Analyses of the cytoskeleton in Saccharomyces cerevisiae.

Annu. Rev. Cell Bioi. Copyright ©

1991

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1991. 7: 633--{j2

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ANALYSES OF THE

Annu. Rev. Cell. Biol. 1991.7:633-662. Downloaded from www.annualreviews.org by Moscow State University - Scientific Library of Lomonosov on 08/29/13. For personal use only.

CYTOSKELETON IN SACCHAROMYCES CEREVISIAE F. Solomon

Department of Biology and Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02 1 39 KEY

WORDS:

tubulin, actin, motility, morphogenesis

CONTENTS INTRODUCTION.............................................................................................................. TUBULIN AND MICROTUBULES ........................................................................................

Tubulins ..... ......... .................................. ................................................................ Consequences of Drug-Induced Defects in Microtubule Structures ............................ Mutant Phenotypes of TUB! and TUB2 .................................................................. Testing Models for Regulation of Microtubule Assembly .......................................... Spindle Pole Bodies ................................................................. ................................. .

. .

ACTIN AND F-ACTIN.......................................................................................................

A Single Actin Gene ............................ ..................................................................... F-Actin Structures in Yeast ...................................................................................... Actin-Associated Proteins .........................................................................................

633 634 636 636 637 638 645 647 647 647 649

Changing Shape........................................................................................................ Moving Organelles ..... .... . . .. . ....... .. .. ... . ..... . .. . ..... ............ .. . .. .. .. ........ . ........ .. .....

653 653 655

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658

TWO MOTILE BEHAVIORS IN YEAST

....

CONCLUSIONS AND PROSPECTS

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.................................................................................. .

..

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..

INTRODUCTION Studies of the cytoskeleton aim at understanding how cells organize their cytoplasm to generate motility and asymmetry. The field has exploited many experimental model systems, a choice mandated by the diversity of interesting motile behaviors. In contrast, studies of other fundamental cellular entities, such as the transcription apparatus, have concentrated on a few organisms that were accessible originally to both biochemistry and genetics, and subsequently to molecular biology. Results of that effort 633 0743-4634/9 1 /1 1 1 5-0633$02.00


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supported the premise underlying the choice of simple model systems: tha the definitive findings obtained in simpler organisms could be readil� transferred in considerable detail to more complex organisms. Several groups have turned to yeast as a model system for the study 0 the cytoskeleton. The advantages of yeast as an experimental organisn include its well-developed classical and molecular genetics, accessibility t( biochemistry, as well as increasingly useful cytology. These advantage have permitted rigorous tests of in vivo function that are much mor, difficult to perform in higher eukaryotes. The disadvantages of yeast seen obvious. Yeast is, after all, an organism that doesn't walk or form tissues indeed, it is an organism that lives readily in suspension. But the limite( repertoire of cytoskeletal functions that yeast does display include fun damental ones. Yeast contains actin and tubulin, assembles them into F actin and micro tubules, and uses them for essential functions. And yeas cells do exhibit motility: they move cytoplasmic components, and the: change shape in patterns both guided by extracellular signals and specifie( by intrinsic determinants. Finally, yeast cells can display a wide variety 0 cytoskeletal structures. For example, even though the micro tubules in wil( type cells are arranged in fairly simple nuclear and cytoplasmic array (Figure lA), deviations from normal stoichiometry of microtubule com ponents (Figure I E-D) or specific mutations (Figures lE-F) can produCi a wide range of structures. Analysis of these aberrant products may lea( to an understanding of the normal morphogenetic pathway of cytoskeleta structures. This review focuses on analyses of two cytoskeletal structures, micro tubules and F-actin, and analyses of two motile behaviors, shape changl and organelle movement. The restricted set of issues were chosen to illus trate the contributions that the study of yeast structures and behavior have made to our understanding of the cytoskeleton in higher cells and t( identify questions that remain. Almost everything reviewed here is fron S. cerevisiae (hereafter just called yeast). Had space permitted, a mor, complete treatment would have included several other yeast studies as weI as the unique and complementary contributions from analogous systems such as S. pombe (Hagan & Hyams 1 988) and A. nidulans (Morris 1 9 89) Other relevant reviews of the progress and promise of yeast have appeare( (Barnes et al 1 99 1 ; Drubin 1 989; Huffaker et al 1 987; Katz & Solomo) 1 989; Pillus & Solomon 1 986b; Solomon 1 989; Stearns 1 990). TUBULIN AND MICROTUBULES

Yeast microtubules form the closed nuclear spindle as well as cytoplasmi, arrays. These structures and the spindle pole bodies, which apparentl:

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Morphogenesis of yeast microtubule structures is affected by alterations in stoi

chiometry or structure of relevant gene products. The micrographs show yeast microtubules visualized by immunofluorescence using antiserum against fJ-tubulin. A. A large-budded wild type cell, with long intranuclear microtubules and short cytoplasmic arrays at the ends. B. Over-expression of fJ-tubulin, visualized shortly after induction, completely depolymerizes

all cellular microtubules. Frequently, fJ-tubulin accumulates as two perinuclear dots, perhaps co-localizing with SPBs (Weinstein & Solomon 1990). C. Later, higher levels of fJ-tubulin accumulate in large, irregular structures that extend throughout the cytoplasm and that are devoid of IX-tubulin (Burke et al 1989; Weinstein & Solomon 1990). D. Co-overexpression of both IX- and fJ-tubulin produces microtubule structures similar to those in wild type cells but thicker (Weinstein & Solomon 1990). E. A conditional mutation in IX-tubulin results in excess cytoplasmic microtubules at or near the perimeter of the cell. Other mutations cause complete disassembly of microtubules (Schatz et al 1988). F. A rare cell in a strain bearing a mutation in SALl-f>, a suppressor of mutations in KARl, appears to contain a single microtubule structure so long that it describes several circumferences (courtesy of E. Vallen

& M, Rose).

serve as microtubule organizing centers in yeast, were first described at the level of electron microscopy (Byers & Goetsch 1 975). The development of an antibody that recognized yeast tubulin (Kilmartin et al 1 982) enabled characterization of tubulin polymers in yeast (Kilmartin & Adams 1 984) by immunofluorescence. Together, the two techniques depict microtubule structures that change during the cell cycle. Intranuclear microtubules are present in virtually every cell and elongate as mitosis progresses. The cytoplasmic microtubules appear to extend toward or even into the for ming buds and grow longer as the buds grow larger. Both nuclear and cytoplasmic arrays appear to originate at the spindle pole bodies (SPBs). The structure and behavior of the SPBs throughout the cell cycle has been characterized at high resolution (Byers & Goetsch 1 975) and reviewed recently in depth (Winey & Byers 1 99 1 ) . Briefly, in unbudded cells, SPBs are present in the nuclear envelope as single plaques. At the earliest stages of bud emergence, the plaque is duplicated, and the

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structures lie side by side near the site of bud emergence in both vegetative cells and in zygotes. As the bud enlarges, the plaques separate to form the poles of the spindle. They lie in indentations in the nuclear envelope. Analyses of the structure and function of yeast SPBs are described in a subsequent section.


Tubulins S. cerevisiae contains three tubulin genes, two encoding a-tubulins (TUB] and TUB3), and one encoding p-tubulin (TUB2). The p-tubulin gene is 70% identical to a chicken p-tubulin gene (Neff et al 1 983). Among p tubulins, the greatest divergence is in the carboxy-terminal domain of the protein and, indeed, the yeast TUB2 gene product is 1 2 amino acids longer than most homologues. The two a-tubulin genes in yeast have about 90% identical amino acid sequences (Schatz et al 1 986a), comparable to the divergence displayed by a-tubulin sequences expressed within other higher eukaryotic organisms. The number of tubulin genes in yeast facilitates rigorous tests of models for cellular regulation of microtubule assembly.

Consequences of Drug-Induced Defects in Microtubule Structures

The microtubule depolymerizing drug nocodazole, which has been used effectively in animal cells to analyze microtubule structure and function, also depolymerizes microtubules in yeast. M oderate concentrations of this drug rapidly arrest cell growth and produce cultures primarily of large budded cells (Jacobs et al 1 988). With a similar time course, the drug induces depolymerization of both cytoplasmic and nuclear arrays of micro tubules, although the nuclear arrays seem more resistant. [Occasionally cultures appear to break through the drug-induced arrest, a remarkable phenomenon that defies the obvious explanations (Jacobs et a1 1 988; Pillus & Solomon 1 986a)]. The arrested cells typically contain two SPBs lying close together, which suggests that SPBs may duplicate but not separate in the absence of microtubules (Jacobs et al 1 988). Even SPBs that are well separated on the nuclear envelope may, after long treatment with microtubule depolymerizing drugs, lie closely apposed on invaginations. These images of a partially collapsed nucleus raise the possibility that maintaining a separation of SPBs may require an intact spindle and that in its absence some other force may bring them together. Drug-induced microtubule depolymerization also affects nuclear move ments associated with budding. Normally, undivided nuclei tend to be close to the mother-bud neck and migrate into the neck as spindle elongation

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proceeds. In nocodazole-treated cells, the undivided nuclei are randomly positioned within the mother, and the alignment of the SPBs with the bud site is perturbed. Several crucial aspects of cellular asymmetry appear to be independent of microtubules: bud enlargement, the position of the bud site, the deposition of chitin, and the association of 1 0 nm-filaments (Byers & Goetsch 1 9 76) with the bud neck. Annu. Rev. Cell. Biol. 1991.7:633-662. Downloaded from www.annualreviews.org by Moscow State University - Scientific Library of Lomonosov on 08/29/13. For personal use only.

Mutant Phenotypes o/TUBl and TUB2

Mutations in fJ-tubulin, encoded by TUB2, have been generated by selec ting spontaneously arising strains resistant to the microtubule depoly merizing drug benomyl (Thomas et al 1 985); by in vitro mutagenesis of the TUB2 gene followed by integration into the wild-type locus (Huffaker et al 1 988); by screening for non-complementers of TUB2 mutations (Stearns & Botstein 1988); and as suppressors of mutations in oc-tubulin (Schatz et al 1988). Among those originally selected for their drug re sistance, several are either cs- or ts - . At the non-permissive temperature ( 1 loq, the cs- cells tend to arrest with large buds. Similarly, among mutants produced in vitro, and then screened for temperature-conditional most are either super-sensitive or resistant to benomyl. These cells also tend to arrest with large buds, with their single nucleus still in the mother. The arrest of cell division apparently does not interfere with normal DNA replication (Huffaker et aI 1 988). Different mutant alleles of TUB2 have significantly different phenotypes (Huffaker et aI 1 988). Some mutant strains have normal nuclear spindles, but either short or non-existent cytoplasmic mierotubules, while others have long cytoplasmic microtubules, but no intranuclear micro tubules. Still other mutants display either no microtubules at all, or have even more cytoplasmic and intranuclear micro tubules than do wild-type cells. These phenotypes suggest that the assembly of different microtubule structures in yeast may be regulated differently. The data also suggest a distinct role for a subset of the microtubules: cells that specific ally lack cytoplasmic microtubules show a pronounced defect in nuclear migration. This defect extends to the mating phenotype: those strains that do not produce cytoplasmic microtubules normally also do not mate efficiently probably because of failure in nuclear fusion (see below). Significantly, the absence of cytoplasmic microtubules has no apparent consequences for either bud growth or secretion-two manifestations of defects in actin. Efficient generation of strains carrying conditional mutations in the TUB] gene as their sole source of oc-tubulin has been achieved using plasmid-shuffie techniques to introduce sequences mutagenized in vitro. Of the 70 mutants in TUB] generated in that fashion (Schatz et al 1 988),

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most are cold-sensitive, between 1 1 to 1 5°C, and arrest with a large bud. When screened by immunofluorescence, these mutants can be divided into three classes of microtubule morphology: too few or no micro tubules; excess micro tubules in both the cytoplasm and the nucleus; and micro tubule structures that are difficult to distinguish from those present in normal cells. Testing Models for Regulation of Microtubule Assembly

The ultrastructure and properties of microtubules are highly conserved evolutionarily. But the organelles into which micro tubules are organized and the functions ascribed to those organelles are quite diverse. A major issue to be resolved is how both the conserved and divergent aspects of microtubule assembly and organization are specified. Three general explanations, derived largely from work on higher cells and studies on assembly of tubulin in vitro, are available. Two of them-the roles of tubulin sequences and autoregulation of tubulin levels-have been tested with some rigor in yeast. The third, the role of minor microtubule com ponents, is the focus of both genetic and biochemical experiments now underway. Both (X- and fJ-tubulins are the products of gene families, and genes with more than superficial primary sequence differences are expressed in the same organism and even in the same cell. One assertion of the multi-tubulin hypothesis is that the conserved elements of tubulin sequence are responsible for the conserved elements of microtubule structure and function, while the divergent elements directly or indirectly specify details of microtubule organization necessary for formation and function of particular organelles. Among fJ-tubulins, the most pronounced sequence divergence is located in the carboxyl end of the protein. The role of that domain has been tested in yeast. A truncated fJ-tubulin, lacking the 1 2 extra amino acids, can be generated by deleting codons 446--457 and replacing them with a stop codon. The wild-type and truncated gene products can be detected and distinguished by specific antibodies (Katz & Solomon 1 988). The truncated fJ-tubulin protein, co-expressed with the wild-type fJ-tubulin protein, is present in every microtubule structure as determined by double immunofluorescence. Haploid cells expressing only the truncated protein are indistinguishable from congenic wild-type strains by several criteria, including growth rate at normal as well as cold and high temperatures, and distribution in the cell cycle. The only phenotype that can be detected is a slight, dominant hypersensitivity to benomyl. These experiments demonstrate that this 1 2 amino acid carboxy-terminus of the TUB2 protein is not essential for normal microtubule functions. A THE MULTI-TUBULIN HYPOTHESIS


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deeper deletion, back to codon 430, produces a ts - phenotype. Shorter chains are not sufficient to support cell viability (Matsuzaki et al 1 988). These results suggest that this divergent domain of the TUB2 protein does not carry out essential functions. Domains of the TUB2 gene have been expressed in animal cells, as parts of chimeric tubulin proteins that contain significant amounts of animal tubulin sequence. Those experiments demonstrate that an animal-yeast chimeric protein, which contains the most divergent domain of the yeast fJ-tubulin gene, the carboxy-terminal 28%, behaves indistinguishably from endogenous animal cell proteins (Bond et al 1 986). These experiments and others like them in which tubulin isotypes are moved from one animal tissue to another (Joshi et al 1 987; Lewis et al 1 987) can not provide unambiguous tests of function, since the endogenous tubulin genes are still expressed. Instead, they test for mechanisms by which divergent tubulins expressed in the same cell may be excluded, partially or completely, for some cell structures. No evidence for such mechanisms is found. Some chimeric constructions containing yeast fJ-tubulin sequences are not detected (Fridovich-Keil et al 1 987), but such a negative result is difficult to interpret. It may mean that the protein is poorly folded because of improper interactions between domains of the polypeptide. The properties of the yeast a-tubulin genes permit even more stringent tests of the multi-tubulin hypothesis (Schatz et aI 1 986a). Strains containing disruptions of one of those genes, TUB3, are viable but show minor phenotypes: hypersensitivity to benomyl and defects in sporulation. In contrast, haploid cells apparently can not survive with a single copy of TUB3 as their sole source of a-tubulin. This result suggests that TUBl but not TUB3 is genetically essential and raises the possibility that these two quite homologous genes encode proteins of different functions, thus fulfilling a prediction of the multi-tubulin hypothesis. But the same behavior could be explained if the levels of expression of these two genes differ significantly, and that is clearly the case. The TUB l protein is about four-fold more abundant than the TUB3 protein (Schatz et al l 986b). This quantitative difference in levels of expression can explain the differences in phenotypes associated with disruptions of the two genes, since all the phenotypes associated with the disruption of one oHubulin gene can be suppressed by over-expression of the other. That is, disruptions of TUBl are viable when TVB3 is over-expressed, and conversely the relatively minor phenotypes associated with TUB3 disruption are suppressed by over-expression of the TUBl gene. Thus these two proteins, though geneti cally distinguishable, are functionally interchangeable. (The fact that a deficit in a-tubulin is lethal and does not just cause slow growth is addressed by quantitative experiments described below).


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The differences in the sequences of TUB1 and TUB3 are dispersed throughout the genes. Of particular interest, however, is the sequence near codon 35. It is in this region that significant differences among many a tubulins occur, analogous to the carboxy-terminal region of f3-tubulins. But TUB1 and TUB3 are identical in this domain, so perhaps they are functionally interchangeable because in the position where divergence would matter the most, these two genes are identical. By exploiting a unique restriction site in this region, it is possible to introduce linkers that insert as many as 1 7 random amino acids to produce a longer protein (Schatz et al 1 987). If this region is crucial to function, such an insertion would disrupt function. In fact, these linker insertions produce a-tubulin proteins, which support normal microtubule function even in the absence of a wild type a-tubulin. The conclusion from the yeast work that specific functions can not be assigned to specific tubulin gene sequences fits well with results from other lower eukaryotes. For example, there are two a-tubulin genes in S. pombe, and conditional mutations in one of them can be suppressed by over expression of the other (not exactly the same experiment as suppression of a disruption) (Adachi et al 1 986). In Aspergillus nidulans, the two f3tubulin genes, only one of which is expressed during conidiation, are also apparently functionally interchangeable (May et al 1 990). Expression of tubulin genes in novel environments, which at the same time replace the endogenous tubulin genes, is the only way to test the true functional capacity of a particular tubulin sequence. The limitation on this experimental design is that it is readily interpreted only when the replacement makes no difference. Any failure to fully substitute for the endogenous gene can be ascribed to other qualitative problems such as codon usage, or quantitative considerations. As described below, even small disparities in the levels of tubulin expression can confer significant phenotypes. QUANTITATIVE

CONTROL

OF

TUBULIN

EXPRESSION:

HOMEOSTASIS

A�D

A second model to explain both quantitative and qualitative aspects of microtubule assembly derives from the convergence of two distinct lines of evidence: the characteristic dynamic instability of nucleated microtubules at steady state (Mitchison & Kirschner 1 984) and the molecular biology oftubulin messenger RNA (Cleveland 1 988). Briefly, micro tubules in cells, visualized with the aid of microinjected labelled tubulin, undergo a rapid transition from growing to shrinking. This same behavior is characteristic of microtubules in vitro under conditions where assembly relies on nucleation from a distinct nucleating element, such as isolated centro somes. The in vitro experiments also demonstrate that the DYNAMIC INSTABILITY


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average length at which a growing microtubule starts shrinking depends upon the concentration of assembly-competent tubulin in the system. If the dependence of length upon concentration also applies to the in vivo situation, then regulation of intracellular tubulin levels becomes important. In fact, experiments from several groups suggest that the levels of tubulin protein may be tied to the state of microtubule assembly by an extra ordinary mechanism: the stability of tubulin messenger RNA varies with drug-induced changes in the pools of assembled and unassembled tubulin. If this mechanism also is responsive to the more subtle and short-lived fluctuations in tubulin concentration that the cell may normally encounter, then it could provide the means by which cells maintain homeostasis with respect to tubulin concentration and thus maintain microtubule polymers at a specified length (Cleveland 1 988). Several predictions of this model are testable in yeast. Diploid strains, hemizygous for one of the tubulin genes, can be constructed and then assayed for various microtubule-dependent functions: growth rate and cell cycle distribution at normal, high, and low temperatures; formation of intranuclear and cytoplasmic microtubule structures; mating and sporu lation; chromosome segregation; and response to microtubule depoly merizing drugs. Diploid strains that are hemizygous for TUB2, that is those that have half the normal complement of [3-tubulin genes, are indistinguishable from congenic wild-type strains by all these assays, except for a slight super-sensitivity to benomyl (Katz et aI 1 990). The absence of a significant microtubule phenotype could be achieved by the sort of homeostatic mechanism described above, which compensated for the absence of a f3-tubulin gene by up-regulation of the RNA or protein gene products to the steady state levels of wild type. In fact, these hemizygous strains have almost half the normal complement of f3-tubulin mRNA and protein and only half the wild-type levels of a-tubulin protein. These cells, then, can contain no more than half the wild-type level of tubulin heterodimer and probably degrade most of those a-tubulin chains that are not dimerized. There is no evidence for a mechanism to maintain diploid levels of tubulin in these cells and no apparent need for such a mechanism. In contrast, hemizygotes of TUBl, the major a-tubulin gene, are not viable. This outcome could be explained if an excess in a-tubulin protein produced in strains hemizygous for TUB2 was not especially lethal, while excess {3-tubulin was extremely lethal. Alternatively, over-expressed levels of either polypeptide might be lethal, but the a-tubulin might not accumu late to the same extent as the [3-tubulin polypeptide, perhaps because of a difference in the mechanism of their degradation. Part of this argument was tested by experiments in which TUB2 was dramatically over-expressed


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by an inducible promoter (Burke et al 1 989). Shortly after induction of TUB2, the cells lose all microtubules. Later, the cells accumulate f3-tubulin protein in extraordinary cytoplasmic structures that resemble large sheets. The cells arrest with large buds and eventually die. In experiments per formed in the same system, over-expression of the a-tubulin gene produced no stable excess of the protein. These results provide a basis for the leth ality specifically associated with f3-tubulin over-expression-the poison ing of microtubule assembly. Strains hemizygous for TUB2 or containing extra copies of TUBl do produce excess a-tubulin protein (Katz et al 1 990; Weinstein 1 990), however, which leads to a re-examination of the details of high-copy overproduction (Weinstein 1 990). Using different constructs and different strains, accumulation of a-tubulin protein can be achieved to levels greater than those observed for f3-tubulin protein. The excess a-tubulin protein does not cause detectable depolymerization of cellular microtubules. The cellular phenotypes of excess TUB I protein also differ from those associ ated with excess TUB2 protein. There is an eventual cell division arrest and loss of viability, but only at much greater protein levels and longer times. When the two genes are over-expressed in the same cell, the micro-. tubule depolymerization and rapid lethality associated with over-ex pression of f3-tubulin alone are suppressed, so that the phenotypic conse quences are the same as for over-expression of a-tubulin alone. M ore modest over-expression of a- and f3-tubulin together is tolerated by these cells, perhaps because there are degradative mechanisms that keep the steady state level of the heterodimer close to that of wild-type cells (Katz et al 1 990). Taken together, these experiments suggest that f3-tubulin is indeed uniquely lethal, perhaps because it can induce microtubule depoly merization even when present in small excess, while a-tubulin has no such effect even at high levels. This loss of microtubules occurs when the steady state level of TUB2 protein is only 50% greater than normal levels, less than the excess expected in TUBl hemizygotes, which are inviable. The results provide a rationale for a cellular mechanism that lowers the concentration of dimeric tubulin toward the normal levels, as observed in strains containing excess copies of tubulin genes. Eventually accumulation of excess tubulin interferes with normal cell division (Katz et al 1 990); Weinstein 1 990) . Finally, these results may explain why TUB 1 hemizygotes and haplid TUBl nulls are not viable rather than just slow growing, even though they contain another functional a-tubulin gene. The amount of a tubulin protein produced by that gene may not be sufficient to prevent the lethal events induced by excess f3-tubulin. The precise mechanism by which excess f3-tubulin kills cells, or depoly-


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merizes microtubules, is not yet known. However, at early times after induction of the TUB2 gene, and after all the microtubules are depoly merized, but before the conspicuous p-tubulin aggregates start to form, an intermediate stage can be detected: staining of one or two dots, at or near the nucleus, with antibodies against p-tubulin, but not against a tubulin, is seen. The number and position of these stained elements suggest that they may be spindle pole bodies, and this raises the possibility that p tubulin in excess interacts anomalously with these microtubule-nucleating structures (Weinstein 1 990). In summary, studies of quantitative control of tubulin expression in yeast do not support models that require maintenance of narrow steady state levels to produce appropriate organization of assembled micro tubules. Instead, cells can function normally with 50% less than normal levels of tuhulin. Too much total tuhulin protein is deleterious, however, and there is evidence of mechanisms that can degrade such excesses. Finally, since even small amounts of undimerized p-tubulin are lethal, cells require regulatory mechanisms that will maintain levels of a-tubulin protein in excess of p-tubulin. A third model advanced to explain the diversity of microtubule organization and function invokes micro tubule-associated proteins that may interact with the conserved mic rotubule lattice to modulate assembly, interactions with other structures, and motility. Attempts to identify such proteins in yeast and to analyze their function have provided promising beginnings.

MICROTUBULE-ASSOCIATED PROTEINS

Genetic approaches Attempts to identify second-site suppressors of tubu lin mutants, genes that may participate in regulating microtubule function, have been disappointing. Typically, the suppressors of mutations in f3tubulin have been in the a-tubulin genes, and vice versa, an interaction that was not in doubt. But alternative strategies already have identified genes of interest. Screens for conditional mutations that produce abnormal segregation chromosomes, potentially the consequence of a defective mitotic spindle, are available (Koshland et al 1 985). Strains showing gain of chromosomes have mutations in the CDC3l gene, which is involved in spindle pole body duplication, and also in two new genes. One of those genes, called IPLl, is essential for vegetative growth, and its deduced amino acid sequence is homologous to that of protein kinases (C. Chan & D. Botstein, personal communication). Strains showing chromosome instability carry mutations in (predictably) tubulin genes and also in three new complementation groups, called CINl, CIN2 and CIN4 (Hoyt et al 1 990). An independent screen for mutations that confer supersensitivity to benomyl identifies the


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same three genes (Stearns et al 1 990). The mutations analyzed in each of these groups are cold-sensitive for growth and tend to arrest with a pheno type similar to that of conditional mutants in tubulins: a large bud and a single nucleus still in the mother cell. Significantly, the arrested cells also have depleted microtubule arrays. The patterns of genetic interactions between CIN genes and tubulin genes are suggestive of specific and direct interactions between the gene products. Some combinations of conditional mutations in CIN and tubulin are lethal, and some mutations in CIN genes suppress mutations in tubulin genes. It is also likely that the three CIN gene products interact with one another. The phenotype of the triple mutant, cinl cin2cin4, is no more severe than any of the three single mutants, consistent with the notion that the gene products participate in one structure. Among the multiple mutations in TUBI described above, several display either no microtubules or excess microtubules at the non-permissive tem perature (Schatz et aI 1 988). The trivial explanations for these phenotypes, too little or too much tubulin, respectively, can be tested directly by measurement of the tubulin complement in these cells. Almost all the mutants that show these quantitative phenotypes in their assembled micro tubule organelles have normal levels of tubulin (L. Connell & F. Solomon, unpublished), thus suggesting a qualitative defect in the regu lation of assembly and disassembly in these strains. Defects in IX-tubulin, which produce i nappropriate levels of microtubule assembly, could be corrected by quantitative or qualitative changes in proteins that regulate microtubule assembly. The in vitro assembly reaction provides precedents for proteins that promote microtubule assembly, but the above assay would not detect proteins that down-regulate microtubule assembly. Yet excess microtubules are associated with some mutant alleles of TUBI and TUB2 and of over-expression of YPTl, while mutants in KARl, KAR3, and CDC3] produce at least abnormally long arrays. These results suggest that maintaining an upper limit on microtubule assembly may be a real problem for yeast and that there is a regulatory mechanism to deal with it. A screen for wild-type genes, which, when expressed in moderate excess, can suppress TUBl alleles displaying too many micro tubules, has identified three such genes, all different from each other and from the tubulins (D. Kirkpatrick & F. Solomon, unpublished). Over-expression of these genes does not rescue mutants that produce too few micro tubules. Such analyses can identify sub-stoichiometric regulators of microtubule assembly. The TUB2 and ACT1 genes are separated by about 1 kb of chromosomal sequence and are divergently transcribed. Between them is YPTl , a homo logue of human c-ras (Schmitt et al 1 986; Segev & Botstein 1 987). When that gene is placed behind a regulatable promoter and shut off, cells die.

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Cell death is accompanied by abnormal enlargement of mother cell bodies, significant lengthening of the cytoplasmic microtubules, and disappearance of the nuclear microtubules. The relationship between the phenotype and chromosomal locus of this gene remains unknown. Biochemical approaches Attempts to isolate significant quantities of yeast microtubule-associated proteins have been frustrated because there is no robust in vitro assembly protocol for yeast microtubules, the approach that has been so productive for animal cells. Another assay, using selective extraction to isolate cellular microtubule structures in their assembled form and then to analyze their components, has been successfully applied to several animal cell types from which in vitro assembly has not been possible. An adaptation of that approach identifies possible microtubule associated proteins from yeast (Pillus & Solomon 1 986a), but in quantities too small to permit molecular analysis. Extensions of that work will depend upon identification of genetically interacting elements that encode those particular proteins. Affinity columns made from stabilized microtubules, using an approach originally worked out for animal cells, select about 30 proteins that bind with specificity. Antibodies have already been raised against two of those proteins, and they decorate yeast microtubules by immunofluorescence (G. Barnes, personal communication). These findings can lead to direct tests of the function of these proteins in vivo. Moreover, the ability to reconstitute spindle activity in preparations from S. pombe (Masuda et al 1 990) raises the possibility of developing functional assays for these elements as well. Spindle Pole Bodies

Microtubule-organizing centers (MTOCs) may influence microtubule organization by providing a nucleating cite, localized to a particular posi tion, and also by suppressing spontaneous microtubule assembly elsewhere in the cell, thus constraining the geometry of the microtubule organelle. There is considerable evidence that SPBs are the MTOCs of yeast. For example, the SPBs are in the appropriate position to act as microtubule organizing centers in yeast; both by light and electron microscopy, they appear to be at the base of nuclear and cytoplasmic arrays. Also, prep arations enriched in SPBs are competent to nucleate microtubule assembly in vitro (Rout 1 990). Genes have been identified that affect the behavior of spindle pole bodies. A screen for haploids, which carry a diploid complement of DNA, identified a temperature-sensitive strain that remained a uninucleate at the restrictive temperature, but that accumulated a large number of spindle pole bodies (Baum 1 988). Both the spindle pole duplication and the ts-


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lethality are associated with a mutant allele of the ESP] gene. A tem perature shift experiment demonstrates that the lethality associated with this mutation can precede the expression of the morphological phenotype. Indeed, analysis of the data suggests that a new SPB is produced every 2.8 h, not much different than the cell cycle time, in linear fashion, rather than geometric duplication. A gene that appears to govern SPB duplication in S. pombe, called cutl +, has been identified and analyzed at the molecular level (Uzawa 1 990) . A comparison of the two genes suggests that they are homologous. Fractionation of S. pombe cells shows that the cutl + protein is stably associated with nuclei, so these gene products are likely associated with, and perhaps even integral components of, SPBs. Also, a yeast gene that encodes a homologue of calmodulin, CDC3] gene, is essential for spindle pole duplication (Baum et al 1 986), but its role in that process is not yet clear. To date, there is no demonstration that the SPBs of S. cerevisiae contain what has otherwise become a ubiquitous component of microtubule organizing centers, y-tubulin. The mipA gene in Aspergillus was identified originally as a suppressor of a f3-tubulin mutation (WeiI 1 986). Its predicted amino acid sequence is about 30% identical to both iY.- and fJ-tubulins, or about as close as those two proteins typically are to one another (Weil 1 986). The y-tubulin is essential for nuclear division in Aspergillus, and at least a significant fraction of the protein localizes to the position of SPBs (Oakely 1 990). Homologous genes have been identified in many other organisms, among them D. mela n ogaster and some mammals (B. Oakley, personal communication), but the presumably inevitable identification of y-tubulin in S. cerevisiae has not yet occurred. A molecular approach to identifying spindle pole components has relied upon human auto-antibodies, which recognize centrosomal structures in animal cells. One such antibody identifies a gene, SPA], that specifies a 59-kd protein in S. cerevisiae (Snyder & Davis 1 988). That protein increases in level in espl-l cells, which have multiple spindle poles. The protein appears to localize to spindle poles in cells by immunofluorescence. Inser tions into the SPA I gene are not lethal, but they do produce slower growth, excess lethality, and chromosome and nuclear division defects. SPBs have been prepared from yeast nuclei and partially purified by monitoring the ability of the fractions to nucleate microtubule assembly (Rout & Kilmartin 1 990). Monoclonal antibodies against those prepara tions already have identified three distinct protein components. Two of them localize to different domains of the SPB (seen by immuno-electron microscopy). The third appears to associate with spindle micro tubules, fragments of which appear to co-fractionate with the nucleating organelles. It is worth noting that there are almost certainly cytoskeletal proteins


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at the other end of micro tubules that regulate their function. If cells are forced to carry nutritional markers on CEN plasmids, which segregate like small chromosomes in a fashion dependent upon the spindle, signs that the system for segregation is saturable become apparent (Futcher & Carbon 1 986). Cells with many plasmids grow more slowly and lose plasmids more frequently than do cells with fewer plasmids. These experi ments raise the possibility that some element is limiting for proper spindle function: CEN-microtubule binding proteins, the number of microtubules, or space on the spindle pole body itself. ACTIN AND F-ACTIN

A Single Actin Gene

The single actin gene in S. cerevisiae, ACT-l , shows considerable sequence homology to actins in animal cells (Gallwitz & Sures 1 980). Functional homology between the ACTI gene product and actins of higher eukaryotes first was established by biochemical criteria. The serendipitous ability of monomeric vertebrate actins to inhibit DNase 1 by high affinity binding was used as an assay to monitor the publication of actin from yeast extracts (Greer & Schekman 1 982). The purified yeast protein and rabbit actin are equally good inhibitors of this enzyme's activity. Like animal actins, yeast G-actin can assemble into F-actin in vitro, and it can interact with muscle myosin to stimulate ATPase activity. F-Actin Structures in Yeast

Yeast cells contain polymerized F-actin in structures that can be visualized by fluorescence microscopy using both anti-actin antibodies and a deriva tive of phalloidin, which recognizes only F-actin (Adams & Pringle 1 984; Kilmartin & Adams 1 984). In fact, the specific binding of phalloidin to the in vivo structures provides further evidence for structural homology between yeast and animal F-actins. The staining appears in two forms, patches and cables. Patches of F-actin do not occur in many cell types, although similar phalloidin staining is detected in embryonic avian red blood cells, another cell type that contains F-actin and that exists in suspension (Kim et al 1 987). By light microscopy, the cables resemble the microfilaments or stress fibers present in the cytoplasm of higher eukaryotes. Are the cables composed of more than one F-actin filament? What is the polarity of the filaments? How is the F -actin organized within the patches? Analysis at higher resolution will help to answer these ques tions and to better understand the functions of the actin cables. Correlations between F-actin localization and shape change are sugges tive of function. In budding cells, many of the cables align with the long


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axis of the mother cell. The patches are concentrated in small and medium sized buds and at the sites of bud formation. The asymmetric distribution of F-actin elements and the differential association of the forms of F-actin with different domains of the actively polarizing cell raise the possibility that actin plays a role in the morphogenetic events associated with budding. However, cells apparently lacking cables do grow (the arguments for participation of F-actin in changes in yeast cell shape are more subtle; see below). In addition, those F-actin structures most closely correlated with motility in animal cells, the contractile ring of mitotic cells, or the ruffling leading edge of cells migrating across a substratum, are absent in yeast. Probing F-actin func tion in yeast relies on genetics; the cytochalasins, which disrupt F-actin structures in animal cells, have no effect in S. cerevisiae. Cells bearing disruptions of ACTl are not viable, so functional analysis depends upon analysis of conditional alleles. These mutants were produced by muta genesis because there was no clear sense of what would constitute a sel ectable phenotype likely to represent a defect in the actin gene (Shortie et al 1 984). The substrate for mutagenesis, a fragment of ACTl that lacked 5'-sequences essential for expression, was chosen so that its reintroduction into the genome by integration at the wild-type locus permitted expression of the mutated gene and simultaneous disruption of the wild-type gene. As a result, recessive phenotypes are manifest immediately. The original screen identified two different mutations, actl-l and actl-2, both tem perature-sensitive (ts-) for growth and both resulting from changes at proline 32. The defects in ACTl produce a wide range of phenotypes (Novick & Botstein 1 985). First, about 90% of the cells are dead after two generations at the non-permissive temperature. Attempts to identify a specific point in the mitotic cycle when cells are sensitive to temperature have failed, which suggests that actin functions may be required at more than one stage. Second, the F-actin structures, as visualized by immunofluorescence microscopy, are significantly disrupted. Third, the mutant cells appear to lyse upon death, and the mutants are hypersensitive to high osmolarity media. Fourth, secretion becomes abnormal. After I h at the non-per missive temperature, the actl-l mutants accumulate vesicles, as do other mutants known to block Golgi function. With the same time course, the intracellular pool of the secreted form of invertase increases. Fifth, mutations in ACTl disrupt a manifestation of cellular polarity. Normally growing cells deposit the cell wall component chitin in discrete structures relative to the site of bud formation. In the mutants, the chitin is found delocalized all over the surface of the cell. CONSEQUENCES OF DEFECTS IN F-ACTIN STRUCTURES


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Determining which of these phenotypes is a direct consequence of F actin disorganization rather than an indirect consequence (because the cells are dying) is problematic. For example, changes in the pattern of chitin deposition similar to those presented by the ACT1 mutants are found in strains bearing mutations in cell division cycle genes growing under restrictive conditions (Roberts 1 983). Even aspects of the F-actin pattern itself are abnormal in cells made sick by treatment with inhibitors of protein synthesis and ATP synthesis (Novick et al 1 989). But recent analyses of several new mutants in ACT1 suggest that the inability to grow in a normal polarized fashion correlates with the severity of the F-actin phenotype of the cells rather than with the severity of the conditional growth phenotype (D. Drubin, personal communication). Actin-Associated Proteins

The study of actin-associated proteins from many cell types suggests that these proteins may be responsible for mediating the extent and form of F actin assembly, the interactions between F-actin and other cellular struc tures, and the motile and structural functions of F-actin polymers. Actin associated proteins in yeast are now accessible because of the success of three experimental approaches: purification of yeast homologue of the animal cell proteins, direct identification of yeast actin-associated proteins based on their affinity for yeast actin, and identification of genes and gene products that suppress actin mutants in vivo. Summarized below are analyses of three yeast homologues of well-known animal cell actin-associated proteins. It is clear that genetic manipulations of each affect actin function, but the function of each protein in yeast is not yet clear.

YEAST ANALOGUES OF ANIMAL ACTIN-ASSOCIATED PROTEINS

Tropomyosin Originally identified as a component of muscle, and then by immunofluorescence as a component of microfilaments in non-muscle cells, tropomyosin is believed to participate in regulating the interaction of actin and myosin. The purification of yeast tropomyosin was based on the observed Mg2+ -dependent binding of animal tropomyosin to F-actin (Liu & Bretscher 1 989b). The physical properties of the yeast and animal proteins are similar, and antibodies against the yeast protein also bind to bovine brain tropomyosin. Those antibodies localize tropomyosin to the yeast actin cables (Liu & Bretscher 1 989a). The gene for tropomyosin, TPM1, is not essential, but its disruption produces loss of the F-actin cables (Liu & Bretscher 1 989a). In some of those cells devoid of tropo myosin, actin is present in thick bars. (It is worth noting that this structure recurs as a consequence of defects in other actin-associated proteins

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described below, and may represent a sort of default status for F-actin.) Conversely, over-expression of the TPMl gene in actl-2 cells at their restrictive temperature restores cables similar to those detected in normal cells, but does not suppress the lethality. These results suggest that the TPMl protein is necessary for formation of actin cables, but that the F actin cables are neither essential nor sufficient for vegetative growth.

Profilin Profilin can interact with G-actin, and could participate in mech anisms for regulating assembly by sequestering the monomeric form. The yeast analogue of this protein, similar to animal profilins on the basis of in vitro assays, was purified, and clones were obtained through mic rosequencing (Haarer et al 1 990). The gene is not essential, but strains bearing a disruption grow poorly; the cells have no F-actin cables, although the patches are present and occasionally a thick bar of actin is found. These cells also have lost their normal asymmetry; they are abnormally round, and the deposition of chitin is delocalized. The cells are frequently multinucleate and have abnormal microtubule arrays; both of these pheno types are almost certainly consequences of the budding defect. Simple interpretations of the primary mutant phenotypes are not available, although there are ways to rationalize an interaction of profilin and G actin with loss of cables in the null strains. But such an interaction needs to be verified in yeast, since at least moderate overproduction of profilin in yeast has no phenotype (B. Haarer & S. Brown, personal communication). Actin-capping protein The animal cell actin-capping protein interacts in vitro with the barbed end of F-actin filaments (Casella et al 1 986). Micro injection of the animal protein into animal cells causes loss of stress fibers of F-actin, consistent with a severing activity of this protein, to form small polymers out of larger ones (Fuchtbauer et al 1 983). The yeast homologue to chicken actin-capping protein is 49% identical (Amatruda et al 1 990). But the phenotypes of strains disrupted in the CAP2 gene, which encode it, are complicated. The gene is not essential for viability, although it is essential for formation of the F-actin cables. The cells show slightly longer doubling times, a shift to larger and more irregular sizes, and chitin deposition becomes delocalized. The yeast version of a micro injection experiment, induction of over-expression, may provide inter esting results. ISOLATION

OF YEAST ACTIN-ASSOCIATEU

PROTEINS BY

AFFINITY

CHRO

To cast a broader net for actin-associated proteins, yeast extracts have been fractionated by affinity-chromatography on phalloidin stabilized F-actin. The association of the candidate-associated proteins with actin structures in vivo was assayed by immunofluorescence (Drubin MATOGRAPHY


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e t aI 1 988). ATP releases a myosin-like protein from these actin columns, while salt elutions identify specifically bound polypeptides of 67 and 85 kd. The analyses of these proteins have produced interesting results and support the validity of the approach. The 67 kd protein, called actin-binding protein 1 , localizes to both cables and patches (Drubin et at 1 990). Sequence analysis demonstrates that the C-terminal domain has considerable homology to the SH3 domains of several animal cell proteins like ezrin and spectrin, which are thought to be involved in associations with the cortical cytoskeleton. There is also genetic evidence that actin-binding protein 1 interacts with actin in the cell: a mutant allele of the non-essential gene that encodes it, SA C6, was identified as a suppressor of the act}-} mutation (Adams et al 1 989). The convergence of these two approaches significantly supports the assumptions underlying each of them. Straightforward tests of the in vivo functions and interactions of domains of this protein are readily available. The 85-kd protein localizes to patches only (Drubin et aI 1 988). Although cells bearing a disruption for this gene are viable, over-expression on a high copy plasmid confers poor growth, especially at high temperatures. The mother cells continue to grow, but the normal patterns of growth are altered. The actin patches normally restricted to the buds are found in mother cells, and the regulated pattern of budding typical of normal cells degenerates (see below). All these results are consistent with the observation that actin may participate in the establishment or expression of polarity. The first systematic suppressor analysis of the temperature-sensitive lethality associated with actl-} identified at least 35 independent loci that restored viability to the m Ulanls at the non-permissive temperature and, when crossed into a wild-type A CT} background, themselves conferred a cs- (cold-sensitive lethality) pheno type (Novick et al 1 989). All of these suppressors of actin (SA C genes) were recessive, but curiously null alleles of the SA C genes tested to date do not suppress. Thus, in these cases it is not loss of function that sup presses the act}-} defect, but perhaps a missense gene product. Yet an independent search for dominant suppressors (Adams et al 1 989) did not reveal any of these complementation groups. At least some of the sac alleles, grown at their non-permissive tem perature in a wild-type strain, produce disruption of the normal actin pattern, formation of heavy bars of actin, and delocalized deposition of chitin. These phenotypes resemble those produced by conditional actin mutations, but unlike A CT], none of the SA C genes is essential . Indeed , GENES THAT INTERACT WITH A CT!


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sorting out which phenotypes are aspects of essential functions, rather than consequences of their loss, is not trivial. Detailed genetic analyses of the SAC genes may demonstrate specific patterns that can help to deter mine which of the phenotypes uncovered by ACT-i mutations involve essential functions. For example, suppression by sac genes is allele-specific for acti mutants, while other combinations are lethal. Genetics interactions have helped to reveal evidence of a complicated relationship between actin and secretion. Among the suppressors of the ts- phenotype of SEC14, a gene that affects post-Golgi secretory functions, is an allele of a gene called RSDi, which turns out to be identical to SACi (Cleves et al 1 989). M utations in RSDi/SACi show delocalized chitin deposition and disorganization of the actin cytoskeleton, e.g. dis appearance of the actin cables and abnormal localization of the actin patches to the mother. But RSDi/SACi mutants also interact with other secretory genes (sec 13 and sec 20) to produce lethal double mutants, even though those two genes are likely involved in an earlier secretory step, transport between the ER and Golgi. The fact that mutants in RSD 1 /SACi affect different stages in the secretory pathway, as well as actin functions, complicates attempts to define a direct interaction b e tween secretion and actin. A property of these suppressors suggests the existence of another inter acting element of as yet unknown function (Adams & Botstein 1 989). The sac2 mutation can interact with actl-i to confer conditional phenotypes, depending upon the genetic background of the strain. The independent modifying gene suggested by these results has been mapped and is called MOXi . The recessive allele moxi-i interferes with sac2 suppression of the acti-i phenotype, but the nature of the MOX I gene product, and whether its effect on actin is direct or part of a pleiotropic function, is not known. In principle, defects in actin that result in improper assembly of F-actin structures could be suppressed by a mutation in a gene specifying other components of the structure. In some models, such mutations would be dominant; the availability of the mutant gene product could be sufficient to restore a wild-type phenotype. Such a dominant suppressor, sac6, was identified from analyses of the spontaneous revertants of the ts - phenotype of actl-i diploids to Ts+ (Adams & Botstein 1 989). Sac6 shows an especially intriguing pattern of interactions. It suppresses only some ACTi alleles, whereas with others it acts synergistically to exacerbate the mutant phenotype. These genetic properties are consistent with the postulated physical interactions upon which this search for suppressors was based. Satisfyingly, the SAC6 gene product is the 67-kd protein identified by affinity elution chromatography, described above (Adams et al 1 989).

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TWO MOTILE BEHAVIORS IN YEAST


Changing Shape

In animal cells, drug interference experiments and · morphological cor relations have established the role of cytoskeletal elements in driving morphological changes. Both intracellular and extracellular determinants can specify the precise shape of the cell. The precise nature of the intra cellular determinants and their interactions with cytoskeletal elements are not known. The issues addressed in analysis of yeast morphogenesis, identifying the cytoskeletal driving force, defining how that motor is coupled to the cell surface, and understanding how sites of shape change are specified, have striking parallels in the analogous issues in animal cells , such as expression of neuronal morphology via growth cones. The advantage in yeast is that genes involved in specifying intracellular deter minants of assymmetry can be identified and their interactions studied genetically. The results may help to illuminate how animal cells change shape as well. As noted above, yeast cells undergo conspicuous shape changes, both during budding and during shmoo formation, the formation of elongated cells in response to mating pheromone. The asymmetric distribution of F actin in budding cells was described above (Hasek et aI 1 987) . Cytoplasmic microtubules in budding cells typically are in a bundle, extending from the nucleus toward the bud site. In cells bearing mutations in CDC4, which form multiple buds at the non-permissive temperature, there are micro tubule bundles extending into each of the buds (Adams & Pringle 1 984). Do either of these morphological correlations reflect a functional role for the cytoskeleton in bud formation? Disruption of microtubules, either by drugs or mutations, causes apparent disappearance of the cytoplasmic bundles of micro tubules, but does not block bud formation, or any of the shape changes associated with mating (Hasek et al 1 987). The inferences from these findings need to be qualified on two grounds. First, the immuno fluorescence assay used here for the state of microtubule assembly is not quantitative and could fail to identify residual microtubules. Second, yeast cells can break through nocodazole blocks to reform microtubules (Jacobs & Szaniszlo 1 982; Pillus & Solomon 1 986a). Therefore, treatment with depolymerizing drugs does not necessarily mean that microtubule structure and function have been lost completely. Analogous experiments to test the role of microfilament function are more difficult to perform. As noted above, there are no drugs that induce microfilament disruption analogous to the effect of benomyl. In ts- actin mutants shifted to the non-permissive temperature, the actin structures rapidly become disorganized. When these cells are treated with mating


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factor, they fail to grow the typical long extensions characteristic of shmoos (D. Drubin, personal communication). They do become pointed, however, which suggests either that actin is not necessary for the initiation of asym metric shmoo formation, or that even at the non-permissive temperature the mutants retain sufficient actin function to carry out this shape change. Studying the effects of microtubule disruption on these cells may suggest assignment of the residual activity for initiating shmoos to microtubules, or to some other cytoplasmic element. Other gene functions affect the initiation of asymmetry in budding cells (Adams et al 1 990; Sloat et al 1 98 1 ). In some conditional mutants in CDC24, no bud forms, the mother cell gets larger, and chitin deposition is delocalized (Sloat et al 1 98 1 ). Those mutants also show abnormal local ization of actin (Adams & Pringle 1 984). Genes that suppress these defects by multicopy over-expression include RSR1, a gene that by sequence is a member of the ras family. Cells bearing deletions of RSRl are viable, but select their bud sites randomly (Bender & Pringle 1 989). The detailed interactions among these genes remain to be resolved, but the complexity already evident suggests that budding, like other morphogenetic processes, relies upon multiple gene products, including some whose functions are pleiotropic and overlapping. Another gene whose function is necessary for expression of polarity is SPA 2. Although originally identified in yeast by application of human anti-centrosomal antibodies, the SPA 2 gene product localizes to the tip of the elongating shmoo in wild-type cells exposed to mating pheromone (Gehrung & Snyder 1 990). Deletion of the SPA 2 gene does not confer a significant defect in vegetative growth, but depresses the efficiency of mating in self-crosses by about 1 00-fold. The defect is not in karyogamy, but rather in shape change. Most of the mutant cel\s when exposed to mating pheromone are round rather than pointed, and their actin patches are distributed randomly all over the cell . In the 20% of the population that becomes oval or pointed, the actin distribution is polarized. The deduced amino acid sequence of the SPA2 protein may specify a coiled coil structure, common to proteins that can form filamentous structures. Yeast cells specify the position of the emerging bud in patterns that are determined by their genomes. In haploid cells (a or a), the new bud emerges in a position axial to the preceding bud, while in diploid cel\s the successive bud positions are bipolar. These patterns are exquisitely sensitive to the expression of mating type information, so that even rapid transitions in that expression are reflected by changes in budding geometry (Rine et al 1 979). Some genes are known to be necessary for maintaining regular patterns, for example,

INTRINSIC DETERMINANTS OF YEAST CELL MORPHOLOGY


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some alleles of cdc24 growing at the permissive temperature show ran domization of bud sites (Sloat et al 1 98 1 ). An ambitious screen for haploid cells that do not give an axial pattern has identified four B UD genes (Chant & Herskowitz 1 99 1) . Mutations in B UDl (identical to RSRl , described above as interacting with CDC24) and B UD2 give random budding pat terns in both haploid and diploid cells, while mutations in B UD3 or B UD4 confer a bipolar pattern on haploid cells, but do not affect the normally bipolar pattern of diploid cells. These phenotypes are consistent with a hierarchy of pattern formation: random, requiring none of the BUD gene products; bipolar, requiring expression of B UD ] and B UD2 ; and axial, which requires the expression of B UD3 and B UD4 as well. Mutations in a fifth locus, B UD5, give random budding patterns in all cell types (Chant et aI 1991). The question remains, how does a cell of a particular genotype localize the machinery required for localized cell growth to the correct position? A screen for mutant strains, which fail to mate because of a failure in localized cell growth toward their mating partner, has identified alleles of the BEMl gene (J. Chenevert et aI, personal communication). Deletion of the gene produces misshapen cells that fail to bud properly. Strains carry ing beml-s alleles are normal for vegetative growth but, in response to mating factor, just expand rather than form an elongated shmoo. The sequence of BEM-l demonstrates the presence of two SH3 domains, the occurrence of which in animal cell proteins correlates with putative associ ations with the cortical actin cytoskeleton. These data suggest a link between a gene product, which may be responsible for initiating polarized growth, and the actin cytoskeletal structures, which may be responsible for driving that growth. At least one of the gene products associated with the I O-nm filaments in the neck, CDC3, may arrive at the bud site before cytokinesis begins (Kim et al 1 99 1 ) and thereby help to define such sites as special. Moving Organelles

Yeast cells are not particularly suited for live cell microscopy because they are small and fairly spherical. But intracellular motility, the movement of chromosomes, has been followed directly (Palmer et al 1 989) in wild-type cells and in cells carrying mutations likely to affect normal mitotic processes. This advance should enable a more detailed analysis of phenotypes to ac company molecular studies. The role of the cytoskeleton in the movement of nuclei themselves can be inferred from the behavior of vegetative cells, described above as part of the discussion of TUB2 mutant pheno types. It is the genetic analysis of nuclear movements during karyogamy, however, that has proven most fruitful to date. The characteristics of


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karyogamy, the fusion of haploid nuclei to make a diploid nucleus, make it a particularly valuable process for analysis of cytoskeletal organization. Briefly, karyogamybegins with the fusion of the two cells and consequent mixing of their cytoplasm. The subsequent fusion of the two nuclei is a microtubule-dependent event, as assessed by the effects of microtubule depolymerizing drugs (Delgado & Conde 1984) and of mutations in TUB2 (Huffaker et al 1988; Thomas1984). The diploid nucleus emerges from the zygote in a bud containing cytoplasm from both parents. Failures in nuclear fusion can be identified by differentially markingthe cytoplasm of the parents, i.e. with a mitochondrial phenotype, and screening for cells that do not express an appropriate combination of parental nuclear and cytoplasmic traits (Conde & Fink 1976; Polaina & Conde 1982). One the great advantages of looking for mutations affecting mating is that there are convenient ways to select for defects, and the processes involved are likely to be interesting ones, but the matingfunction itself is not essential for cell viability. Analysis of KARgenes has provided valuable insight into mechanismsof intracellular motility in yeast and in other organisms. OF GENES AFFECTING KARYOGAMY The mutant karl-1 allele was originally identified by the assay described above (Conde & Fink 1976). Only one of the parents needs to bear the mutation in order to have a significant effect on the efficiency of mating. This unilateral behavior suggests that the KARlgene product is required by both nuclei, and that either the function of the gene product is required before cell fusion, or that the gene product is so firmly associated with one parental nucleus that cytoplasmic mixing does not enable functional complementationof the defect in the other nucleus (Dutcher & Hartwell 1983). In fact, a low level of nuclear fusion persists in the presenceof the karl-1 allele, but the defect can be enhanced by mutations in other genes--KEM1, 2, and 3 (Kim Fink 1990). The properties of mutations in the KEMgenessuggest that the gene products are diffusible and that they are involved in other microtubule functions. The K.4R1 gene has been cloned and conditional mutations introduced into it by in vitro mutagenesis (Rose & Fink 1987). The phenotypes those mutations reflect defects in microtubule function related both to vegetative growth and normal karyogamy. At the permissive temperature, the mutant strains display abnormal chromosomesegregation. At the nonpermissive temperature, the cells arrest fairly synchronouslywith a single large bud containing an unduplicated SPB. Further, EManalysis showed that the spindle plaques are abnormally convex and lie on deep invaginations of the nuclear envelope (Rose & Fink 1987). Uponarrest at nonpermissive temperature, the cytoplasmic microtubules are much longer ANALYSES


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than normal, often wrapping around the interior of the cell and, in some cases, appear to be independent of an association with the spindle pole. Haploids bearing karl mutations and treated with mating pheromone also produce an abnormally long microtubule array. The complex phenotypes associated with KARl defects may be the result of action of the gene product directly at the SPB. A lacZ fusion with KARl, expressed in yeast and localized with antibodies to fJ-galactosidase, is associated with SPBs (E. Vallen, personal communication). If the KARl gene product is involved in assembly of a normal SPB, the long microtubule arrays evident in the mutants can be explained as a consequence of defec tive nucleation. If cells do control the steady state level of microtubule assembly, then SPBs capable of nucleating fewer than normal microtubules might give rise to longer than normal microtubules. These three aspects of the KARl gene product-its localization to SPBs, its effects on karyogamy and on mitotic growth-can be assayed in a variety of mutations made by inserting and deleting sequences. Those experiments identify domains involved in mitotic and karyogamy functions, as well as domains involved in localizing the fusion protein to either nuclear membrane, or to the site of the spindle pole body itself. Perhaps the most striking result of these experiments is that they dem onstrate that the karyogamy function and mitotic function of the protein depend upon separate domains of the protein (E. Vallen et aI, submitted). The original mutant allele of the KAR3 gene, kar3-l (Conde & Fink 1976), gives a unilateral defect in karyogamy. The frequency of failed nuclear fusion events increases 30-fold in crosses of mutant cells with wild type cells. However, the mutation has no apparent effect on vegetative growth. The KAR3 gene has been cloned (Meluh & Rose 1990); analysis of its sequence suggests that it is a member of the kinesin family of microtubule-interacting motors. One domain of the protein is homologous to microtubule-binding domains identified in other proteins. Another domain has a potential ATP-binding site, consistent with a motor. These two domains, as in other kinesins, are joined by a stretch of coiled coil. That the KAR3 protein might be a microtubule motor does not fit easily with the unilateral phenotype of the kar3-l mutant. After all, one would not expect a motor to be so firmly attached to one ncleus that it could not diffuse in mixed cytoplasm. Nor should the action of a motor be restricted to the period before the nuclei fuse. Indeed, disruptions of KAR3 show no effect on karyogamy when crossed with wild-type cells; that is, the null mutant is not unilateral. These results can best be explained if the kar3-l mutant protein is defective in such a way that it poisons the operation of the wild-type gene product. That explanation is supported by mapping of the kar3-l defect to the ATP-binding domain. A protein that could


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bind normally to microtubules, but not serve as a motor, might compete effectively with the functional motor. Mutants do produce some effect on growth rate, which gives rise to a high proportion of non-viable cells. KAR3-lacZ fusions localize to microtubules and probably produce more stable microtubules. It is interesting to note that kar3-} gives longer microtubules too, again suggesting a need for down-regulation of micro tubule length. A third gene that causes a defect in karyogamy, BIK}, has now been characterized as a microtubule-associated protein in the more conventional sense (Berlin et al 1990). At least part of the BIK 1 protein localizes with spindles, although there is a fair amount of staining elsewhere. Disruptions of the BIK} gene produces abnormally short microtubu\es, or even cells that have no microtubules. Moreover, the N-terminal domain shows sig nificant homology to the microtubule-binding domain of the tau protein. All of these are properties that one might expect of a microtubule-associ ated protein, which interacts with microtubules to stabilize them. Con sistent with that conclusion, karyogamy is normal if either nucleus bears the wild-type BIK} gene.

CONCLUSIONS AND PROSPECTS

This highly selective progress report of analyses of the yeast cytoskeleton should have made clear the strengths of this system. First, testing hypothe sis in vivo can be accomplished with relative technical ease, since manipu lations of genes, ablating or enhancing expression, altering sequence, replacing whole genes, go quickly in yeast. Second, certain motile behaviors, wisely chosen, succumb to genetic and moleclar dissection rap idly, so work on issues like nuclear fusion and morphogenesis already has led to a better understanding of these complex processes. Third, the development of biochemical and cytological techniques for yeast has suc ceeded in making the results of the genetic analyses more concrete and more readily comparable to results in other systems. It is not clear that the conclusions regarding the cytoskeleton from yeast will extend to higher eukaryotes with the same fidelity as have the analyses of transcription or cell cycle. As frequently noted above, provocative homologies continue to arise, ranging from sequences of cytoskeletal pro teins to the properties of motile behaviors. But matters may be different in metazoa. Diversity among tubulin isoforms or specific actin-associated proteins may have more nearly essential functions. F-actin and micro tubules may participate differently in driving shape change or moving nuclei. The experiments required to resolve these issues in animal cells face

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significant technical obstacles, but they can only be facilitated by more detailed knowledge of the yeast system.


ACKNOWLEDGMENTS

I thank D. Botstein (Stanford), L. Hartwell (Washington), L. Pillus (U. C. Betkeley), and M. Rose (Princeton) for valuable conversations; A. Adams (Arizona), G. Barnes (Stanford), A. Bender, K. Corrado, and J. Pringle (Michigan), S. Brown and B. Haarer (Michigan), C. Chan (Texas), J. Chant, J. Chenervert, and I. Herskowitz (U.C.S.F.), D. Drubin (U.C. Berkeley), B. Oakely (Ohio), and M. Rose for sharing unpublished work; and several members of our laboratory (L. Connell, M. Falconer, S. Guenette, D. Kirkpatrick, M. Magendantz, V. Praitis, and B. Weinstein) for their comments. Work on yeast in our laboratory is supported by a grant from the National Institute of General Medical Sciences. Literature Cited

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Functional analyses of the C-terminal half of the Saccharomyces cerevisiae Rad52 protein.

Biogenesis of the vacuole in Saccharomyces cerevisiae.

DNA of Saccharomyces cerevisiae.

Allantoate transport in Saccharomyces cerevisiae.

GABA transport in Saccharomyces cerevisiae.

Multivariate analyses of cellular carbohydrates and fatty acids of Candida albicans, Torulopsis glabrata, and Saccharomyces cerevisiae.

The flavoproteome of the yeast Saccharomyces cerevisiae.

Ribosome biogenesis in the yeast Saccharomyces cerevisiae.

The pheromone signal pathway in Saccharomyces cerevisiae.

The mitochondrial unselective channel in Saccharomyces cerevisiae.

Microtubule proteins in the yeast, Saccharomyces cerevisiae.

Peptidase activities in Saccharomyces cerevisiae.

Peroxisome biogenesis in Saccharomyces cerevisiae.

Oxalurate transport in Saccharomyces cerevisiae.

Allantoin transport in Saccharomyces cerevisiae.

The ribosomal proteins of Saccharomyces cerevisiae.

Proteomic analyses reveal that Sky1 modulates apoptosis and mitophagy in Saccharomyces cerevisiae cells exposed to cisplatin.

Iron-reductases in the yeast Saccharomyces cerevisiae.

Chromosome Duplication in Saccharomyces cerevisiae.

Urea transport in Saccharomyces cerevisiae.

Glucose repression in the yeast Saccharomyces cerevisiae.

Glycolysis mutants in Saccharomyces cerevisiae.

Saccharomyces cerevisiae growth media.

Dichlorofluoromethane inactivates Saccharomyces cerevisiae.