioles have not been found in vertebrate erythrocytes; however, thin sections of amphibian and reptilian erythrocytes reveal centrioles in regions near the nucleus (Gambino et al., 1984; Euteneuer et a / . , 1985).

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FIG. 8. Blood clam erythrocytes and their cytoskeletal system. (a) Living erythrocytes, seen by phase contrast. Most are ellipsoidal, but occasionally singly pointed cells are observed(arrowhead1. (b-d) Erythrocyte cytoskeletons produced by Triton X-100lysis of cells under microtubule (MT)-stabilizingconditions. (b) A pair of centrioles is associated with the marginal band (MB) in every cell, appearing as adjacent dense dots in phase contrast (arrowhead). N, Nucleus. (c) Uranyl acetate-stained cytoskeleton whole-mount, transmission electron microscopy (TEM). MB-associated centrioles (Ce)are densely stained. The MB is, in this case, twisted into a figure eight, with the membrane skeleton (MS) more readily visible where overlapped near the crossover point. N, Nucleus. (d) A centriole pair in thin-section, TEM. In typical elliptical cells, centrioles are usually located at or near one end of the ellipse. In pointed cells, centrioles are invariably located at the point. (a,b, and d) Noetia ponderosa; (c) Anadara transversa. From Cohen and Nemhauser (1980) and Nemhauser et al. (1983);reproduced from the Journal of CellEiology, 1980,86,286-291; and 1983, %, 979-989, by copyright permission of the Rockefeller University Press.

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FIG. 9. Proposed model for experimentally induced marginal band (MB) reassembly in blood clam erythrocytes. (a) Microtubules (MTs) initiate at the centriole-containing organizing center and grow toward the distal end of the cell in diverging directions. (b) MTs pass each other distally and continue growth around the periphery. (c) MTs pass each other proximally, closing the MB. The resulting MB contains MTs of mixed polarity. From Nemhauser et al. (1983); reproduced from the Journal of Cell Biology, 1983,%, 979-989, by copyright permission of the Rockefeller University Press.

E. MB FUNCTION I N MATURE CELLS Retention of mature erythrocyte shape after MB disassembly was initially interpreted as indicating nonparticipation of MBs in cell shape maintenance (Barrett and Dawson, 1974; Behnke, 1970a). This was somewhat illusory, however, because cells were examined under the static conditions of microscopic examination. Challenged with mechanical stress (fluxing through capillary tubes) or hyperosmotic conditions, both dogfish and blood clam erythrocytes pretreated so as to lack MBs behave quite differently from those containing MBs ( Joseph-Silverstein and Cohen, 1984,1985), as summarized in Fig. 10. Here, mature cells with and without MBs at physiological temperature were prepared by MB disassembly at low temperature followed by rewarming with or without colchicine. Mechanical stress produced a significant proportion of deformed cells only in cells lacking MBs (Fig. 10, sequence a-c versus a-d). The same was true for cells without MBs at low temperature, as compared with those containing taxol-stabilized MBs (Fig. 10, a-g versus a-f). These results indicate that MBs can restore and/or stabilize mature erythrocyte shape, regardless of temperature. However, no quantitative information is available on the extent to which MBs resist an imposed force, the yield point or range at which MB deformation begins, or the rapidity of MB recovery and restoration of cell shape. The relationship between MB thickness and the effect of imposed forces is similarly unexplored.

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FIG. 10. Marginal band (MB) function in mature erythrocytes. General effects of mechanical stress (cells with fist) and hyperosmotic conditions ( t 0s) on cells containing or lacking MBs, as observed in dogfish erythrocytes. t T, Physiological temperature (20-22°C); T, 0°C; tax, taxol; col, colchicine. See text for a detailed explanation.

In experiments testing MB influence on cellular responses to hyperosmotic conditions ( Joseph-Silverstein and Cohen, 1984), significantly greater numbers of cells “shriveled” if they lacked MBs (Fig. 10, a-b versus a-e). This indicates that the MB acts as an internal frame supporting the cell surface from within, again resisting distortion of mature cell shape. An interesting feature of the shriveling response was its all-or-none

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nature; that is, cells lacking MBs were either relatively normal in morphology or drastically shrunken and distorted, without intermediate stages. One possible explanation is that there is resistance to shrinkage until cellular contents suddenly burst outward through a localized weak spot in the MS and the bilayer. This would parallel the mechanism of hypoosmotic lysis as currently understood for both mammalian and nonmammalian erythrocytes (Lieber and Steck, 1982a,b;Lieber et al., 1987).The capacity of the MB to support and deform the cell surface from within has also been evident in other studies with mature dogfish erythrocytes. These cells acquire pointed ends during long-term incubation in Elasmobranch Ringer’s solution at physiological temperature, a phenomenon useful for testing the causal relationship between cell and MB pointedness. These experiments are included in the discussion of the MS (Fig. 16, Section V). The mechanical properties of MBs (Section II1,B) indicate a functional mechanism based on flexibility, resistance to bending, and the capacity to return to equilibrium shape by the equalization (i.e., redistribution) of strain. These features would all appear to be independent of MT polarity. Therefore, they are compatible with the observed variability of MT polarity within erythrocytes of an individual organism (Euteneuer et d., 1985), and with the possibility that different modes of MB biogenesis (Section IV) or reassembly can achieve the same mechanical end in different species and cell types. TO FRIEDRICH MEVES F. A TRIBUTE

This discussion would be incomplete without reference to the work of Meves, who began study of the “marginal ring” in salamander erythrocytes in 1903, equipped only with a light microscope. Challenged by skeptical contemporaries, Meves showed convincingly that the MB was not a cytological artifact by partially isolating it and by devising special staining procedures that revealed MB substructure. The following admirable observations and conjectures are taken from his classic 1911 paper (translated from the German): One can perceive parallel lines within the ring which become more distinct after the cell body has lost its hemoglobin. The marginal ring now appears to be composed of many ultrafine filaments running in parallel or, what is just as possible, of a single, uninterrupted filament which is wound into a skein along the edge of the blood disk. In the pole areas the filaments often maintain larger intervals between each other; the skein, if one exists, is looser in these areas. . . . In blood cells treated with a gentian violet solution, one frequently observes loop formations at one or both poles of the marginal ring, which are probably caused by torsion of the ring. . . . It is well known that the red blood corpuscle can change its shape passively due to a mechanical influence, be it within the body or without; however, it assumes its original shape as soon as the force ceases. This is made

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possible by the elasticity inherent in the marginal ring, which allows it to return to its natural state.

IV. Marginal Band Biogenesis and Function during Erythrocyte Morphogenesis The morphogenetic sequence for nucleated vertebrate erythrocytes and accompanying cytoskeletal properties are summarized in Fig. 7. The first of three major stages encompassed is jattening, or conversion of the spheroidal erythroblast to a flattened disk concomitant with MB biogenesis (Fig. 7, cell 1-2) (Barrett and Scheinberg, 1972; Barrett and Dawson, 1974; Dorn and Broyles, 1982). The second stage is elliptogenesis, in which the flattened discoidal cell becomes ellipsoidal (Fig. 7, cell 2+3), and the third is maturation, during which cell morphology and properties of the erythrocyte cytoskeletal system change toward greater stability (Fig. 7, cell 3+4) (Barrett and Scheinberg, 1972; Barrett and Dawson, 1974; Dorn and Broyles, 1982, Kim et al., 1987). Details of the sequence and specific timing of major structural and biochemical events are not yet well known. Thus, the separation between elliptogenesis and maturation indicated in Fig. 7 may be somewhat artificial in that at least some properties associated with maturation probably begin to appear during elliptogenesis. Moreover, it is not yet clear whether a discoidal stage precedes the generation of ellipsoids in all species (Twersky et al., 1990). However, such definition and separation of stages are convenient here for purposes of discussion. A. MB BIOGENESIS AND THE MECHANISM OF CELLFLATTENING

MB biogenesis is considered a causal factor in the flattening of nucleated erythrocytes. This was evident in the work by Barrett and Dawson (1974) on chick bone marrow cells, in which newly flattened discoidal cells exposed to low temperature reverted to spheres (Fig. 7, cell 2-2b). These spheres reflattened upon restoration of physiological temperature (here, 37"C), even if cytochalasin B was present (Fig. 7, cell 2b+2c), but such reflattening was blocked by colchicine (Fig. 7, cell 2b+2d). However, exposure to colchicine did not cause MB disassembly in newly flattened discoidal cells maintained at physiological temperature (Fig. 7, cell 2-2a). One fascinating observation was the initiation of flattening while cultured erythroblasts were still physically associated as postmitotic daughter cell pairs (Barrett and Scheinberg, 1972), a phenomenon worthy of further investigation.

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Information on properties of immature flattened discoidal cells derives principally from studies of chicken erythrocytes, which have cold-labile MBs even after maturation. Far less is known about discoid cells of other species, such as amphibians (Dorn and Broyles, 1982), in which the MB of mature elliptical cells is stable at low temperature (Fig. 7, cell 444a). An early electron microscopic study of MB biogenesis in chick primitive erythrocytes showed that small MT bundles first appeared near the cell surface in “basophilic erythroblasts” at about 2 days of incubation (Small and Davies, 1972), with complete MBs in “polychromatophilic erythroblasts” at 3 days. More recently, anti-tubulin immunofluorescence has been used to study MB biogenesis in chicken erythroblasts of both the primitive (Kim et al., 1987) and definitive series (Murphy et al., 1986), the latter using bone marrow of chicks made anemic by phenylhydrazine. In this case, MTs were initially organized radially about centrosomes, were subsequently reorganized into cytoplasmic “wreaths” without centrosome association, and finally became tightly bundled MBs at the periphery. In immature primitive erythroblasts, centriole-containing MTOCs were observed at the cell periphery (Kim et al., 1987). Here, however, assembling MTs formed bundles running throughout the cytoplasm, but were not organized into wreaths prior to MB formation. Disagreement on this intermediate stage might be due to a real difference between primitive and definitive MB biogenesis in chicken erythrocytes, but it could also reflect differences in experimental procedures or in specific stages examined. Neither of these studies attempted direct correlation of cytoskeletal structure with cell morphology, so it is unclear whether cells containing the cytoplasmic MT bundles or the MT wreaths had just begun to flatten, or whether they were already flattened (discoidal). When these observations are compared with ones made on amphibian erythroblasts (Ginsburg et al., 1989), the picture becomes more complex. The axolotl larval spleen develops initially as a closed sac containing differentiating nucleated erythrocytes, including spheres, disks, and ellipses. However, it also contains a significant percentage of unusual singly or doubly pointed erythroid cells, with correspondingly singly and doubly pointed MBs. Similar pointed erythrocytes have been noted previously in the circulation of phylogenetically diverse species including the chicken (Lucas and Jamroz, 1961), skate (Cohen, 1986), and slender salamander (Cohen, 1982), but never in such large numbers. Many of the pointed splenic erythroblasts were found to contain a pair of centrioles close to a pointed end, suggesting MTOC function and the hypothetical sequence for MB biogenesis illustrated in Fig. 1 I (Ginsburg et al., 1989). Here, a centrosome nucleates MTs that ultimately form two bundles growing in opposite directions around the periphery (Fig. 1 la). The bundles meet to produce a

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FIG. 1 1 . Proposed model for marginal band (MB) biogenesis and cellular morphogenesis in amphibian erythroblasts. Stages of microtubule (MT) initation and growth from an organizing center (a and b) are similar to those for blood clam MB reassembly (Fig. 9). The cells are initially spheroidal (a), with doubly pointed and singly pointed cells (b and c) following as intermediate stages. MB closure produces a discoidal cell (d). Adapted from Ginsburg et al. (1989), “Cellular morphogenesis and the formation of marginal bands in amphibian splenic erythroblasts,” Cell Motility and the Cytoskelefon U ,159-168, with permission of WileyLiss Div. of John Wiley & Sons, Inc.

doubly pointed cell (Fig. 1lb), followed by closure of the distal (Fig. 1Ic) and then proximal ends (Fig. 1Id). Closure could not involve annealing, because the meeting MT ends would all have the same polarity, but it might be effected by cross-bridging between MTs of the two bundles and their continued peripheral growth in opposite directions [as suggested for blood clam MB reassembly (Fig. 9)]. A critical step in testing this proposal would be the establishment of an in uitro amphibian erythroblast culture system in which the complete differentiation sequence could be followed in individual cells. Although this has not yet been accomplished, the production of singly and doubly pointed cells during amphibian erythrogenesis has been verified in recent time-course studies of Xenopus during recovery from phenylhydrazine-induced anemia (Twersky et al., 1990) (Fig. 12). In considering MB biogenesis, it is important to remember that there may be different modes of MB construction in different species. Thus, intermediate stages in amphibians may prove to be dissimilar to those in chickens, with the end products (i.e., MBs) nevertheless structurally and functionally equivalent. Ultimate formation of a mechanically functional MT bundle is a common feature of MB biogenesis, regardless of the particular structural path taken. The properties of a potential erythrocyte MT-bundling protein such as syncolin are thus of particular interest. In dividing chicken erythroblasts, syncolin distribution differed from that of tubulin. However, it became associated with MTs of the forming MB at later stages, and was localized exclusively to the MB of mature cells (Feick et al., 1991). As noted by Feick e f al., it remains to be determined whether syncolin is an

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FIG. 12. Examples of pointed cells appearing in large numbers in the circulation of Xenopus laeuis during recovery from experimentally induced (phenylhydrazine) anemia. (a) Doubly pointed; (b) singly pointed with a long point; (c) singly pointed with a shorter point. Such cells are rarely observed in normal animals. Phase contrast video microscopy of glutaraldehyde-fixed cells. From Twersky et a / . (19%).

initial causal factor in the bundling of erythroblast MTs or whether it functions to further stabilize existing MT bundles. Study of erythrocyte tau distribution during MB biogenesis would similarly shed some light on its possible role as a bundling protein, and clarify its temporal relationship to syncolin binding. In both splenic amphibian erythroblasts and chicken primitive-series erythroblasts (Ginsburg et al., 1989; Kim et al., 1987),centriole-containing MTOCs are located closer to the cell periphery than in many other cell types. These MTOCs presumably function in MB biogenesis both to nucleate MTs and to influence their spatial distribution, as appears to be the case during MB reassembly in blood clam erythrocytes (Nemhauser et al., 1983). MTs nucleated by centrosomes in interphase cells exhibit steadystate “dynamic instability” (e.g., Mitchison and Kirschner, 1984; Schulze and Kirschner, 1986), with some growing at the same time that others rapidly disassemble. A mechanism can thus be envisioned in which peripheral MTOC localization reduces the angle at which some growing MTs approach the cell surface, permitting them to follow the surface contour and to make lateral stabilizing contacts with it. These MTs would continue to elongate at the expense of others that had grown in a different direction (i.e., toward the cell interior) and remained dynamically unstable. With respect to additional molecular mechanisms involved in MB biogenesis, synthesis of MB-specific cp6 tubulin is coincident with that of hemoglobin (Murphy et al., 1986). Nevertheless, the cp6 isotype can copolymerize with other tubulins in uitro and can participate in the formation of interphase MT arrays and functional mitotic spindles after transfection into living cultured cells that normally do not contain it (Joshi et al., 1987; Baker et al., 1990). Conversely, assembly of non-MB (brain) tubulin within MT-depleted chicken erythrocyte cytoskeletons produces MB-like structures containing very long MTs (Swan and Solomon, 1984). Questions thus arise as to how and why the divergent cp6 isotype is produced at the time of MB assembly.

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Assessment of the significance of MB-specific tubulin isotypes demands consideration of the real environment in which the erythrocyte cytoskeleton functions. Assembly of MB-like structures from brain tubulin in cytoskeletons in uitro (Swan and Solomon, 1984), or even of incorrect isotypes into a normal-looking MB in a living cell (if achieved), tells us little about functional properties of the cell. Even mature erythrocytes with no MB have relatively normal morphology (Fig. 7, cells 4b and 4d) when left unperturbed! The significance of cp6 may well lie in the mechanical requirements for long, stable MTs that can effect cell flattening, and ultimately provide critical cytoskeletal responses in the bloodstream (Fig. 10). This is in accord with the suggestion that MTs with reduced cp6 content are less stable; while they may help generate cell flattening, their eventual disassembly would leave more stable cp6-containing MTs to constitute the mature MB. This could account, in part, for reduced MT numbers in the mature cells of some species (Baker et al., 1990). The real question, then, is whether MBs containing incorrect isotypes would be fully functional in the living animal, since even slight alterations of MB mechanical responsiveness might lead to lethal blockage of capillaries. Similarly, incorporation of cp6 into normal-looking, functioning mitotic spindles in cultured cells tells us little about true function. Incorrect mitotic distribution of only an occasional chromosome during early development in the living animal could be lethal. Thus, the consequences of natural selection with respect to both erythroblasts and mitotic cells would be strict developmental programming and the evolutionary conservation of tubulin isotypes. Is this testable? Developments in viral transfection of chicken erythroblast lines, identification and manipulation of tubulin genes, and achievement of terminal erythrocyte differentiation in culture suggest that in vitro production of nucleated erythrocytes containing variant tubulins in their MBs may soon be possible. Initially, experimental comparison could be made between their mechanical/rheological properties and those of normal cells in uitro. The ultimate goal, however, would be functional comparison of cells with normal versus genetically manipulated cytoskeletal systems in the living animal, either by transfusion of in uitro-produced cells into the bloodstream or by engineered production of genetically variant erythrocytes during otherwise normal development. The mechanism by which MB biogenesis causes cell flattening is a separate unresolved problem. As a point of departure, the relatively simple hypothetical model illustrated in Fig. 13 can be considered. Here, the forming MB is depicted as a tight multiply wound coil that gradually expands outward like the spring winding mechanism of a clock. Such expansion does not require MT growth; rather, the release of bending strain by the initial coil drives the expansion. The MT slides along itself as

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a

tight coil expanded coi I FIG. 13. An “expanding coil” mechanism as a possible basis for marginal band (MB) function during erythrocyte flattening. In this structurally oversimplified version, an initially tight microtubule (MT) coil (a) expands outward (b) by the release of bending strain, establishing a plane of flattening. A mechanism of this type would require only mechanical properties already attributed to the MB of mature cells, as discussed in the text.

the diameter of the ring increases, without requiring motile cross-bridges. Reduction in the number of MTs appearing in MB cross sections, observed in some species, is a natural consequence of the process. As presented in pure form in Fig. 13, the model is no doubt unrealistic. MT distribution during MB biogenesis in chick or amphibian erythroblasts is not a neat tight coil, but rather a looser arrangement of curved MT bundles, or a wreath, as noted earlier in this section. However, the same basic mechanism might be at work, with curved MTs or MT bundles pressing outward until meeting MS resistance. Once such pressure began to establish a plane of flattening, it is likely that all additional MTs would find their way into the same plane, since this plane would permit the greatest outward movement and maximal MT straightening. One appeal of such a mechanism is simplicity: It requires only mechanical properties already attributed to the MB of mature cells, with no need to invoke predetermined peripheral “guidetracks.”

B. ELLIPTOGENESIS Little is known about the mechanism by which the discoidal MBcontaining cell becomes elliptical. Isolated MBs tend to assume either a circular or slightly elliptical configuration (Fig. 6c), whereas MBs are usually highly elliptical in situ (Fig. 6a). It is unlikely that isolated MB

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circularization involves loss of MB structure, because it occurs regardless

of the particular isolation medium or technique used (Bertolini and Monaco, 1974; Cohen, 1978b; Waugh and Erwin, 1989; Sanchez et al., 1990). This indicates that the MB alone does not generate ellipticity. In our model (Fig. 4), ellipticity is imposed by the MS through application of force asymmetrically across the flexible MB. Experiments and calculations by Waugh and Erwin (1989) indicate that a force of -12.5 x dyn is needed to deform a circular newt MB into an appropriately elliptical one. They determined that sustained support of this force by the nucleated erythrocyte membrane (presumably, the MS) would be possible if the membrane had greater rigidity than that of the mammalian definitive erythrocyte [shear modulus ( p ) = 0.07 dyn/cm versus -0.01 dyn/cm]. If not, then a mechanical contribution to ellipticity by other cytoskeletal elements, possibly IFs, would be required. Actually, membrane microaspiration experiments yielded a p value of 0.07 dyn/cm or greater for nucleated erythrocytes of representative bony fish, amphibians, reptiles, and birds (Waugh and Evans, 1976),but the extent to which MB presence influenced the data is unknown. Clarifying information might well be obtained by repeating microaspiration studies on experimentally produced erythrocytes lacking MBs versus ones containing MBs ( Joseph-Silverstein and Cohen, 1984). A direct experimental approach to the mechanism of elliptogenesis is inherent in the work of Dorn and Broyles (1982). These workers used density gradients to separate developing erythrocytes during metamorphosis in the bullfrog Rana catesbiana, obtaining essentially pure preparations of disks versus ellipses for hemoglobin analysis. Similar preparations should be valuable for comparing cytoskeletal molecular components before and after elliptogenesis, and possibly for attempts at obtaining synchronous elliptogenesis in uitro. With respect to the latter, it is important to note that the mechanisms involved in both flattening and elliptogenesis are intrinsic; they do not require external forces generated in the bloodstream. This is demonstrated by the terminal differentiation of avian erythroblasts in cultures (Beug ef al., 1982)and of larval amphibian erythroblasts confined within the developing spleen in uiuo (Ginsburg et al., 1989). Thus, nucleated erythrocyte morphogenesis anticipates functional requirements for flow conditions. C. MATURATION The term “maturation” encompasses the emergence of several cytoskeletal properties, one of which is retention of cell shape at low temperature in species that have cold-labile MBs (Fig. 7, cell 444b). It is not

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known whether such stabilization follows elliptogenesis or is concomitant with it, but “setting” of the MS to differentiated morphology is apparently involved (Barrett and Scheinberg, 1972; Barrett and Dawson, 1974; see Section V,C). In other species, MBs of mature cells are cold stable. The basis for such differences has not really been explored, but lowtemperature lability of dogfish and blood clam MBs (Fig. 7, cell 4+4a) shows that there is no correlation with homeothermy. Tubulin isotype composition may be a contributingfactor, as indicated in work on chicken erythrocytes (Joshi et al., 1987). Another characteristic of maturity in at least some species is reduction in the number of MTs per MB cross section. In chicken erythrocytes, for example, the number diminishes from -50 (average)in early erythroblasts to 10 in mature cells (Small and Davies, 1972). Such reduction does not occur universally, however; early erythroblasts of the salamander Triturus cristatus contain about the same number (average, 109) as mature erythrocytes (Small and Davies, 1972). A third feature of maturation is that, in appropriate species, essentially all MTs reassemble into a peripheral MB upon rewarming following lowtemperature MB disassembly (Fig. 7, cell 4a+4c). That this occurs in the original plane of flattening can be inferred from the absence of morphological changes in the cells during reassembly (Cohen et al., 1982a). In contrast, at early stages of MB biogenesis in immature cells, not all MTs reassemble in their initial locations (Kim et al., 1987). One explanation adduced for MT reassembly in the original plane of flattening is that factors controlling spatial organization reside in that plane near the cell periphery. This possibility has been examined in vitru by depleting chicken erythrocyte cytoskeletons of native tubulin at low temperature, then reassembling MTs within them using MAP-free brain tubulin (Swan and Solomon, 1984). MTs were found to be localized at or near the cytoskeleton periphery in MB-like fashion. However, such localization in itself does not demonstrate the presence of peripheral determinants unequivocally. MT coils closely resembling MBs can be induced to assemble in synaptosomes (Gray et al., 1982),artificially produced cell fragments that do not normally contain such coils, and in which the presence of preorganized peripheral determinants is highly unlikely. Peripheral MT localization in both cases is more simply explained as a mechanical effect of MT elongation coupled with resistance to bending (see Fig. 13 and Section IV,A). The presence of spatial determinants in nucleated erythrocytes is indicated more convincingly by the finding that the total length of reassembled MTs (i.e., the number of MT windings) was independent of tubulin concentration (Swan and Solomon, 1984).Since not all MB MTs are in contact with the MS, this result implies either that determinants are present both at the MS interface and deeper within the extracted cytoskeleton, or that more localized pe-

-

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ripheral determinants control both MT location and length (or, number of windings). One additional feature of mature erythrocyte morphology is calcium lability. Exposure of mature flattened elliptical cells to the ionophore A23187 rapidly converts them to spheres (Fig. 7, cell 4-4e). This occurs both in Xenopus (Gambino et al., 1985), a species with cold-stable MBs, and in the smooth dogfish (Bartelt, 1982),a species with cold-labile MBs, accompanied by loss of much cytoskeletal structure and, in dogfish erythrocytes, ultimate cell lysis. Experimentally induced lysis of Xenopus erythrocytes in the presence of Ca2+,or exposure of erythrocyte cytoskeletons to a Ca2+-containingcytosolic fraction, causes rapid depolymerization of MB MTs. This effect appears to require calmodulin and other as yet unidentified cytosolic factors (Gambino et al., 1985). It is noteworthy that the presence of calmodulin has been demonstrated in dogfish erythrocyte extracts (Bartelt et al., 1982).In Xenopus erythrocytes, Ca2+induces the partial proteolytic degradation of spectrin, suggesting that selective proteolysis of major cytoskeletal proteins may be the cause of Ca2+ ionophore-induced conversion of mature ellipsoids to spheres (Gambino et al., 1985). Similar conversion of mature nucleated erythrocytes to spheres is induced by prolonged contact with surfaces such as slides or coverslips. It is not known how rapidly such contact initiates cytoskeletal changes. Thus, the interpretation of data reported by many laboratories is complicated by the fact that living erythroid cells have been allowed to attach to surfaces prior to experimental treatments (e.g., Granger and Lazarides, 1982; Gambino et al., 1984; Murphy et al., 1986; Joshi et al., 1987; Kim et al., 1987; Koury et al., 1987). V. The Membrane Skeleton

The MS of nucleated erythrocytes has been less thoroughly studied than that of nonnucleated definitive mammalian erythrocytes. In earlier work (Cohen, 1978b), the noncommittal term “trans-MB material” was applied to a rough network spanning the MB in TEM whole mounts of nucleated erythrocyte cytoskeletons. Although it was suggested that this network might contain actin and spectrin-like molecules, it was initially assumed to contain cytoplasmic elements as well, collapsed onto the planar grid substrate. Subsequently, it became clear that most of this material was associated with the plasma membrane external to the MB, and the terminology was refined to cell “surface-associated cytoskeleton (SAC).” At the same time, “membrane skeleton” came into wider use for the corresponding

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structure in mammalian erythrocytes, and thus “MS” is adopted in this review as generic. A. STRUCTURE

The paradigm here is the MS of mammalian definitive erythrocytes (Fig. 14), essentially their entire cytoskeletal system. In stretched-out nega-

tively stained MS preparations, details of the spectrin-actin network are visible (Byers and Branton, 1985), and in deep-etch replicas the mammalian erythrocyte MS appears as a relatively homogeneous interwoven fabric of spectrin, actin, and associated proteins (Coleman et al., 1989). Comparable high-resolution views of the MS in nucleated erythrocytes of nonmammals have not yet been obtained, but fundamental structural equivalence is generally assumed based on similarities in protein composition and function. In intact nucleated erythrocytes, the exterior MS surface is believed to interface with the plasma membrane bilayer throughout the cell, with its interior surface contacting the MB in the plane of cell flattening. The MS is not visualized directly in thin sections of whole nucleated erythrocytes, in which it is obscured by electron-dense hemoglobin, but its presence is indicated by the fact that MB MTs are never found in direct contact with the membrane bilayer. The MS is readily visible, however, in thin sections of cells simultaneously detergent-lysed and fixed, or in TEM whole

--

FIG. 14. General molecular structure of the membrane skeleton (MS), as derived principally from studies of mammalian erythrocytes. The MS is essentially a spectrin ( S ) tetramer network, with foci composed of actin (a) oligomers and band 4.1 protein. The MS is bound to and spectrinthe membrane bilayer through interaction between 4.1 and glycophorin (G), bound ankyrin (ank) and the anion transporter (AT). The general model is thought to apply to the MS of nucleated erythrocytes of nonmammalian vertebrates as well, with variation in spectrin subunits (af-p). as discussed in the text.

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mounts (e.g., Cohen et al., 1982a, 1990).The question arises as to whether the MS might contain a specialized structural/molecular “track” that guides MB reassembly and possibly MB biogenesis during erythrocyte differentiation. Indeed, Granger and Lazarides (1982), using shadowed replicas of sonicated chicken erythrocytes, made the intriguing observation that structural tracks resembling ridges between grooves were continuous with MTs at the inner plasma membrane surface. In itself, however, this is not compelling evidence that tracks exist, since they could be an artifactual imprint resulting from MS pressure against the MB during cell preparation and fixation. Thus, it remains to be determined whether such tracks are real by looking for them in erythrocytes predepleted of MTs at low temperature, or rewarmed to room temperature with MB reassembly blocked by colchicine or other inhibitors. COMPOSITION B. MOLECULAR The molecular components of the erythrocyte MS have been studied extensively in recent years, and details can be found in a number of excellent reviews (e.g., Geiger, 1983; Lazarides, 1987; Lazarides and Woods, 1989; Steck, 1989). The molecular organization of the mammalian erythrocyte MS (Fig. 14) is thought to be similar to that of the nucleated erythrocyte, since nonmammalian equivalents of spectrin (e .g., Jackson, 1975; Cohen et al., 1982a; Bartelt et al., 1982, 1984; Repasky et al., 1982; Glenney et al., 1982), actin, ankyrin (Moon and Lazarides, 1984), band 4.1 (Granger and Lazarides, 1984, 1985; Ngai et al., 1987), and band 3 (anion transporter) (Woods et al., 1986) are present. Terminology regarding the spectrin family proteins has been and continues to be somewhat confusing. Like other representatives, that of the nucleated erythrocyte is a heterodimer, with subunits of Mr -240,000 (a) and -220,000 (p), as observed on SDS-PAGE. The Mr 240,000 a subunit resembles the a subunit of mammalian nonerythroid spectrin (i.e., a-fodrin, or a-“brain spectrin”) (Levine and Willard, 1981) more closely than that of mammalian erythrocytes, and is here designated af.This was demonstrated by Bartelt et al. (1982, 1984) for dogfish erythrocytes on the basis of calmodulin binding and reactivity with antibodies, and by several other laboratories at about the same time (e.g., Repasky et al., 1982; Glenney et al., 1982). Nonmammalian erythrocyte afbinds calmodulin with high affinity, as does a-fodrin, but the a subunit of mammalian erythrocyte spectrin does not. Comparison of nucleotide/amino acid sequences has shown >90% homology between these calmodulin-binding a subunits, as compared with 0 and 0

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a

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;

20

.-.-S>

0

a

8 E 4 d0

a,

a

80

s

4

s

0

A

Time (min)

o pFLl

+ FL

pMD45

pFL1-ca

FIG. 7. Localization of purified and cloned native or targeted luciferase. Mitochondria ( I mg/ml) isolated from wild type (pFLI) or yeast cells transformed with the native (pMD45) or fusion gene (pFL1-ca) were incubated on ice with trypsin (10 pg/ml). Wild-type mitochondria were supplemented with soluble luciferase (FL, 10 ng/ml). At the indicated times, trypsin inhibitor (0.2 mg/ml) was added and aliquots from the suspension were analyzed for luciferase activity at saturating substrate concentrations, or separated into soluble and particulate fractions and then analyzed. (A) Time course of proteolysis. arb., arbitrary units. (B) Residual activity in untreated (con) or treated (15 minutes, try) samples.

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VI. Light Emission by Luciferase in Biological Systems Light emission from cloned luciferase in situ depends on a variety of factors which may affect the amount of product present in the cell, its intrinsic catalytic ability, and the local conditions under which the enzyme is operating. IN WHOLECELLS A. LIGHTEMISSION

The total amount of luciferase produced in the cell depends on the efficiency of transcription and translation, determined mainly by the type of promoter used with the cloned gene (see Gould and Subramani, 1988; Wood, 1989; Aflalo, 1990; Schneider et al., 1990), and other regulatory elements added upstream (e.g., enhancers) or downstream [e.g., poly(A) sequences] to the gene (Wood, 1989; de Wet et al., 1987)or mRNA (Gallie et al., 1989). The efficiency of bulk expression is best assessed by quantitative determination of mRNA, or that of the product peptide in immunoblots from solubilized extracts of whole cells. A reasonably good correlation is generally found between these two parameters. The level of bioluminescence in transgenic plant tissues closely reflects that of transcription of the luciferase gene (Scheider et al., 1990). Moreover, the overexpressed luciferase appearing in the microparticulate fraction of yeast cells is active and represents most of the total activity (Aflalo, 1990). Finally, studies’of the translation and stabilization of luciferase in X.laeuis oocytes (Kutuzova et al., 1989) indicate that the enzyme is correctly assembled in a conformation compatible with eukaryotic cellular environment. Early characterization studies of the cloned enzyme in bacterial extracts indicate that it is undistinguishable from mature luciferase. Indeed, the kinetic pattern of light emission, its spectral distribution, and sensitivity to inhibitors are similar for both enzymes in the presence of bacterial extracts (Wood, 1989). These conclusions may be somewhat extended to the chimeric luciferase targeted to mitochondria, as shown in Fig. 8. The dependence of soluble luciferase activity on [LH2] is significantly affected by the presence of mitochondria (see Fig. 8 insets). Moreover, in the presence of neutral detergents, the differences in the kinetic behavior of both systems are further reduced (unpublished observations). It seems, then, that the intrinsic catalytic properties of the enzyme are rather insensitive to moderate variations in the primary structure at either end of the sequence. Additional experimental evidence is needed to confirm this preliminary proposal, which is relevant to the design of genetically engineered luciferase.

301

BIOLOGICALLY LOCALIZED FIREFLY LUCIFERASE

16 12 o8 '' OO

400 800 1200 1600 2000

IE-l

I

100

10

1000

10 I IE-l

16 12 0' 8 ..

I€-2

'0

20

40

60

00

100

I E-3 IE-3 IE-2 IE-l

4 0 I

10

f

100

FIG. 8. Dependence of light production of soluble and mitochondria-bound luciferase on ATP and luciferyl adenylate (LH2). Isolated mitochondria containing 20 pg of protein and/or 0.2 ng of purified luciferase were assayed for light production activity. The reaction was initiated by injection of LH2. The peak height (I, closed symbols) and steady light emission (I,,, open symbols) are reported. The samples assayed were soluble firefly luciferase (FL) alone (circles), FL supplemented with wild-type mitochondria (triangles), and FL bound to mitochondria isolated from cells transformed with pFL1-ca (squares). (A and B) Dependence on [ATP]. [LH2] = 0.1 mM. (C and D) Dependence on [LH2]. [ATP] = 2 mM. (Insets) Dependence of the ratio Z, :I,, on substrate concentration. Reproduced with permission from Atlalo, C. (1990), Biochemistry 29,4758-4766. Copyright 0 1990 American Chemical Society.

The activity of cloned luciferase in situ may, in addition, be significantly affected by the accessibility of substrates and other environmental conditions occurring locally. Preliminary light measurements with intact cells containing cloned luciferase indicate that the most likely limiting factor is the intracellular concentration of luciferin diffusing in from the medium (Gould and Subramani, 1988; Aflalo, 1990). The bioluminescent activity of transformed cells with exogenous luciferin increases at low pH, due to a better permeability of protonated LH2 through the cell membrane. In animal cells, this is further improved by the addition of dimethylsulfoxide, or nigericin in the presence of potassium (Gould and Subramani, 1988). It is expected that the acidic LH2 molecule will accumulate at equilibrium in

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the more basic of two aqueous phases separated by a membrane. Some features of light emission by transformed yeast cells are presented in Fig. 9. Upon addition of LH2, a slow increase in light output is observed, representing the build-up of intracellular [LH2] (not shown). At saturation, the light emission shows the characteristic flash pattern indicating bound product inhibition. However, this occurs at a much slower rate than with soluble luciferase, due to the kinetic limitation imposed by the slow diffusion of luciferin. Oxygen is present at saturating concentrations (-250 p M at 25°C) in solutions equilibrated with air. However, actively respiring cells rapidly consume all the oxygen in standing suspensionsand reduce the light output according (Fig. 9B-D). After additional incubation in anaerobic conditions, a fast flash is generated upon reoxygenation. The peak height is dependent on the length of the anaerobic incubation (Fig. 9A,3), indicating that the inhibitory product is slowly released from the inactive enzyme. The flash emission does not occur with cells with a low respiratory capacity (see Fig. 9A,2, for yeast grown in the presence of glucose). The results indicate that even under severe anaerobiosis, the local [ATP] is not a limiting factor for light production. Thus, the average [ATP] in normal cells (in the millimolar range) apparently saturates the light production by luciferase in intact cells. However, these data illustrate the use of luciferase as a localized oxygen sensor. Bioluminescence of intact transformed cells is currently used as a qualitative tool to detect positive transformation, at relatively low sensitivity to cell-free extracts. At this stage, one can only speculate on its quantitative aspects. However, further studies on the catalytic behavior of the native enzyme and its localized fusion products in various environments (e.g., cell-free extracts) will help to better define the factors affecting the light output in situ. This will provide additional insights on localized processes in the cell. At present, straightforward measurements of ATP are possible at low concentrations only, and the use of the probe is limited by its calibration. A strict control of the conditions for the calibration and operation of the localized probe is possible only in isolated systems, in which luciferase is exposed to the medium, as illustrated in the following section.

B. MONITORING LOCAL[ATP] AT THE MITOCHONDRIAL SURFACE 1 . Local versus Bulk [ATP] Measurements

Low [ATP] emerging from isolated mitochondria can be determined by directly monitoring the light output from the outer membrane-bound enzyme or soluble luciferase added to wild-type mitochondria as a control. The light output by luciferase in these conditions is linearly related to

BIOLOGICALLY LOCALIZED FIREFLY LUCIFERASE Time after LH2 addition (rnin)

A 20

25

30 35 -40 45

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1 1 -

Time after LH2 additon (min)

55

10

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15

TI

\

c

Mixing:

Time after LH2 addition(min)

off

on

303

D

\ 2.5~ \ \

Time after LH;! addition (min)

Mixing'off on

FIG. 9. Light production by cloned luciferase and oxygen consumption in intact yeast cells. Yeast cells transformed with pFL1-ca (fusion gene) or pMD45 (native gene inducible by galactose) were grown in the presence of glucose (repressor of mitochondria proliferation) or a nonfermentable carbon source. The cells were harvested (log phase), washed in water, and resuspended in Na-MES (Na-morpholino ethane sulfonate), pH 5.5. The luciferase reaction was initiated by addition of 50 p M luciferyl adenylate (LH2) and vortexing to saturate the suspension with oxygen. (A) Light output from cells transformed with the native or fusion gene of luciferase and grown as indicated: I , fusion, lactate; 2, fusion, glucose; 3, native, galactose. Arrows indicate reoxygenation. (B) Native, galactose. Effect of cell concentration (in multiples of that in A3) on the light output and oxygen consumption, simultaneously recorded using a microelectrode. (C) Fusion, lactate. (D) Native, galactose; Same as (B), with continuous magnetic stimng, as indicated at the bottom. Reproduced with permission from Aflalo, C. (1990), 0 Biochemistry 29, 4758-4766. Copyright 1990 American Chemical Society.

[ATPI up to 1-2 pM (see Fig. 8B). The steady-state [ATP] generated by the mitochondria and consumed by exogenous soluble HK has been assessed in both systems (Fig. 10). In the absence of HK, no significant difference could be detected in the kinetics of ATP formation in the rapidly stirred mitochondria1suspensions, nor in the calibration of both the localized and soluble luciferase using exogenous ATP. However, while in the control system the steady-state [ATP] can be reduced to undectable concentrations at high enough levels of HK (Fig. lob), the light output from mitochondria-boundluciferase is reduced to a finite value (Fig. 10a) under identical conditions. This value is further lowered in the presence of atractyloside, which blocks the transport of nucleotides through the mito-

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10

I

I

30

40

I

I

a

b

HK

HK

1.

ti

mit0 ATP

t ADP

t

ATP

I

atrac

mitt! ltTP

f

ATP

t

ADP

FIG. 10. Light emission from localized or soluble luciferase during oxidative phosphorylation coupled to hexokinase. Mitochondria (40 pg/ml) isolated from transformed (pFL1-ca) (a) or wild-type (b) yeast cells were suspended in a stirred medium containing 0.6 M sorbitol, 20 mM tricine (pH 7.8), 20 mM KCI, 3 mM Mg-tricine, 5 mM Pi, 10 m M K-succinate, 0.1 mM luciferin, and 1 mglml bovine serum albumin; (b) 10 ng/ml purified luciferase was added. The indicated additions (in small volume) were 0.1 p M ATP, 1 p M ADP, and 10 pg/ml atractyloside, and at the upper arrows, yeast hexokinase was added from serial dilutions so that its concentration doubled each time (2-64 U/ml).

chondrial inner membrane and thus inhibits oxidative phosphorylation. The residual activity reflects the contribution of adenylate kinase to ATP formation in the intermembrane space. These results indicate that when ATP is efficiently depleted in the medium by HK (as detected by soluble luciferase),the ATP emerging from the mitochondria is still available to the localized probe. Thus, at least for high catalytic rates of ATP consumption in solution, the coupled heterogeneous system may be limited by diffusion of nucleotides to and from the surface of mitochondria.

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2 . Dependence on the Rate of ATP Formation

The extent of the actual limitation by diffusion is directly related to the catalytic ability of the concurrent enzymic steps. Thus, upon increasing the specific (i.e., local) activity of phosphorylation, more ATP should accumulate at steady state near the outer membrane, as suggested by the effect of atractyloside (Fig. 10a) and other inhibitors of oxidative phosphorylation. On the other hand, increasing the overall (i.e., bulk) rate of phosphorylation in the system by adding more mitochondria should not affect significantly the flux of ATP emerging from the membrane, and thus leave the local accumulation of ATP unaffected. This rationale is illustrated in the experiment presented in Fig. 1 1. The inital rate of phosphorylation in the absence of HK (see Fig. 10) was found to depend on the concentrations of added ADP and mitochondria. The experiments in Fig. 10 were repeated in the presence of tripled amounts of ADP or mitochondria, resulting in either case in a proportional increase in the observed initial rate. The steady-state concentrations of ATP at variable HK efficiencies were replotted as a function of l/[HK] in

B

A

-T

1 .o

1.o

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m

r I-

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n

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"

0.4

'

'

"

0.6

llHK ImllUi

0.8

0.4

. '

1.0

llHK Iml/U~

FIG. 11. Titration of steady-state ATP at the surface of mitochondria or in the bulk medium. Experimental conditions were as in Fig. 10. (A) Mitochondria bearing the luciferase fusion product. Circles, [mito] = 40 pg/ml, [ADP] = 1 p M ; triangles, [mito] = 120 pg/ml, [ADP] = 1 p M ; squares, [mito] = 40 pg/ml, [ADP] = 3 p M . (B) Wild-type mitochondria supplemented with purified luciferase. Symbols and concentrations are as in (A).

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order to derive their extrapolated limit at infinitely high ATP consumption in the medium. The values obtained with the local probe are finite and depend on added [ADP], but not on the mitochondria concentration ([mito]) (Fig. 11A). The control experiments with wild-type mitochondria and soluble luciferase yielded zero extrapolation values (Fig. 11B). Therefore, with excess HK the nucleotide in the medium is all in the form of ADP, and the extrapolated values with the local probe represent the net accumulation of ATP near the membrane when its concentration in the bulk medium is zero. The results strongly suggest that the accumulation of ATP near the mitochondria depends on its flux through the membrane (i.e., per unit area) rather than on the total rate of its appearance in the bulk medium, indicating that a true local event is being monitored by the probe.

3. Modeling A similar behavior can be simulated by reducing the reactions to a simple model, including two pseudo-first-order catalytic reactions coupled through the diffusion of intermediates. In the first one, the sequences of steps-including the transfer of ADP to internal mitochondrial compartments, its phosphorylation, and the export of ATP-is assumed to occur as a single catalytic step located at the surface of spherical particles in suspension. Control experiments have shown that the initial rate of ATP formation is linearly dependent on both [ADP] and [mito] in the ranges used in this work. The second reaction of dephosphorylation of ATP in the bulk medium by HK is similarly related to [,4TP] and [HK]. The diffusion of nucleotides between the bulk medium and the surface can be described as a mass transfer whose rate is assumed to be proportional to the difference in the concentrations of the transferred nucleotide in both compartments and the concentration of surfaces (i.e., [mito]). For the sake of simplicity, it is assumed that the nucleotides diffuse in a near-planar unstirred layer sticking to the mitochondria surface, and that the volumes of both mitochondria and the diffuse layer are negligible compared to that of the bulk medium. The model is schematically outlined in Fig. 12a, and the pertinent equations describing the system are formulated in Fig. 12b, assuming an identical mass transfer coefficient of ADP and ATP. One can solve the system of differential equations for steady state, and by introducing [AXP] as the sum of endogenous and added nucleotides (ADP + ATP, represented as 100% in the ordinate of Fig. 10). A simple expression for the local [ATP] at infinite HK concentration may be reached [Eq. (21) in Fig. 121.

BIOLOGICALLY LOCALIZED FIREFLY LUCIFERASE

307

a

kmit kdit kHK

=

=

kox A [mito] D A [mito]/6 k"[HK]

b

d[ADP]b

=

-

d[ATP]b

kmlt

[ATPIs,axtrap.= k d l f + k ml t

=

kox D16 + ko x

. [AXP]

(21)

FIG. 12. Model for coupling two catalytic steps in a heterogeneous system. The indices s and b represent surface and bulk, respectively. R and 6 represent the radius of the (spherical) mitochondria and the width of a hypothetical unstirred layer, respectively. A is the specific area of mitochondria (cm2/mgof protein), k,, and k" are second-order rate constants, and D is the diffusion constant. The second-order mass transfer coefficient is D/6. [Eqs. (13)-(16)] Rate laws for each nucleotide species. [Eq. (17)] Mass conservation in the bulk medium. [Eqs. (18)-(19)] Steady-state solutions for Eqs. (13)-(16). [Eq. (20)] Steady-state rate of ATP appearance in the medium in the absence of hexokinase (HK) [Eq. (21)]

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Since both kmit and kdjf are proportional to the concentration of mitochondria, it follows that the fraction of ATP accumulated at the surface is proportional to [AXP] and independent of [mito], in accordance with the results in Fig. 11. The ratio kmit/kmit + kdif can thus be determined experimentally, as the slope of the extrapolated values versus the total nucleotide concentration. In the absence of HK, the phosphorylation of added ADP may be represented by a single process with a composite pseudo-first-order rate constant (mass transfer and surface catalysis occurring in sequence) (Easterby, 1981), as shown in Fig. 12 Eq. (20). This value, proportional to the concentration of mitochondria, can be determined experimentally as the slope of the observed initial velocity of phosphorylation versus added [ADP]. Next, the values of kmit and kdif can be derived by solving the system of equations [Eqs. (20)-(21)] as formulated in Fig. 12b. It turns out that the ratio kdiflkmit is in the range 15-20 so that diffusion does not contribute significantly as a rate-limiting step with stirred suspensions in the absence of HK. However, as the rate constant for HK increases (kmit -e kdif 5 ~ H K ) ,diffusion becomes more limiting and the relative accumulation of ATP at the surface increases. While this relatively simple model does not reflect the physical reality, it is useful to describe the effective properties of the system. A more exact (and elaborated) mathematical model has been developed (Aflaloand Segel, 1991),which describes the experimental data with less “unrealistic” assumptions. The steady-state value of local [ATP] in the presence of excess HK is 5-10% of the total nucleotide concentration added to isolated mitochondria. This value represents a lower limit for the true concentration gradient, since HK may selectively associate with the outer membrane of yeast mitochondria (Krause et al., 1986; but see Kovac et al., 1986). Another aspect which was not considered is the microdistribution of luciferase on the outer membrane. Indeed, the membrane-bound enzyme would detect only the fraction of “vectorial” ATP (emerging from the porin molecules) which has diffused laterally along the membrane. In contrast, “scalar” ATP added to the medium would be simultaneously and equally accessible to the membrane probe, in the absence of a net flux for its formation or utilization. The resolution of this requires a finer resolution for luciferase localization, using, for example, immunogold labeling of mitochondria preparations (Douma et al., 1985; Hines et al., 1990). Finally, a substantially higher diffusion limitation should also be expected in the more intricate (and unstirred) cellular environment. The commonly observed aggregation of mitochondria around ATP-consuming sites in some cells represents a trivial solution for the cell to overcome such a limitation.

BIOLOGICALLY LOCALIZED FIREFLY LUCIFERASE

309

MI. Experimental Approaches and Perspectives A. LIGHTMEASUREMENT Various light-measuring instruments may be used for the detection and quantitation of luminescence (for a review of commercially available radiometers, see Stanley, 1986). They include photon counters, which evaluate the number of quanta emitted in a predetermined lapse of time, and luminometers, which measure light energy flux as an electrical current. Many modern radiometers can operate in either mode. Light detection itself is achieved using two main kinds of devices. The photomultiplier is based on a photocathode which acts as an electron emitter when illuminated. The photoelectrons are captured and multiplied by a succession of anodes under increasing voltage. The response (pulse or current), proportional to the incident light over a wide dynamic range, may be amplified up to 108-fold.The second device is the photodiode, or charge-coupled device (CCD), on which each incident photon creates a single electron-hole pair on a siliconjunction. The resulting signal can be amplified to alesser extent than in the photomultiplier, resulting in less sensitivity. On the other hand, photodiodes are sturdier and more compact and may be miniaturized. Recent advances in CCD technology have led to the development of array detectors consisting of two-dimensional matrices of light-sensitive elements (pixels), which enable quantitative imaging at high resolution (500 x 500 pixels/cm*) and real-time measurement capability when coupled to image intensifiers (Hooper et al., 1990). Relatively high sensitivity can be reached using digital image processing of stored signals, including background subtraction, integration, and averaging of the collected data. These techniques have been used to assess the expression of luciferase in single mammalian cells infected with a recombinant vaccinia virus (Hooper et al., 1990), or plant protoplasts after the insertion of (luciferase) mRNA by electroporation (Gallie et al., 1989). Further improvement of the sensitivity and microscopic video imaging is needed to enable real-time analysis of light production at subcellular spatial resolution, which is the ultimate challenge for this rather expensive and sophisticated technology. The use of spectroluminometers has also been described for analysis of the color of light emitted upon varying environmental factors or with different luciferases isolated or cloned from P. plagiophthalamus (Seliger et al., 1964; Wood et al., 1989b). Another approach for semiquantitative light detection involves direct exposure of two-dimensional objects (Petri dishes or microtiter plates) to photographic films (Kricka and Thorpe, 1986; Wood and DeLuca, 1987). This is particularly useful for rapid screening procedures of recombinant colonies or plaques expressing luciferase. Moreover, the film darkening

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may be quantified by scanning densitometry. This method has also been used to show the spatial distribution of luciferase luminescence from whole recombinant tobacco plants (Ow et al., 1986; Schneider et al., 1990). Liquid scintillation counters may also be used for routine light measurements. In these widely available instruments, the light is collected by two photomultipliers, which can be operated independently, giving a total count from both detectors (chemiluminescence mode), or in conjunction through a coincidence circuit, yielding counts representing luminescent events recorded simultaneously by the detectors (scintillation mode). The rate recorded in the coincidence mode is proportional to the square of the light emitted (Kimmich et al., 1975), in contrast with the first mode which directly measures light intensity, as with common radiometers (Nguyen et al., 1988). While increased sensitivity (i.e., signal-to-background ratio) may be attained by measuring steady light emission over a long time, this method is not suitable for monitoring sudden changes of light output in response to the addition of reagents, since the time required to insert the vial in the counting chamber is relatively long. Presently, the time evolution of luminescence can be assessed at the highest sensitivity as a space-average phenomenon with the light-emitting units in stirred solutions or suspensions. The configuration of the light detection chamber is important for the optimization of light collection from the luminescent object. The light-sensitive (planar) surface should be as close as possible to the object, since the light flux decreases drastically with the distance. Thus, when a single detector is used, light emitted from the side of the object opposite the detector is lost unless it is reflected back to the detector by a surface of appropriate geometry. Some commercially available luminometers are equipped with mixing devices which insure a homogeneous distribution of the light emitters and a fast dilution of added reagents, and help to sustain an adequate oxygen concentration in respiring biological suspensions (see Figs. 9C and D).

B, PRACTICAL CONSIDERATIONS 1. Use of FL as a Probe for Metabolites Concentrations

The quantum yield of luciferase bioluminescence [i.e., photon : molecule of luciferin consumed = 0.88 (McElroy and Seliger, 1961)l is one of the highest known in photochemistry. The turnover rate for catalysis is rather low compared to other enzymatic reactions (0.01-0.03 sec-', saturated). The combination of these features represents the basis for using luciferase as a probe, since the light signal can be measured at

BIOLOGICALLY LOCALIZED FIREFLY LUCIFERASE

31 1

high sensitivity with a relatively slow consumption of the substrate being monitored. In addition, the reaction is very specific toward ATP, and light production is only marginally affected by other nucleoside di- or triphosphates (Lee ef al., 1970). Both the peak and steady-state light intensities are proportional to the amount of luciferase in the reaction. The fast-evolving peak value is routinely used to determine the native or recombinant enzyme concentration in biological samples at high sensitivity and with relatively low interference from concurrent reactions (e.g., ATP or 02 consumption) likely to occur in the extracts. Of course, the most attractive feature of this analytic system is the linear relationship between light output and the concentration of the limiting substrate, sustained in a wide range (10-'2-10-6 M for ATP). The linear range can be somewhat expanded by lowering the catalytic rate at the expense of sensivity. This may be done by lowering the cosubstrate concentration or by adding competitive inhibitors (respectively to the limiting substrate being monitored). Another approach is to relieve the bound product inhibition through the addition of neutral surfactants or polar additives (see Kricka and DeLuca, 1982). Under these conditions, in addition to ATP, a wide range of metabolites can be determined when luciferase is coupled to enzymes which use them as cosubstrates in ATPgenerating or -consuming reactions. Moreover, the activity of such enzymes in biological extracts can be similarly monitored in the continuous mode by following the time course of ATP formation or consumption. Light evolution as a response to variation in ATP concentration occurs generally fast enough (-0.5 seconds) to permit such an application. The reader is referred to a comprehensive review of these aspects by Lundin (1982). A major drawback of this method is that a linear response is elicited only by relatively low (i.e., submicromolar) [ATP]. This does not permit the study of enzymes or more complex biological systems under physiologically relevant conditions with respect to ATP whose average concentration in cells is in the millimolar range. Another limitation with fast-kinetics systems may be the time dependence of light production by luciferase. Lemasters and Hackenbrock (1979) have developed calibration and data manipulation procedures to deal with the dependence on [ATP] in the continuous monitoring of oxidative phosphorylation in isolated mitochondria. Moreover, pre-steady-state kinetic data for luminescence can be converted to the real variation of ATP in fast reactions by calculation using kinetic models based on the study of purified enzyme (Aflalo and DeLuca, 1987, 1988; Slooten and Vandenbranden, 1989; C. Aflalo, unpublished observations). Nevertheless, the straightforward use of luciferase as a

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probe for ATP (or other substrates) concentration is limited to controled conditions in which the latter is kept in the micromolar range. This may be achieved only with isolated or permeabilized systems, or alternatively, if luciferase were to be directed to cellular compartments with either intrinsically low ATP content or a high ATP consumption ability.

2. Genetic and Chemical Engineering of Luciferase In principle, with appropriate structural and mechanistic information from luciferase, it may be possible to modify the enzyme so it will be more suitable for probing ATP in a cellular environment. The aim would be to decrease the apparent affinity for ATP in order to maintain a linear relationship between the steady light output and [ATP] in the physiological range. However, it may be anticipated that a modification of the active site by covalent derivatization or mutagenesis of specific residues could also affect other characteristics of the enzyme relevant to its use as a probe. Ideally, the modification should exclusively shift the luciferin activation equilibrium (step 1 in Section 111,B,3) to the left, with no effect on the specificity toward ATP (or luciferin), or the low turnover rate which enables noninteracting continuous measurements. This prospective approach is hindered by the uncertain validity of active site-directed chemical modification data acquired before the primary structure of the enzyme was resolved. Three partial sequences of peptides derived from luciferase chemically derivatized with reactive substrate analogs have been reported (Travis and McElroy, 1966; Lee and McElroy, 1971b; Lee et al., 1981).None of these putative active site-relatedpeptides could be recognized in the translated sequence for cloned luciferase (Wood, 1989). So far, a first attempt for mutagenesis of nonessential but catalytically related (Alter and DeLuca, 1986) cysteine residues to alanine did not affect the activity of luciferase expressed in bacteria(D. C. Vellom, personal communication). Thus, it seems that some aspects of the early characterization of the enzyme involving active site-directed chemical modification need reinvestigation in conjunction with the genetic approaches currently used to study the color of light emitted by different beetle luciferases (Wood, 1990; Wood et al., 1989b). The latter are oriented to map the luciferin binding site. A parallel study is needed for the pyrophosphate (in ATP) binding site, which is more relevant to the proposed modification. The process of the cloning and direction of luciferase to foreign cells and compartments involves genetic manipulation of the coding sequence, and may, a priori, result in a change in the enzyme properties. There is no available experimental evidence for that in eukaryotic cells. However, with bacteria transformed with luc, a large amount of antiluciferase reac-

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tive protein may be detected in an insoluble fraction which contains no bioluminescent activity. The effect of short deletions from either the 3' or 5' end of the coding region in luc cloned in bacteria was studied systematically. The total light production in extracts is drastically reduced when as few as eight residues are deleted from either the amine or carboxy terminus (Wood, 1989). The decrease in activity with longer deletions is correlated with the amount of luciferase in soluble extracts, determined by Western blot analysis. These results suggest that the deletions affected the stability of luciferase in the bacterial cell environment rather than the intrinsic catalytic ability of the truncated products. The terminal fragments may contribute to the solubilization of the enzyme or prevent interactions between hydrophobic domains on different molecules normally located in the core of the protein.

C. PERSPECTIVES 1 . Luciferase as a Probe for Protein Metabolism

A complete picture for gene expression should include information on the various processes leading to the establishment of the final phenotype. These include the transcription and translation of genetic material, targeting (processing) and assembly of the resultant polypeptide chain(s), and the catabolic degradation of the mature protein. This pathway is generally studied through the chemical determination of translated protein in extracts by radioactive (pulse-chase labeling) or immunochemical methods, which provide sensitive means to determine the amount of protein. However, this approach does not provide information on the functionality of the polypeptides investigated. Catalytic ability is the best criterion for correct folding and assembly of translated enzymes, but it is often quantitated with much lower sensitivity compared with the chemical methods. Therefore, correlation between structural and functional expression is difficult to test with most systems, especially in the case of low-level gene expression, which is often required for viable cells. Numerous applications of cloned luciferases as reporter genes have been reviewed by Gould and Subramani (1988), Wood et al. (1989a), and Wood (1990). All take advantage of the unique light emission ability of the enzyme from firefly (or other organisms) in the biological systems investigated. The ease and sensitivity for determination of light emission confer to luciferase a significant advantage over different probes for protein metabolism. Indeed, bioluminescence from cloned luciferase may be quantitated at noninvasive levels of expression. This insures a low interference of the enzyme with other cellular systems. In genetic applications, as opposed to the use of the enzyme as a chemical sensor, the limiting

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factor for light emission is the concentration of the enzyme itselfin biological samples supplemented with saturating substrates. The focus of these applications is the regulation of active enzyme synthesis, and the posttranslational or degradation aspects have been only marginally considered. A few studies of the stability of the enzyme in foreign environments have emerged which use the enzyme activity (Kutuzova et al., 1989). However, the information gathered by activity measurements alone may be incomplete and must be confronted with chemical determinations as mentioned above. An example of discrepancy between both approaches in an overexpression bacterial system was given in Section VI,B,2. Studies of the stability of cloned luciferase to heat or related stress assessed by both the methods indicate that the enzyme is not degraded, but becomes insoluble as the activity is lost (Nguyen et al., 1989). This illustrates the use of the enzyme as a promising tool to study the molecular mechanisms of thermolability and resistance to stress. This can be extended to wider studies of the interaction of the enzyme with the cellular machineries involved in protein traffic and processing. The possibility of formation of a tightly bound product (e.g., L-AMP in Fig. 2) enables the possible isolation of a translocation intermediate, since the tightly folded polypeptide chain will not cross the membrane (see Section 11,A). Finally, the analysis of experimental cases in which the fusion protein failed to be localized in an active form may provide important information on the cellular import machinery. 2 . Targeting Luciferase to Cellular Compartments New fusion genes may be designed to direct luciferase to various cellular (micro)compartments.The fusion genes should be constructed by isolating appropriate restriction fragments encoding for leader sequences directing natural proteins to specific cellular compartments in cells (see Section 11,A). These can be ligated to the FL gene, and the resulting fusion genes can be inserted into shuttle plasmids behind selected promoters. These plasmids may be propagated in bacteria. The selection of positive transformants is easily performed by screening colonies for bioluminescence using luminometric or photographic standardized assays (Wood and DeLuca, 1987). After reisolation of the plasmids, they can be used to transform eukaryotic cells. Care should be taken to choose appropriate lengths of the fused sequences to minimize their possible interaction with the luciferase moiety until the final assembly of the protein. Also, the peroxisomal targeting sequence at the carboxy terminus of luciferase should be deleted or modified to avoid ambiguity in the final localization of fusion proteins. The use of fusion genes has been proposed (Froshauer et al., 1988) to determine membrane protein topology, normally assessed by the interac-

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tion of proteases or antibodies with hydrophilic domains exposed to the medium. The approach is based on the creation of a chimeric gene consisting of the coding sequence of a soluble enzyme fused in frame with fragments of varying length encoding for the membrane protein. Thus, the soluble enzyme domain should appear on the same side of the membrane as the point to which it was fused in the original membrane protein. Walter and associates (Green et al., 1989) have applied this approach with soluble galactokinase fused to various fragments of yeast arginine permease, a plasma membrane protein including multiple trans-membrane segments. They used the glycosylation of galactokinase in the lumen of microsomal vesicles to determine its location relative to the membrane. One can take advantage of the characteristics of luciferase in a similar system to assess the topology, since the membrane-bound enzyme responds differently to substrate addition according to its relative location. While a flash emission of high intensity is expected with the enzyme exposed to the medium (saturating [ATP] and [LHz], pH 7.8), a very low light output should develop slowly with the enzyme inside the vesicles, due to the low accessibility of substrates (see Section V1,A). Of course, this “activity-oriented” approach may (and should) be complemented by the physical demonstration of exclusive localization of the protein in the correct compartment with the exclusive topology. This may be conveniently done by immunogold labeling (Keller et al., 1987; Douma et al., 1985) of whole-cell preparations for electron microscopy. It remains to be demonstrated that the luciferase activity will be retained in the various fusion proteins during or after their translocation through biological membranes and compartment-specific processing. Experimental evidence to date has shown that the enzyme is active in peroxisomes (de Wet et al., 1987), cytoplasm (Gould et al., 1987), mitochondria (Aflalo, 1990), and chloroplasts (Schneider et al., 1990). An intriguing and highly speculative prospect would be the design of chimeric proteins consisting of FL fused to peptide domains known to interact specifically with proteins or structures localized in the cell. For example, mellitin-luciferase or troponin-luciferase chimerae would bind to calmodulin or actin filaments, respectively. Such an approach may provide significant refinements to the biological localization of the probe. However, the probability of adverse interaction with the target is expected to rise due to steric hindrance from the large luciferase domain. 3. Prospects for Local [ATP] Measurements Most isolated cells have a stable adenylate concentration which may be modulated experimentally to some extent. The steady-state average concentration of cellular ATP is determined by the ratio of its rate of formation

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(mainly by oxidative phosphorylation and glycolysis) to that of consumption by various energy-requiring systems. Thus, modulation of the activity of these systems by addition of exogenous substrates or inhibitors may be used to control the total ATP content of cells. Morever, it is possible to modulate [ATP] in selected compartments by specifically affecting localized processes (see Section 11,D). Although a most significant reduction of [ATP] in compartments sustaining a high rate of its consumption is conceivable, it remains doubtful that one could reach ATP levels suitable for linear analysis by luciferase in most compartments of viable cells. Indeed, the calculated [ATP] in cells is higher than the latter by three orders of magnitude. As a noninteracting probe, it is expected that the enzyme will detect only freely diffusible ATP in its microenvironment as opposed to the possibly channeled ATP which is committed in the respective reactions of its production and utilization. An interesting prospect for localized luciferase is the assessment of free ATP in concentrated cellular phases as the mitochondria1 matrix, or muscular filaments which have a potentially high nucleotide binding capacity. The use of luciferase as a local probe for ATP seems more adequate in isolated and/or reconstituted systems artificially depleted from adenine nucleotides. Channeling of ATP may be demonstrated when ATP is preferentially utilized in a selected system from a specific source, and the operation of the selected system is relatively unaffected by ATP generated or consumed by other systems. This rationale was illustrated in Sections IV,A and VI,B in an artificial and a biological system, respectively. In both cases, tunneling of ATP between the site of its formation and the proximal luciferase was due to its slow transfer by diffusion to a remote compartment, in which it is efficiently consumed. In intact hepatocytes, a similar reasoning has been used to propose the establishment of [ATP] gradients (Aw and Jones, 1985). In erythrocytes (Mercer and Dunham, 1981) and smooth muscle (Lynch and Paul, 1987), ion transport by Na+, K+-ATPase at the plasma membrane appears to be tightly coupled to glycolysis, and relatively independent of the bulk (cellular) [ATP]. The reduction of ion transport rate under aerobic conditions in the absence of glycolysis cannot be explained only by diffusional restrictions for ATP transfer between mitochondria and the plasma membrane, in view of the high concentration of bulk cellular ATP, and its expected diffusivity, although significant gradients of ADP could be predicted by the same models (Lynch and Paul, 1988).Thus, the mode of interaction between the coupled systems must be elucidated in more detail. To date, a single attempt was made to demonstrate the direct transfer of ATP between kinases (Dillon and Clark, 1990), as for NADH with dehydrogenases (Srivastava and Bernhard, 1986a,b). Such a mechanism may

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provide an alternative explanation for the experimentally inferred channeling of nucleotides in cells. Some systems have been reconstituted in vitro from isolated catalytic components to better assess the coupling mechanisms and the kinetic features resulting from physical association (see Masters, 1981; Keleti and Ovadi, 1988; Wilson, 1988). The use of luciferase colocalized with ATP-consuming systems, in conjunction with soluble (delocalized) luciferase, represents an independent means to assess the fate of ATP in such a reconstituted coupled system. Under the experimental conditions used to test for direct transfer (Srivastava and Bernhard, 1986a,b), the concentration of free ATP would be extremely low. 4 . Monitoring Local Events in Cryptic Compartments As mentioned in Section VI,A, the most probable limiting factor for light production in normal cells is the local luciferin concentration. A relatively simple model system for light emission in situ is the soluble enzyme expressed in bacteria. The study of light production in permeabilized cells should help to define the contribution of some environmental factors. This situation is essentially analogous to the model system described in Section IV (effect of colocalized enzyme activity). The next step would be the determination of internal ATP and luciferin concentration (diffusing in from the medium) in intact cells under different metabolic states expected to affect the limiting conditions. This can be done in extracts from cells rapidly separated from the suspension medium. The light output from the localized enzyme in these conditions may be confronted with these measurements in order to better define the limitation on light production imposed by these factors. For example, the addition of glucose to cells incubated at low pH in the presence of citrate causes a marked transient reduction in the steady light output (see also the effect of citrate itself, shown in Fig. 9,C and D). On the other hand, the addition of uncouplers results in a large increase in light production, followed by an irreversible inhibition. These preliminary data indicate that the luciferin-limited variation of light output may reflect repartition of luciferin associated with proton movements. At saturating luciferin concentrations, local changes in pH or divalent cation concentration may also affect the intensity and the color of light emitted by the enzyme. Thus, light emission by luciferase restricted to defined compartments may indicate specifically a wide range of events occurring locally, which are not detectable by conventional means. A systematic study of the dependence of luminescence on various factors with the enzyme in solution may help to design calibration procedures for the localized enzyme.

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With luciferase localized in various defined cellular compartments, a survey of metabolism in situ may be initated through assessment of the local concentration of reactants using the different probes. This long-range perspective would be applicable only after adequate calibration procedures are developed for the probe in cryptic compartments. So far, our present experimental and theoretical modeling abilities fall short of this ideal use of luciferase. However, the unique properties of bioluminescent enzymes and the possibility to localize them specifically at various cellular sites enable novel approaches to integrative studies of the structure and function of biological systems. These may provide valuable information on assembly processes and local events previously inferred indirectly by conventional methodologies. The systematic analysis of the latter should promote the development of more focused theoretical treatments for metabolic function in situ, which could, in turn, be tested directly in model systems. Finally, these models may be useful to elucidate complex problems in cellular metabolism in which the use of local probes is not presently applicable. ACKNOWLEDGMENT This chapter is dedicated to the memory of Marlene DeLuca, whose spirit, it is hoped, is reflected here. The support of the Israel Ministry of Integration to the author is gratefully acknowledged.

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INDEX

A

Abudefdf marginatus, 137 Accessory cells, appearance, 214-216 Activated B lymphocytes, 12 Agriolimax reticulatus, 134 a- and P-chloride cells in freshwater-adapted fishes, 204-207 a cells, hypertrophy, 212-214 Ameca splendens, 137 Amphibia, presence of MGPB, 140-141 Amphiuma, 39,47 Ampullaria canaliculata, 133 Anadara transversa, 55 Anagasta kuehniella, 128, 129, 151 Anas platyrhynchos, 142, 150 Annelida, presence of MGPB, 125-127 Anolis carolineus, 141 Antibodies as probes for study of plant cell walls, 234-240 applications, 240 purification and specificity, 237-240 raised against cell wall components, 234-237 Aphanius dispar, 137, 138-138, 140 Apical cavity of chloride cells, 193-196 Arion circumscriptus, 133 Arion ater rufus, 133 ATP, probe for ATP in model systems, 287-2% [ATPI at mitochondria1 surface, monitoring local, 302-308 ATP formation in FL, dependence on rate, 305-306 [ATP] measurements of FL

local versus bulk, 302-304 local, prospects, 315-317 Autoimmune aggression in mammals and the MGPB, 163-169 Auxin-induced metabolic turnover of cell wall polysaccharides, 246,247 Aves, presence of MGPB, 142

B

B cell differentiation pathway and immunoglobulin transport immunoglobulin secretion in plasma cells, 29-30 J chain, role of, and assembly of polymeric immunoglobulin, 30 L chains, role of, and appropriate conformation for secretion, 30-31 posttranslational modifications and secretion, 30 intracellular retention of secretory immunoglobulin, 13-19 cysteine tailpiece-dependant retention, 16-19 KDEL sequences and salvage pathway, 14-16 membrane immunoglobulin transport during B cell ontogeny, 20-27 ER degradation versus cell surface transport, 20-22 membrane immunoglobulin recycling, endocytosis and down-regulation, 27-28 325

326

INDEX

membrane immunoglobulin retention in plasma cells, 29 transport of membrane IgM, 22-27 overview, 2-13 activated and memory B lymphocytes, 12 mature IgMIIgD-expressingB lymphocytes, 11-12 plasma cells and end stage of B lymphoid ontogeny, 13 pre-B lymphocytes, 6-7 pre-B-specific surrogate immunoglobulin L chains, 7-10 pre-B to B-cell transition, 10-11 pro-B lymphocytes, 4-6 studies, 1-2 summary, 31-33 8-cells, degeneration, 209-212 (1+3),(1~4)-8-~-Glucan, 243,252-254 effect of in maize coleoptile segments, 253 Balanus eburneous, 128 Barbus aeneus, 140 Barbus conchonius, 166 Basolateral tubular invaginations, 196- 199 Batrachoseps, 72,78 Biologically localized firefly luciferase, see Firefly luciferase Bip gene, in pre-B cells, and intracellular retention, 14-16 Blood-organ bamers, different, 112-1 13 Blood-testis barrier, I 1 1-1 12, see also Male germ cell protective barrier concept of, 157-158 efficacy of in physiological, pathological and experimental conditions, 160-163 terms, 113-1 14 Bombyx mori, 128 Bound steroid hormone hypothesis, 161 Branchiobdella pentadonta, 125 Branchiostomafloridae, 136 BTB, see Blood-testis barrier Byfo arenarum, 140, 141 Bufo byfo, 141 C

Carassius auratus, 140 Carassium carpio, 140

Cell flattening, MB biogenesis and mechanism, 59-64 Cell surface, 193-199 apical cavity, 193-196 tubular system, 196-199 Cell surface transport versus ER degradation, 20-22 Cell wall components, antibodies raised against, 234-237 Cell wall functions, diversity, 233-234 Cell wall polymers, location and metabolism, 240-246 enzymes, 246 polysaccharides, 241-245 (1+3),( 1+4)-P-D-Glucan, 243 other polysaccharides, 244-245 polygalacturonic acids, 243-244 xyloglucan, 241-243 structural glycoproteins, 245 Cellule ramificati, 114 Cellular biogenesis and organization endogenous enzymes, use of as reporters for local concentrations of reactants, 278-280 involvement of physical processes and local catalysis in cellular metabolism, 275-278 protein traffic and assembly, 270-272 structural basis for organization of metabolism, 272-275 Cellular compartments of FL, leading cloning, expression and localization of native luciferase, 2%-297 retargeting to other cellular organelles, 297-299 targeting luciferase to, 314-315 Cellular organelles of FL, retargeting to other, 297-299 Cephalochordata, presence of MGPB, 136 Ceratostoma foliolaturn, 133 Cerithium adansonii, 133, 151 Chlamydomonas cell walls, 236 Chloride cells and accessory cell during transfer of sea-water adapted fishes to fresh water, 217-221 and accessory cells in stenohaline seawater fishes, 216-217 Chloride cells, general ultrastructural features

327

INDEX cell surface, 193-199 apical cavity, 193-196 tubular system, 196-199 endoplasmic reticulum, mitochondria, and cytoskeleton, 202-204 golgi apparatus and vesiculotubular system, 200-202 Chordata, presence of MGPB, 136-143 amphibia, 140-141 aves, 142 cephalochordata, 136 c yclostomata, 136- 137 mammalia, 142-143 reptilia, I4 1-142 teleostei, 137-140 Clarias gariepinus, 140, 170 Cloning, expression and localization of native luciferase, 2%-297 Cnidaria, presence of MGPB, 120-122 Cobitis taenia, 207 Coturnix coturnix, 142 Crustacea, presence of MGPB, 127-128 Cyclostomata, presence of MGPB, 136- I37 Cyprinodon variegatus, 214 Cyprinus carpio, 137 Cysteine tailpiece-dependant retention of secretory immunoglobulin, 16- 19 Bip-dependent model, 19 incomplete assembly model, 19

D Deroceras reticulatum, 133 Desmosomes and gap and septate junctions between barrier-forming cells, 150- 152 desmosomes, 150-151 gap junctions, 151 septate junctions, 151-152 Diapause barrier, 130 Dicotyledons, 249-252 other polysaccharides, 25 1-252 xyloglucan, 249-25 1 Digitonin, use of as lysis buffer, 24 Diodora nubecula, 135 Dipetalonema dessetae, 116, 123 Dithiothreitol- and protease-solubilized ZP, 100 Dolichos biflorus. 249

Down-regulation, and membrane IgM, 27-28 Dugesia biblica, 122-123, 124, 174, 176 E

Echinodermata, presence of MGPB, 135-136 Eisenia foetidus, 125 Electron microscopy and ZP, 99-104 Elliptogenesis of MB cells, 64-65 End stage of B cell ontogeny, 13 Endocytosis, and membrane IgM, 27-28 Endogenous enzymes, use of as reporters for local concentrations of reactants, 278-280 Endoplasmic reticulum, mitochondria, and cytoskeleton, 202-204 Environment, modifications of and mitochondria-rich cells a-and p-chloride cells in fresh water-adapted fishes, 204-207 chloride cells and accessory cell during transfer of sea-water adapted fishes to fresh water, 217-221 chloride cells and accessory cells in stenohaline seawater fishes, 216-217 ultrastructural features of cells during transfer of euryhaline fishes from fresh water to sea water, 207-217 accessory cells, appearance, 214-216 degeneration of p-cells, 209-212 hypertrophy of a cells, 212-214 Enzyme characteristic of FL, 282-287 initial and steady-state kinetics of light production, 285-287 molecular mechanism of light emission, 282-283 structure, 284-285 Enzymes, 246 antibodies raised against plant cell wall, 234, 235 ER degradation versus cell surface transport, 20-22 Erythrocytes, nucleated, cytoskeletal system, see also Nucleated erythrocytes mammalian primitive, 76-79

328

INDEX

mature, marginal band, 43-59 morphogenesis, 69-67 Euryhaline fishes, ultrastructural features of cells during transfer of from fresh water to sea water, 207-217 Excurrent duct epithelium of testes, MGPB in, 152-155 F

Fertilization, ZP, functions after, 86-89 during, 86 summary, 89 Firefly luciferase (FL), biologically localized cellular biogenesis and organization endogenous enzymes, use of as reporters for local concentrations of reactants, 278-280 involvement of physical processes and local catalysis in cellular metabolism, 275-278 protein traffic and assembly, 270-272 structural basis for organization of metabolism, 272-275 experimental approaches and perspectives light measurement, 309-310 perspectives, 313-3 18 luciferase as probe for protein metabolism, 313-314 monitoring local events in cryptic compartments, 317-318 prospects for local [ATP] measurements, 3 15-3 17 targeting luciferase to cellular compartments, 3 14-3 15 practical considerations, 310-313 use of FL as probe for metabolites concentrations, 3 10-3 I2 genetic and chemical engineering of luciferase, 312-313 leading into cellular compartments cloning, expression and localization of native luciferase, 2%-297 retargeting to other cellular organelles, 297-299

light emissions by in biological systems, 300-308 in whole cells, 300-302 monitoring local [ATP] at mitochondria1 surface, 302-308 dependence on rate of ATP formation, 305-306 local versus bulk [ATP] measurements, 302-304 modeling, 306-308 overview enzyme characteristic, 282-287 initial and steady-state kinetics of light production, 285-287 molecular mechanism of light emission, 282-283 structure, 284-285 history and applications, 280-281 probe for ATP in model systems, 287-2% continuous monitoring using immobilized luciferase, 288-291 mathematical modeling with soluble and localized luciferase, 291-296 kinetic analysis of FL reaction, 291-292 kinetic behavior of immobilized enzymes, effect of diffusion, 292-294 quantitation of light output from (Co) immobilized luciferase, 295-296 Fishes, fresh water-adapted, a-and P-cells, 204-207 FL, see Firefly luciferase Free steroid hormone hypothesis, 161 Fruit softening, 257-259 Fundulus heteroclitus, 191,204

Gallus domesticus, 142 Gap junctions, desmosomes, 151 Germination of plant cell walls, 255-256 Gill cells of teleost fishes, see also Teleost fishes gill morphology, 192-193 Gloosphonia complanata, 127

329

INDEX Glycine m a , 249 Glycoproteins, arrangement of in ZP filaments, 105 Glycoproteins, structural, 245 Glycoproteins, ZP, 92-95 ZP1,92 ZP2,92-94 ZP3,94-95 source, 95-96 Gobio gobio, 207 Golgi apparatus and vesiculotubular system, 200-202 Gramineae, 252-254 (1 + 3),(1 + 4)-P-D-GlUCan, 252-254 other polysaccharides, 254 Growth regulation of plant cell walls, 246-254 dicotyledons, 249-252 other polysaccharides, 251-252 xyloglucan, 249-25 1 gramineae, 252-254 (1 + 3),(1 4)-P-~-Glucan,252-254 other polysaccharides, 254 preliminary study approaches, 248-249

-

Immunoglobulin transport in B cell development, see B cell lymphocyte differentiation pathway and immunoglobulintransport Immunological approaches to plant cell walls, see Plant cell walls, immunological approaches In vitro Sertoli cells, 118-1 19 Initial kinetics of light production, 285-287 Insecta, presence of MGPB, 128-132 Intermediate filaments (IFs) in nucleated erythrocytes, 75 Inter-Sertoli septate junctions, MGPB formed by, 133 Intracellular retention of secretory immunoglobulin, 13-18

J J chain, role of, and assembly of polymeric immunoglobulin, 30

K H

H chain and Bip in pre-B cells, intracellular retention, 14-16 Heliothis virescens, 128 Helix aspersa, 134 Hirudo medicinalis, 127 Hormones and chloride cells, 225-227 Hyalophora cecropia, 15 1 Hydra littoralis, 176 Hydra oligactis, 176 Hydra viridis, 120-122, 173 Hydrophobicity, role of, and associated proteins, 22-27 Hypertrophy of a-cells, 212-214 I

IgM/IgD-expressing B lymphocytes, mature, 11-12 Immunoglobulin secretion in plasma cells, 29-30

KDEL sequences and salvage pathway, 14-16 Kinetic analysis of FL reaction, 291-292 Kinetic behavior of immobilized FL enzymes, effect of diffusion, 292-294 Kinetics of light production, initial and steady-state, 285-287

L L chains, role of and appropriate conformation for secretion, 30-31 pre-B-specific surrogate immunoglobulin, 7-10 Lacerta muralis, 141 Lacerta sicula, 141 Leaf abscission of plant cell walls, 256-257 Lebistes reticulus, 206 Lectins important to study of plant cell walls, 237,238

330

INDEX

Levantina hierosolyma, 116, 132, 133, 134, 159 Life cycle of plant cell walls, 233 Light emission, molecular mechanism, 282-283 Light emissions by in biological systems, 300-308 in whole cells, 300-302 monitoring local [ATPI at mitochondria1 surface, 302-308 dependence on rate of ATP formation, 305-306 local versus bulk [ATPI measurements, 302-304 modeling, 306-308 Light measurement of FL, experimental approaches and perspectives, 309-310 Light production, initial and steady-state kinetics, 285-287 , Limulus polyphemas, 249 LIS-solubilized ZP, 99-100 Lithobius forjicatus, 116, 128 Locusta migratoria, 128, 129, 131 Lonchuria striata, 142 Luciferase genetic and chemical engineering, 3 12-3 13 immobilized, continuous monitoring using, 288-291 localized, mathematical modeling, 291-2% kinetic analysis of FL reaction, 291-292 kinetic behavior of immobilized enzymes, effect of diffusion, 292-294 quantitation of light output from (Co) immobilized luciferase, 295-2% Luciferase as probe for protein metabolism, 313-31.4 monitoring local events in cryptic compartments, 317-318 prospects for local [ATP] measurements, 315-317 targeting luciferase to cellular compartments, 3 14-315 Luciola cruciata, 2% Luciola mingrelica, 296 Luidia clathrata, 115

Lumbricus terrestris, 125 Lymnaera stagnalis, 116, 133, 134-135, 154, 159, 171

M Macrobrachium rosenbergii, 127 Macropus eugenii, 76 Male germ cell protective banier (MGPB) background blood-organ barriers, different, 112-113 MGPB, methods helping to arrive at concept, 115-1 18 Sertoli cell for somatic cells in testis in different phyla, 114-1 15 Sertoli cell in vitro, 118-1 19 terms, 113-1 14 conclusions, 173- 177 function autoimmune aggression in mammals and the MGPB, 163-169 efficacy of barrier in physiological, pathological and experimental conditions, 160-163 MGPB, medical aspects, 171-173 MGPB, role of in maintaining spermatozoan immortality, 169- 171 secretory activity of sertoli cell, 159-160 along phylogenesis annelida, 125-127 chordata, 136-143 cnidaria, 120-122 crustacea, 127-128 echinodermata, 135-136 insecta, 128-132 mollusca, 133-135 myriafoda, 128 nematoda, 123 platyhelminthes, 122- 123 porifera, 19-120 structure desmosomes and gap and septate junctions between barrier-forming cells, 150-152 desmosomes, 150- 151

33 1

INDEX gap junctions, 151 septate junctions, 151-152 MGPB in excurrent duct epithelium of testes, 152-155 Sertoli cell and occluding junctions, 143-147 Sertoli cell junctions during growth, annual reproductive cycle, and spermatogenic process, 148-150 sites of different MGPB in testes, 157-159 testicular vascular system, 155-157 Mamestra brassicae, 116, 128, 130 Mammalia, presence of MGPB, 142-143 Mammalian primitive erythrocytes, 76-79 Mammals, autoimmune aggression and MGPB, 163-169 Marginal band (MB) biogenesis and function during erythrocyte morphogenesis, 59-67 elliptogenesis, 64-65 maturation, 65-67 MB biogenesis and mechanism of cell flattening, 59-64 Marginal band reassembly, experimentally induced, 53-56 Mastela vison, 145 Mature erythrocytes, marginal band, 43-59 experimentally induced MB reassembly, 53-56 MB function in, 56-58 molecular components, 49-53 other MB-associated proteins, 50-53 tubulin, 49-50 mechanical properties, 47-49 structure, 44-47 tribute to Friedrich Meves, 58-59 Mature IgMIIgD-expressing B lymphocytes, 11-12 Mauremys caspica rivulata, 125 MB, see Marginal band Membrane immunoglobulin ER degradation versus cell surface transport, 20-22 recycling, endocytosis and down-regulation, 27-28 retention in plasma cells, 29 transport, 22-27 transport during B cell ontogeny, 20-27

Membrane skeleton (MS) of nucleated erythrocytes, 67-75 biogenesis, 70-71 mechanical properties, 71-75 molecular composition, 69-70 structure, 68-69 Memory B lymphocytes, 12 Mesocricetus auratus, 148 Metabolites concentrations, use of FL as probe, 310-312 Metabolism of FL, structural basis for organization, 272-275 Meves, Friedrich, tribute, 58-59 MGPB, see Male germ cell protective barrier Mitochondria-rich gill cells of teleost fishes, see Teleost fishes Model system for coupling catalytic steps in a heterogenous system, 306-308 Molecular components, of mature erythrocytes, 49-53 proteins, other MB-associated, 50-53 tubulin, 49-50 Molecular mechanism of light emission, 282-283 Mollusca, presence of MGPB, 133-135 Monodelphis domestica, 39,76,77 Mouse egg extracellular coat, structure, see Zona pellucida MS, see Membrane skeleton Mustelus canus, 42 Myriafoda, presence of MGPB, 128 Myxine glutanosa, 136 N

Nematoda, presence of MGPB, 123 Noetia ponderosa, 55 Notophthalmus viridescens, 49 Nucleated erythrocytes, cytoskeletal system conclusions, 79-80 intermediate filaments, 75 mammalian primitive erythrocytes, 76-79 marginal band biogenesis and function during erythrocyte morphogenesis, 59-67

332

INDEX

elliptogenesis, 64-65 maturation, 65-67 MB biogenesis and mechanism of cell flattening, 59-64 mature cells, marginal band of, 43-59 experimentally induced MB reassembly, 53-56 MB function in, 56-58 molecular components, 49-53 other MB-associated proteins, 50-53 tubulin, 49-50 mechanical properties, 47-49 structure, 44-47 tribute to Friedrich Meves, 58-59 membrane skeleton, 67-75 mechanical properties, 71-75 molecular composition, 69-70 MS biogenesis, 70-71 structure, 68-69 phylogenic and physiologic portrait, 39-43 study, 37-38

0 Oligocottus maculosis, 217 Onchorhynchus keta, 170 Oocyte, growing, ZP of, 100-104 Oreochromis mossambicus, 210,217 Oreochromis niloticus, 116, 139 Oryzias latipes, 137, 139 Oryzias niloticus, 137

P Photinus pyralis, 28 1, 296 Phragmatopoma lapidosa, 127 Phylogenesis, MGPB annelida, 125- 127 chordata, 136-143 amphibia, 140- 141 aves, 142 cephalochordata, 136 cyclostomata, 136-137 mammalia, 142-143 reptilia, 141-142 teleostei, 137-140 cnidaria, 120-122 crustacea, 127-128

echinodermata, 135- 136 insecta, 128-132 mollusca, 133-135 myriafoda, 128 nematoda, 123 platyhelminthes, 122- 123 porifera, 19-120 Pylogenic portrait of nucleated erythrocytes, 39-43 Physical processes and local catalysis in cellular metabolism, involvement, 275-278 Physiologic portrait of nucleated erythrocytes, 39-43 Placobdella costata, 125, 127 Plant cell walls, immunological approaches antibodies as probes for study, 234-240 applications, 240 purification and specificity, 237-240 raised against cell wall components, 234-237 conclusions, 261-263 diversity of cell wall functions, 233-234 future prospects, 261-263 growth regulation, 246-254 dicotyledons, 249-252 other polysaccharides, 251-252 xyloglucan, 249-251 gramineae, 252-254 (1 + 3),(1 + 4)-P-D-GlUCan, 252-254 other polysaccharides, 254 preliminary study approaches, 248-249 life cycle, 233 location and metabolism of cell wall polymers, 240-246 enzymes, 246 polysaccharides, 241-245 (1 + 3),(1 + 4)-P-D-GlUCan, 243 other polysaccharides, 244-245 polygalacturonic acids, 243-244 xyloglucan, 241-243 structural glycoproteins, 245 other aspects, 259-261 selective breakdown, 254-259 fruit softening, 257-259 germination, 255-256 leaf abscission, 256-257 structural characterization of plant cell wall, 234

333

INDEX Plasma cells and end stage of B lymphoid ontogeny, 13 Plasma cells, membrane IgM retention, 29 Plasma cells, immunoglobulin secretion, 29-30 J chain, role of, and assembly of polymeric immunoglobulin, 30 L chains, role of, and appropriate conformation for secretion, 30-31 posttranslational modifications and secretion, 30 Platyhelminthes, presence of MGPB, 122- 123 Plafysamia Cynthia, 151 Poecilia latipinna, 154 Poecilia reticulata, 137, 140 Polygalacturonic acids, 243-244 Polymers, cell wall, location and metabolism, 240-246 enzymes, 246 polysaccharides, 24 1-245 structural glycoproteins, 245 Polysaccharides, 241-245,251-252,254 (1 + 3),(1 --* 4)-/3-~-Glucan,243 other polysaccharides, 244-245 polygalacturonic acids, 243-244 xyloglucan, 241-243 Porifera, presence of MGPB, 119-120 Posttranslational modifications and secretion, 30 Pre-B cells, fate of membrane immunoglobulin, 20-22 Pre-B to B-cell transition, 10-1 1 Pre-B lymphocytes, 6-7 Pre-B-specilic surrogate immunoglobulin L chains, 7-10 Pro-B lymphocytes, 4-6 Protease- and dithiothreitol-solubilized ZP, 100

Protein traffic and assembly of FL, 270-272 Proteins, other MB-associated, 50-53 Pyrophorus plagiophtalarnus, 281,282, 309 Q

Quantitation of light output from (Co) immobilized luciferase, 295-296

R Rana catesbiana, 65 Rana espculenta, 140, 155 Rana ridibunda, 141, 155 Rana temporaria, 141 Recycling, membrane immunoglobulin, 27-28 Reptilia, presence of MGPB, 141-142 Retentioddegradation and transport of immunoglobulin, choices between, 31-33 Rhamnogalacturonan 1 antibodies, 244 Ribulose biphosphate carbox ylase (RUBISCO), 299

S Saccharornyces cerevesiae, Bip gene in, 15 Saccharomyces cerevisae, 297 Salamandra salamandra, 140 Salmo gairdneri, 154, 166,227 Salmo salar, 223 Salrno trutta, 226 Salmonids, smoltilication and mitochondria-rich cells, 221-225 Salvelinus fontinalis, 154 Schistocerca gregaria, 129, 130 Scophthalmus maximus, 216,219 Secretion in plasma cells, immunoglobulin, 29-3 1 Secretory immunoglobulin, intracellular retention, 13-18 cysteine tailpiece-dependant retention, 16-18 KDEL sequences and salvage pathway, 14-16 Septate junctions, desmosomes, 151-152 Sertoli cell in vitro, 118-1 19 junctions during growth, annual reproductive cycle, and spermatogenic process, 148-150 occluding junctions, 143-147 secretory activity, 159-160 for somatic cells in testis in different phyla, 114-115 Smoltification in salmonids, and mitochondria-rich gill cells, 221-225

334

INDEX

Somatic cells, sertoli cells in testis in different phyla, 114-1 15 Sparus aurata, 174 Spermatozoan immortality, role of MGPB in maintaining, 169-171 Staphylococcus species, 237-238 Steady-state kinetics of light production, 285-287

Stenohaline seawater fishes, chloride cells and accessory cells, 216-217 Structural characterization of plant cell wall, 234

Testes, sites of different MGPB in, 157- 159

Testicular vascular system, 155-157 Tetragonolobus purpureas, 248 Tilapia aurea, 214 Tilapia specimens, 166 Transitional and immature B cell stages, 10-11 Triatoma infestans, 128, 130, 152 Triturus carnifex, 140 Triturus cristatus, 66 Tubulin, 49-50

T

U

Taeniopygia guttata, 142 Teleost fishes, mitochondria-rich gill cells, ultrastructure chloride cells, general features cell surface, 193-199 apical cavity, 193-1% tubular system, 196-199 endoplasmic reticulum, mitochondria, and cytoskeleton, 202-204 Golgi apparatus and vesiculotubular system, 200-202 conclusions, 227-228 environment, modifications a-and p-chloride cells in fresh water-adapted fishes, 204-207 chloride cells and accessory cell during transfer of sea-water adapted fishes to fresh water,

Ulex europaeus, 248 Ultrastructural features of cells during transfer of euryhaline fishes from fresh water to sea water, 207-217 accessory cells, appearance, 214-216 degeneration of p-cells, 209-212 hypertrophy pf a cells, 212-214 Ultrastructure of zona pellucida, 96-105 arrangement of glycoproteins in ZP filaments, 105 electron microscopy, 99-104 LIS-solubilized ZP,99-100 oocyte, growing, ZP of, 100-104 protease- and dithiothreitol-solubilized ZP, 100 general considerations, 96-98 summary, 105

217-221

chloride cells and accessory cells in stenohaline seawater fishes, 2 16-2 17

ultrastructural features of cells during transfer of euryhaline fishes from fresh water to sea water, 207-217 accessory cells, appearance, 2 14-2 16

degeneration of p-cells, 209-2 12 hypertrophy of a cells, 212-214 gill morphology, 192-193 hormones and chloride cells, 225-227 and smoltification in salmonids, 221-225 Teleostei, presence of MGPB, 137-140

v Vesiculotubular system and golgi apparatus, 200-202 Virgin splenic B cells, 16-17 Vitamin-A deficient diet, effect on spermatogenesis, 162-163 Viviparus uiuiparus, 134 W

Whole cells, light emissions in biological systems, 300-302

INDEX X Xenopus laevis, 61,62,67, 141, 296 Xiphophorus helleri, 166 Xylanase-gold conjugates, use, 245 Xyloglucan, 241-243,249-251 effect of antibodies in azuki and oat segments, 250,251 Xyloglucan oligosaccharide, 250

z Zona pellucida (ZP), structure characteristics, 89-96 general considerations, 89-91 source of ZP glycoproteins, 95-96 summary, % ZP glycoproteins, 92-95 ZP1.92

335

Zp2.92-94 ZP3,94-95 conclusions, 105-108 description, 85-86 functions, 86-89 after fertilization, 86-89 during fertilization, 86 summary, 89 ultrastructure, 96-105 arrangement of glycoproteins in ZP filaments, 105 electron microscopy, 99-104 LIS-solubilized ZP, 99-100 oocyte, growing, ZP of, 100-104 protease- and dithiothreitol-solubilized ZP, 100 general considerations, 96-98 summary, 105 Zonula adherens, 150-151 ZP, see Zona pellicuda

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