International Review of Cytology: A Survey of Cell Biology, Volume 155

VOLUME 155

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

1949-1 988 1949-1 984 19671984-1 992 1993-

ADVISORY EDITORS Aimee Bakken Eve Ida Barak Howard A. Bern Robert A. Bloodgood Dean Bok Stanley Cohen Rene Couteaux Marie A. DiBerardino Donald K. Dougall Charles J. Flickinger Nicholas Gillham Elizabeth D. Hay Mark Hogarth Anthony P. Mahowald M. Melkonian Keith E. Mostov

Audrey Muggleton-Harris Andreas Oksche Muriel J. Ord Vladimir R. Pantic M. V. Parthasarathy Thomas D. Pollard Lionel I. Rebhun L. Evans Roth Jozef St. Schell Manfred Schliwa Hiroh Shibaoka Wilfred Stein Ralph M. Steinman M. Tazawa Yoshio Watanabe Alexander L. Yudin

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

Jonathan Jarvik Department of Biological Sciences Carnegie Mellon University Pittsburgh, Pennsylvania

VOLUME 155

ACADEMIC PRESS San Diego New York

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Copyright 0 1994 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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United Kingdom Edition published by Academic Press Limited 24-28 Oval Road, London NWl 7DX International Standard Serial Number:

0074-7696

International Standard Book Number:

0-12-364558-1

PIUNTED IN THE UNITED STATES OF AMERICA 94 95 9 6 9 7 98 9 9 E B 9 8 7 6

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

vii

Phylogeny and Ontogeny of Chemical Signaling: Origin and Development of Hormone Receptors G. Csaba I. 11. 111. IV.

Introduction ........................ Signal Molecules and Receivers in Unicellular Organisms Ontogeny of Hormone Receptors . Conclusions ....................... References ........................

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Growth Factor-Induced Cell Migration: Biology and Methods of Analysis Marianne Manske and Ernesto G. Bade I. II. 111. IV.

Introduction ... Analysis of Cell Migration ............. Growth Factors That Modu .................................. Conclusions and Outlook .................................................................. References ................................................................................

49 53 67 81 81

Physiological and Biochemical Aspects of Cytoplasmic Streaming Teruo Shimmen and Etsuo Yokota I. introduction ................................................................................ V

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CONTENTS

vi II. 111 . IV. V.

Mechanism of Motive Force Generation .................................................. DemembranatedCell Models of Characeae and Characteristicsof Cytoplasmic Streaming Extracellular Factors Affecting Cytoplasmic Streaming ................................... Concluding Remarks ...................................................................... References ................................................................................

98 110 120 129 131

Cell and Molecular Biology of Flagellar Dyneins David R . Mitchell I. II. 111. IV. V. VI . VII . VIII.

Introduction .................................................. Dyneins in Flagellar Motility .... .................... Outer Row Dyneins ............ ................ Inner Row Dyneins: 57 Varieties Distinct Roles for Outer and Inner Row Dyneins The Cross-Bridge Cycle ........ Dynein Regulation .............. ................. Conclusions .................... References ..........

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141 142 146 162 166 168 170 173 175

Morphological and Functional Reorganization of Human Carcinomas in Vitro Petra Kopf.Maier. Birgit Kolon. and Markus Bugenings I. II. 111 . IV.

Introduction ................................................... Cell Culture Systems for Growing Human Carcinomas ...... High-Density (Organoid) Culture .................... Concluding Remarks ........... References ..........

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

181 182 187 245 252 259

Numbers in parentheses indicate the pages on which the authors' contributions begin

Ernest0 G. Bade (49),Arbeitsgruppe Zellbiologie-Tumorbiologie,Fakultat fur Biologie, Universitat Konstanz, 78434 Konstanz, Germany Markus Bugenings (181), InstitutfiirAnatomie,freie Universitat Berlin, D- 14195BerlinDahlem, Germany G. Csaba (1), Department of Biology, Semmelweis University of Medicine, 1445Budapest, Hungary

Birgit Kolon (181), lnstitut fur Anatomie, Freie Universitat Berlin, D- 14195 BerlinDahlem, Germany

Petra Kopf-Maier (181), lnsfifut fur Anatomie, Freie Universitat Berlin, D- 14195 BerlinDahlem, Germany Marianne Manske (49),Arbeiisgruppe Zellbiologie-Tumorbiologie,Fakultat fur Biologie, Universitat Konstanz, 78434 Konstanz, Germany David R. Mitchell (141), Department of Anatomy and Cell Biology, and Program in Cell and Molecular Biology, State University of New York Health Science Center, Syracuse, New York 13210 Teruo Shimmen (97), Department of life Science, faculty of Science, Himeji lnstitute of Technology, Harima Science Park City, Hyogo 678-12, Japan Etsuo Yokota (97), Department of life Science, Faculty of Science, Himeji Institute of Technology, Harima Science Park City, Hyogo 678-12, Japan

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Phylogeny and Ontogeny of Chemical Signaling: Origin and Development of Hormone Receptors G.Csaba Department of Biology, Semrnelweis University of Medicine, 1445 Budapest, Hungary

1. Introduction A. Communication in the Lowest Level of Phylogeny Unicellular organisms live in aqueous environment and thus the molecules of the medium determine the environment itself. These molecules can be beneficial for the cell (e.g., nutrients) or harmful (e.g., toxins). It is essential that an encounter with these molecules result in a change in future behavior, a memory, because this will give the cell an advantage in its search for beneficial molecules and in its avoidance of harmful ones. The communication is unilateral in this case, the cell detecting its environment and orienting itself with the help of signals (reception). Since the environment of the unicellular organism is very variable, it is not possible to have previously defined-fixed-receptors on the plasma membrane. Only the continuous extension and withdrawal of structures can provide the sensitivity required in the plasma membrane. The encounter of receptor structures and signal molecules fixes the reception. Everything points to the fact that only the subunits of the plasma membrane are genetically coded in unicellular organisms (Koch er al., 1979), and the way in which they are assembled is accidental. Following an encounter with signaling molecules, the number of structures that receive the signals increases in the plasma membrane (or the structures are produced as a complex of subunits); with the help of this “memory,” the ability to respond promptly is assured (Csaba, 1986a). The lifespan of the unicellular organism is brief and can be measured in hours or minutes. This means that receptor memory has value for the species as a whole if it is transferred to following generations. Protozoa Inlernalionol Review of C.vIolog.v, Vol. 155

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Copyright 0 1994 by Academic Press,lnc. All rights of reproduction in any form reserved.

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are able to transfer information acquired by imprinting to their progeny (Csaba e? al., 1982~); the presence of this memory was observed over a long period (in a thousand generations at least). The unicellular organism does not have the ability to transfer cultural information as do the higher ranks of animals or human beings. Nevertheless, the transmission of receptors (memory) that develop in an encounter performs the same role as transmission of cultural information. It is probable that the formation of receptors in the membrane is preceded by other activities because isolated intracellular compartments have to find each other and this requires the ability to recognize each other. When a lysosome containing enzymes approaches and fuses with the phagosome which enters the cell, there is a process of recognition. For this process to take place, glycoprotein structures are required in the intracellular membranes. These structures build signals and signal-receiver units (marker and receptor-like structures), which are considered to be the ancestors of the plasma membrane receptors. The unicellular organism can detect signals and synthesize signal molecules to be secreted into the environment (Le Roith e? al., 1980, 1981, 1982; Roth et al., 1982). Interestingly, ancestors of signal molecules that have a hormonal function in higher animals have been demonstrated in unicellular eukaryotes, though their exact function is still unclear. Insulin or an adrenocorticotropic hormone produced by a protozoan might be a side product of the protein synthesis of a similar hormone which has the capacity to influence either the cell itself in an autocrine way or to affect other unicellular organisms. Whether or not an autocrine function exists, the protozoan has a complete endocrine system because it is able to produce a hormone that interacts with the membrane receptors. Since the unicellular organism has its own second messenger system (Kuno et al., 1979; Schultz e? al., 1983; Nakaoka and Ooi, 1985; Nagao er al., 1981; Kovhcs and Csaba, 1987b, 1990c; Kovacs e? al., 1989a), the hormone is also able to induce a response. However, it is possible that the material secreted to the environment can influence other related cells, as happens in the case of Dictyostelium discoideum (Ray and Lerner, 1987). Here secreted cAMP is the signal which induces grouping of amebas because the amebas have cAMP receptors. Similarly, special hormone-like materials (pheromones) are secreted during the conjugation of protozoa (Nobili e? al., 1987). Probably the receptors are developed earlier than the signal molecules (hormones) at this stage of phylogeny, because the ability for receptors to recognize signals and molecules is the critical element in the cell-environment relationship. In the first phase of development, the communication is such that the cell “understands but does not speak”; in the second phase, hormone-like materials appear and the cell “both speaks and under-

ORIGIN AND DEVELOPMENT OF HORMONE RECEPTORS

3

stands.” At the same time, if we agree with Blalock’s hypothesis (Bost et ul., 1985a,b), this suggests that the DNA strand coding for the receptor also contains a code for the hormone (Schwabe, 1990). 6 . Fundamentals of Communication in Multicellular Organisms

At the unicellular level, the receptor becomes stronger in the presence of signal molecules because it is not preprogrammed. However, it is usual for the receptors of multicellular organisms to be preprogrammed (Csaba, 1986a,b). This means that the types of receptors appearing on the surface (or intracellularly) are determined at the gene level in each cell. Similarly, the determination of which cell will synthesize a certain kind of hormone is also programmed. These traits are derived from the multicellular character of the organism, which is structured so that the signal and the receiver are built on the information of the same genome. However, if there is a community of cells, certain regulator molecules are able to influence only special groups of cells. Although the signal molecule (hormone) may be present in the whole organism, only the receptor possessing the proper cells will respond to it. In this way the gene program permits inquiry by receptors. In this case, the interaction between the signal molecule and the receiver is completed in a closed system. This does not exclude the subsequent emission of some signal molecules, as in the case of the emission and reception of pheromones. Thus the older system of signaling persists.

II. Signal Molecules and Receivers in Unicellular Organisms A. Receptors in Protozoa and Microbes

When substances which function like hormones in the higher ranks of animals are added to the environment of protozoa and suitable indexes are applied, the reaction of the unicellular organism can be described. Phagocytosis in Tetruhymena was successfully induced by histamine, which acts as a phagocytosis hormone in higher ranks of phylogeny, and also by serotonin (Csaba and Lantos, 1973). The sequence of the phagotrophic effect is similar to the order described in higher animals. Hormones influencing the carbohydrate metabolism of higher cells, insulin (Csaba and Lantos, 1975a) and epinephrine (Csaba and Lantos, 1976) are

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able to induce sugar metabolism in Tetrahymena while they are ineffective in several other reactions (Csaba and Lantos, 1975b). The morphogenetic hormone of higher animals, thyroxine and its precursors, can influence the division of Tetrahymena. Other hormones and hormone-like substances are able to induce positive or negative chemotaxis in Tetrahymena (Nobili et al., 1987; Csaba and Kovacs, 1994a). Hormones that have no target reaction in Tetrahymena cause alterations in protein synthesis, and so they are not ineffective (Csaba and Ubornyak, 1981). From these facts and other works (Castrodad et al., 1988; Renaud et al., 1991; De Jesus and Renaud, 1989; Quinones-Maldonado and Renaud, 1987; Wyroba, 1989; Zagon and McLaughlin, 1992), we can conclude that Tetrahymena is affected by the hormones of higher animals. At the same time, the biological view requires us to pose the question inversely, since it seems that it is not the hormones of higher animals but those substances that became hormones and signal molecules during evolution that have the capacity to evoke some specific reaction in Tetrahymena. Tetrahymena has no endocrine system and the hormone is only one substance of many which the cell is able to recognize; the presence of this material in its environment or its interaction with the cell membrane elicits some reaction by the cell. In this way perhaps some materials were selected as hormones from the materials surrounding the protozoa if they did not already have another evolutionary purpose, and if they were able to bind to receptors to transmit information into an intracellular system. It is probable that a material suitable for becoming a hormone has to fulfill many additional conditions. The information carried by a signal molecule becomes valuable and usable only when a cell has a receptor for the molecule. Such receptors are present in the plasma membrane of unicellular organisms (Kovacs and Csaba, 1990a). For a protozoan it is essential to recognize the environment, to select the useful molecules as nutrients, and to avoid the harmful ones (Leick and Hellung-Larsen, 1992). The existence of nutrient receptors (Lenhoff, 1968, 1974)for amino acids or polypeptides makes this possible. Tetrahymena recognizes an amino acid and the hormone derived from the amino acid as similar molecules. This cell also has the ability to differentiate the hormone, the analog of the hormone, and its antagonist (Csaba and Lantos, 1975c; Csaba and Darvas, 1986) (Fig. 1). The nutrient receptor detects the binding of similar molecules by an altered response. Tetrahymena possesses not only nutrient receptors but mating receptors (Rosati and Verni, 1991) and it is postulated that the latter have a role in the development of hormone receptors (pheromone receptors). The development of membrane receptors that are rigidly determined at the gene level is impossible because of the lifestyle of the protozoa. This organism lives in a changing environment that includes a wide variety

5

ORIGIN AND DEVELOPMENT OF HORMONE RECEPTORS P.C.1

sc C=l .o

0.6

FIG. 1 Effect of histamine and histamine antagonists on the phagocytosis of Tetrahymena related to the control as 1 .O (broken line). P.C., Phagocytic coefficient; C, control (untreated); C-H, histamine-treated cells; CICim, cimetidine; C/s, chloropyramine; C/D, tripelennamine; and SC, significance to control. The scale represents the range of scattering. In the case of cells that were not treated with histamine earlier, only the histamine treatment elevated the phagocytotic capacity. (Reproduced from Csaba and Darvas, 1991, with permission. Copyright The Faculty Press, Cambridge, England.)

of surrounding molecules. This makes the presence of plastic structures in the plasma membrane important because these structures are able to form the most diverse configurations and thus are able to recognize the molecules of the surroundings. It seems to be essential to keep the receptor-type structures in the membrane to facilitate recognition. The hormone receptors have to be able to recognize everything; this capacity supports the formation of permanent structures (receptors) more than of other molecules. The location of recognition structures in the plasma membrane does not seem to be accidental at this level of evolution because this component of the cell maintains the cell’s connection with the environment. Though Tetrahymena is a first-class model cell for studying receptor evolution, the effects of hormones have been demonstrated not only in Tetrahymena but in Amoeba (Csaba et al., 1985c; Mayers and Couillard, 1990; Couillard et al., 1989) and even in fungi and bacteria (Le Roith et al., 1987; Lummis, 1992). Since the protozoa are representative of a lower level of evolution, we have to take into account the fact that the molecules which are hormones of the higher animals appear in some form in a lower rank of evolution. That is why it is assumed that in Tetrahymena, the precursors of hormones have greater importance than the higher rank hormone itself (Csaba and Nemeth, 1980). It has been shown that the less complex molecules of the thyroxine series-triiodothyronine, diiodothyronine, and monoiodothyrosine-are able to induce the multiplication of cells better than the thyrox-

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ine itself. These studies also support the theory of the origin of nutrient receptors. The low concentration of the base molecule of the thyroxine series, tyrosine, is as effective as diiodothyrosine. The difference is that diiodothyrosine works like a hormone, which means that the effect decreases in low doses and there is a fall following the peak because of toxicity. Tyrosine works as a nutrient; it induces a more intensive response (actually the division of cells) in the highest concentration applied. It is conceivable of course that this phenomenon could be observed where the complexity of the hormone is greater, (consider the jump from tyrosine to thyronine in the example of the thyroxine series, while in the case of the serotonin series, the hormone serotonin itself is most effective).

6. Hormones in Protozoa and Microbes The recognition of hormones by protozoa led to the study of endogenous hormones in unicellular organisms and microbes. In Tetrahymena pyriformis, the following substances were demonstrated: insulin, somatostatin, adrenocorticotropin (ACTH), @-endorphin,relaxin, vasotocin, and calcitonin (Le Roith et al., 1987);from the steroid hormones, dehydroepiandrosterone (DHEA) and estradiol (Csaba et al., 1985a); and from the amino acid-type hormones, serotonin and adrenalin (Blum, 1967; Brizzi and Blum, 1970; Janakidevi et al., 1966; Csaba and Kovacs, 1994a). In microbes thyrotropin (TSH) was detected in Clostridium perfringens, and chorionic gonadotropic hormone (HCG) was detected in many bacteria. Neurotensin, somatostatin, calcitonin, and insulin were detected in Escherichia coli (Lenard, 1992). The presence of neurotensin, insulin, and somatostatin was proved in other bacteria the same way. Neurotensin is present in Caulobucter crescentus and in Rodopseodomonas palustrus; insulin is present in Aspergillus fumigatus, Halobacterium solinarium, and in Bordatella pertussis; while somatostatin was demonstrated in Bacillus subtilis. Among fungi, calcitonin, arginine, and vasotocin have been detected in Neurospora crassa; cholecystokinin in Candida albicans; and farnesol-type mating hormones were detected in Saccharomyces cerevisiae (Le Roith et al., 1987). There are references to the presence of prostaglandin in Tetrahymena but this is uncertain (Csaba and Nagy, 1987). The presence of thyroxine and triiodothyronine could not be proved though it was checked (Csaba and Nagy, 1987). The presence of hormones does not mean that they are secreted by the cell. This is shown clearly by DHEA, which is not present in the medium of Terrahymena though the cells can be induced to secrete DHEA by dexamethasone (Csaba et al., 1985a). Tetrahymena also secretes insulin and serotonin into its medium (Csaba and Kovacs, 1994a).

ORIGIN AND DEVELOPMENT OF HORMONE RECEPTORS

7

As in the case of the hormone receptors discussed earlier, the question arises whether the hormones of higher animals also have hormone-like functions in Tetrahymena or whether they are simply the side products of amino acid, polypeptide, or steroid synthesis. It is probable that nature made several trials in the highly developed ciliates and those molecules that have not been detected have not been sought. The absence of triiodothyronine and thyroxine contradicts this, but the presence of such a nonhormone but steroid-type molecule as digoxin supports this premise (Csaba et al., 1984b; Darvas et al., 1985b). Considering that Tetrahymena has receptors, the presence of autocrine regulation is also imaginable, as is a paracrine-like intercellular regulation. In this case the Tetrahymena hormones have value in communication, and this ability, combined with receptors and hormones, may serve as a basis for the evolution of hormone and receptor alike. According to Roth and his co-workers, who played a pioneer role in the detection of hormones, there are well-conserved regions in the hormones of Tetrahymena and higher animals, and for this conservation the presence of a function is required (Le Roith er al., 1987). On the basis of this, it is conceivable that the molecules fulfill a communicative role. However, the excessively high number of molecules with this role in unicellular organisms raises doubts about their function. C. Specificity of Hormone and Receptor

Tetrahymena receptors have a good ability to discriminate. Receptors of the wall-less mutants of Neurospora crassu bind insulin in a specific way (Fawell et ul., 1988; Fawell and Lenard, 1988; McKenzie et al., 1988; Kole et al., 1991).Amebas possess specific opioid receptors and naloxone inhibits them ( Josefsson and Johansson, 1979). The biological mirrorversion of naloxone has no capacity to inhibit these receptors. Pseudomonus maltojilia has human chorionic gonadotropin (hCG) binding sites, and their affinity and specificity are similar to the HCG receptors of the human ovary. There are TSH binding sites in Yersiniu enterocolica and in Escherichia coli, and one can replace the labeled hormone with the unlabeled hormone. Paracoccidiodes brasiliensis has high-affinity binding sites for estrogen (Le Roith et af., 1987). Also, Saccharomyces cerevisiae is able to bind estrogens, and the biochemistry of the binding protein is similar to the steroid receptors of higher animals. Candida albicans binds corticosterone and its affinity lies between the affinities of cortisol binding protein (CBG) and the glucocorticoid receptor (Loose and Feldman, 1982). Trypanosoma cruzi has beta adrenergic receptors with radioligand binding criteria characteristic of mammalian cells (de Castro and Oliveira, 1987).More-

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over, the opioid receptor of Tetrahymena has a sequence homology with the identical receptors for leeches and rats (O'Neil et al., 1988; Zipser er al., 1988). All of this is meant to show that there are binding sites in protozoa which are similar to the binding sites of higher animals in several respects, although we have to consider the fact that the binding sites for steroids were found in cells that live in host organisms and thus the cells are not isolated from the influence of the host. Among the hormones of unicellular organisms, the alpha-type yeast mating factor has similarities to the gonadotropin-releasing hormone of mammals. Administration of this substance induces the production of hormones in the pituitary of mammals (Loumaye et al., 1983). Terrahymenu insulin is able to influence the level of blood glucose in vertebrates (Le Roith er al., 1983). On the basis of all these facts, the specificity of Tetrahymena hormones is acceptable and demonstrates that these molecules, which are present at a low level of phylogeny, can influence, at least in some part, the regulation of higher animals. D. Second Messenger Systems

The hormone itself and the presence of the membrane structures which bind it do not ensure the responsiveness of the cell. The system will only work properly when one terminal of the receptor performs its mediator function in a way similar to the epidermal growth factor (EGF) or insulin receptors, or when second messenger systems are present. It has been well known for a long time that cAMP serves as a starvation signal in bacteria and that the presence of these molecules is able to induce the assembly of Dicryosrelium discoideum (Roy and Lerner, 1987). Dictyostelium has cAMP receptors and there are different types of Gproteins which are able to generate and induce the liberation of different second messengers such as CAMP, cyclic guanosine monophosphate (cGMP), inositol trisphosphate, and calcium (Snaar-Jagalska et al., 1988a,b; Kesbeke and Van-Haastert, 1988; Kesbeke er al., 1988). It is probable that the structure of the G-protein is also similar to the identical molecule in higher animals. It has been shown that the isolated a-subunit is similar to the a-subunits of G-proteins of vertebrates in its function and physical parameters. In Trypanosoma cruzi, where the effect of isoproterenol on cell division was observed, a synchronous cAMP increase was also reported (de Castro et al., 1987). The presence of adenylate cyclase, the effect of CAMP, and the participation of the guanosine triphosphate (GTP) regulatory protein were demonstrated in Saccharomyces cereuisiae (Levitzki, 1988; Becker er al., 1988; Engelberg et al., 1989). Protein kinases and tyrosine kinase, which has a role in the function

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ORIGIN AND DEVELOPMENT OF HORMONE RECEPTORS

of receptors, were also found. The GTP binding protein was also detected in Neurospora crassu (Hasunuma and Funadera, 1987; Hasunuma et al., 1987a,b; Furukawa et ul., 1987). There were mutants of Neurospora in which the concentrations of both the cyclic phosphodiesterase and the CAMPwere decreased and the 24-hr oscillation of CAMP was also characteristic. In this mutant, white light also decreased the level of cGMP. It is clear that second messengers are present at the lowest levels of phylogeny, at the unicellular level of organization. In the protozoan Tetrahymenu, the work and cooperation of several second messenger systems was observed. They are the adenylate cyclase-CAMP system (Nagao et al., 1981; Csaba and Sudar, 1978; Kovacs and Csaba, 1986a; Kovacs, 1986; Csaba et al., 1976, 1978, 1987a), the guanylate cyclase-cGMP system (Kovacs et ul., 1989a), and the calcium-calmodulin system (Kovacs and Csaba, 1987a,b) (Figs. 2 and 3). Hormonal effects modify the level of second messengers working in the systems. Inositol phospholipids were also detected (Kovacs and Csaba, 1990~). Considering that (1) all three members of a typical endocrine system are present in unicellular organisms because the hormones are there and they are secreted, (2) the identical receptors are there, and (3) the second

I--

-A

0

C

FIG. 2 Guanylate cyclase activity after insulin imprinting in Tetrahymena. There is a highly significant difference ( p t

0'

10-10

I 10-~

10-0

10-6

10-7

lW

z

cc insulin(M)

FIG. 7 FITC-insulin binding of the nuclear (.) and plasma ( + ) membranes of Tetrahyrnena

treated (imprinted) with different concentrations of insulin 48 hr earlier. Control was 100% fluorescence intensity. All points are significant ( p