the spindle pole body of yeast

Chromosoma (1994) 103:369-380 C H R O M O S O M A �9 Springer-Verlag 1994

Chromosoma Focus

The spindle pole body of yeast Michael Snyder

Department of Biology, P.O. Box 208103, Yale University, New Haven, CT 06520-8103, USA

Received: 18 February 1994 / Accepted: 18 March 1994

Abstract. Microtubule organizing centers play an essential cellular role in nucleating microtubule assembly and establishing the microtubule array. The microtubule organizing center of yeast, the spindle pole body (SPB), shares many functions and properties with those other organisms. In recent years considerable new information has been generated concerning components associated with the SPB, and the mechanism by which it duplicates. This article reviews our current view of the cytology and molecular composition of the SPB of the budding yeast, Sacchatvmyces cerevisiae, and the fission yeast, Schizo- saccharomyces pombe. Genetic studies in these organisms has revealed information about how the SPB duplicates and separates, and its roles during vegetative growth, mating and meiosis.

Introduction

A wide variety of cellular processes including mitosis, cell motility, intracellular transport and morphogenesis depend upon microtubules. In most instances, the microtubule organizing center (MTOC) nucleates microtubule assembly and thereby establishes the microtubule array (Brinkley 1985). Microtubules nucleated by the MTOC have an intrinsic polarity. The "plus" end, which is the site of rapid microtubule assembly and disassembly, lies distal to the MTOC and the "minus" end, which exhibits slower rates of microtubule assembly and disassembly on free microtubules, lies proximal to the MTOC (Allen and Borisy 1974; Summers and Kirschner 1979; Bergen et al. 1980; Heidemann and Mclntosh 1980).

Although the primary role of the MTOC in microtubule assembly has been widely conserved, the morphology of the MTOC varies considerably among different organisms. For instance, in mammals and many other organisms the MTOC is called the centrosome, and com- prises a pair of centrioles surrounded by electron dense pericentriolar material (Brinkley 1985). However, the MTOC of many plant cells lacks centrioles but contains electron dense material similar to the pericentriolar material of animal cells.

In yeast and many other fungi, the MTOC also lacks centrioles and is called the spindle pole body (SPB) (Byers 1981). The SPB plays important roles during vegetative growth, conjugation and meiosis. This article reviews the morphological features, components and roles of the SPB of the budding yeast, Saccharomyces cerevisiae, about which considerable information has been generated over the last several years. Comparisons with fission yeast, Schizosaccharomyces pombe, are also provided although much less is known about the SPB in this organism. For other recent reviews covering the SPB see Winey and Byers (1993), Rose et al. (1993) and Page and Snyder (1993). A comprehensive review covering the morphology and behavior of the SPB in a variety of fungi is presented by Heath (1981).

Morphology of the yeast SPB

The SPB of S. cerevisiae is a disk-like structure embedded in the nuclear envelope (Robinow and Marak 1966; Moens and Rapport 1971; Byers and Goetsch 1973). It is composed of three electron-dense layers (Fig. 1): a cen-

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Table 1. Components involved in SPB function in Saccharomyces cerevisiae

SPB components Protein Gene Essential Location

name comments Arrest phenotype a

Reference

90 kDa Not cloned In inner and outer plaques

42 kDa SPC42 Yes Central plaque Nufl/Spcll0 NUF1/SPCllO Yes Between inner and

central plaque

Calmodulin CMD1 Yes Binds to Nufl/Spc110

Cdc31 CDC31 Yes Half bridge/ bridge; Ca+ binding protein

Karl KARl Yes Half bridge? Binds Cdc31

Components associated with SPB Nuf2 NUF2

80 kDa Not cloned

Spal SPA1

Kar3 KAR3

Cikl CIK1 No

N.A.

N.A. N.A.

Nuclear microtubules often detached from the SPB; aberrant nuclear morphology Single enlarged SPB without satellite

Single enlarged SPB without satellite

Yes Coiled-coil protein. Large budded arrest In mitosis on nuclear side of spindle, like 80 kDa Associated with N.A. microtubules adjacent to inner plaque

No Chromosome segregation Aberrant spindles; and karyogamy multiple nuclei defects

No Minus end-directed High fraction of motor protein; large budded cells pheromone induced with closely

separated SPBs Putative Kar3 light Like kar3A chain; pheromone induced

Rout and Kilmartin (1990, 1991) Rout and Kilmartin (1991) Kilmartin et al. (1993); Mirzayan et al. (1992); Rout and Kilmartin (1990) Davis (1992); Sun et al. (1992); Geiser et al. (1993)

Baum et al. (1986); Spang et al. (1993); Biggin and Rose, personal communication Conde and Fink (1976); Rose and Fink (1987); Vallen et al. (1992); Biggin and Rose, personal communication

Osborne et al. (1994)

Rout and Kilmartin (1990)

Snyder and Davis (1988)

Meluh and Rose (1990); Poliana and Conde (1982); Rose and Endow, personal communication Page and Snyder (1992); Page et al. (1994) with closely

Inte Cdc31, Karl Nuclear Membrane ~ , ~ ~

Central Plaque ~ '~ ' t 1~ ~ ' ~ " ~ ' ' " Half Bridge

Inner plaque " 1 ~ r z ~ ~ } ' ~ liOkD/NUF1

IHHHN , 4 90 o Discontinuous Microtubules 80 kD NUF2

Pole-to-Pole Microtubules fl Ht"1 rA H Fig. 1. Diagram of the S, cerevisiae spindle pole body (SPB) and probable locations of known SPB components. Adapted from Rout and Kilmartin (1990) and Page and Snyder (1993)

tral plaque lies in the plane of the nuclear envelope and is flanked by a parallel inner plaque on the nucleoplasmic side of the SPB, and an outer plaque on the cytoplasmic side. Nuclear microtubules, including those of the mitotic spindle apparatus, emanate from the inner plaque; cytoplasmic microtubules project from the outer plaque. Finally, a structure termed the "half bridge" lies in the plane of the nuclear envelope beside the central plaque. Following SPB duplication a bridge connects the two unseparated SPBs (see below).

The inner and outer plaques may be connected to the central plaque by filamentous spacers. Short filaments extending between the outer and central plaques have been identified in whole cells (Robinow and Marak 1966; see also Fig. 9, Byers and Goetsch 1975), and short rods have been found between the inner and central plaque in isolated SPB preparations (Kilmartin et al. 1993; see below).

The SPB of S. pombe has some general similarities to that of S. cerevisiae but there are a number of differences.

Table 1 (continued)

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SPB components Protein Gene

name Essential Location

comments Arrest phenotype ~

Reference

Components involved in SPB duplication Cdc28 CDC28 Yes

Cdc31 CDC31 Yes

Karl KARl Yes Mps 1 MPSI Yes

Mps2 MPS2 Not known

Ndc I NDC1 Yes

Components involved in SPB separation Cdc4 CDC4 Yes

Cdc34 CDC31 Yes

Cin8 C1N8 No. cin8A kiplA is lethal

Protein kinase

See above

See above Protein kinase homolog No homology

Nuclear envelope/ ER component

Large domain homologous to Gfl transducin repeats Ubiquitin conjugating enzyme Kinesin-related protein

Unduplicated SPB with satellite Single enlarged SPB without satellite Like cdc31 Large SPB with enlarged half bridge One normal SPB+ one separated defective SPB Like raps2

Side-by-side SPBs

Side-by-side SPBs

Cin8 ts kip lA strain; side-by-side SPBs

Kip 1 K1P1 No Kinesin-related Cin8 ts kipA strain; protein side-by-side SPBs

separated SPBs Byers and Goetsch (1973)

Byers (1981)

Rose and Fink (1987) Winey et al. (1991)

Winey et al. (1991)

Thomas and Botstein (1986); Winey etal. (1993)

Byers and Goetsch (1973); Fong et al. (1986); Yochem and Byers (1987) Goebl et al. (1988)

Hoyt et al. (1992); Roof et al. (1992); Saunders and Hoyt (1992) Hoyt et al. (1992); Roof et al. (1992) Saunders and Hoyt (1992)

a Arrest phenotype of conditional alleles is indicated. For Kar3 and Cikl the deletion allele exhibits a temperature sensitive growth defect. N.A. conditional mutants not available

During interphase, the S. pombe SPB is an electron dense disk-shaped structure, 220 nm in diameter, which lies adjacent to, but not in, the nuclear envelope (McCul- ly and Robinow 1971). As described for S. cerevisiae, several layers are apparent in the interphase S. pombe SPB (McCully and Robinow 1971), although the SPB morphologies are not identical. Some amorphous material lies near the S. pombe SPB inside the nuclear envelope. Interestingly, the S. pombe SPB changes its shape and location during the cell cycle. During mitosis, the SPB becomes intimately associated with the nuclear envelope and assumes a longer bar or dumbbell shape (McCully and Robinow 1971; Tanaka and Kanbe 1986). Amorphous material near the nucleoplasmic side of the SPB is still evident, and microtubules originate from this site.

In both budding yeast and fission yeast as in many other fungi, the nuclear envelope does not break down during mitosis and the entire spindle apparatus is con- tained within the nucleus (Byers 1981a; McCully and Robinow 1971; Tanaka and Kanbe 1986). Relative to other fungi, S. cerevisiae is unusual in that the SPB is permanently embedded in the nuclear envelope throughout the cell cycle; in many other fungi such as S. pombe, the SPB is clearly cytoplasmic during interphase. To account for this difference Heath (1981) speculated that in S. cerevisiae, the process by which the SPB becomes ex- ternalized may be lost or suppressed. In any event, the location of the S. cerevisiae SPB in the nuclear envelope makes it ideally suited for nucleating both nuclear and

cytoplasmic microtubules. In fact, the S. cerevisiae SPB contains both nuclear and cytoplasmic microtubules throughout the cell cycle (Byers 1981a) and in vitro microtubule assembly experiments suggest that it is competent for assembly throughout this time (Byers and Goetsch 1978; Hyams and Borisy 1978). This is not the case for S. pombe. The SPB does not contain associated microtubules during interphase (Hagan and Hyams 1988). Instead, cytoplasmic microtubules are organized from a novel and poorly characterized MTOC at a cortical region specified by the previous site of cytokinesis (Hagan and Hyams 1988). At the G2/M transition, microtubule disassembly from this MTOC occurs and the SPB becomes competent for microtubule assembly and organization of the mitotic spindle apparatus (see below).

Components of the yeast SPB

Several components of the S. cerevisiae SPB have been identified (Fig. 1, Table 1). Monoclonal antibodies generated to subcellular fractions enriched for the SPB have identified three proteins (Rout and Kilmartin 1990, 1991). A 42 kDa polypeptide lies in the central plaque, and a 90 kDa polypeptide localizes to both the inner and outer plaques. Since microtubules originate from these latter regions, it will be of interest to determine whether the 90 kDa protein directly or indirectly participates in nucleation of microtubule assembly.

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A 110 kDa polypeptide called Nufl/Spc110 localizes both between the inner and central plaques and throughout the nucleus (Rout and Kilmartin 1990; Mirzayan et al. 1992). An elegant series of experiments has shown that the 110 kDa polypeptide serves as the fibrous molecular connector extending between the inner and central plaques (Kilmartin et al. 1993). The gene encoding this polypeptide has been cloned (Mirzayan et al. 1992; Kilmartin et al. 1993). The protein sequence is predicted to be 944 amino acid residues in length and contains a long central coiled-coil domain of 627 amino acids. A series of deletions that remove varying amounts of the central coiled-coil region were constructed; yeast strains deleted for most of this region survive, but their microtubules originate closer to the central plaque than they do in wild-type strains (Kilmartin et al. 1993). The distance between the microtubule ends and the central plaque de- creases as larger segments of the coiled-coil domain are deleted; the correlation is not precise raising the possibility that other components may reside in the spacer region. Since filaments have also been reported in the region between the central and outer plaques (Robinow and Marak 1966; see also Fig. 9, Byers and Goetsch 19)5), coiled-coil proteins that localize to this region may also ultimately also be discovered.

The carboxy-terminal region of the 110 kDa polypeptide has recently been shown to bind calmodulin in vitro, (using gel blot overlay assays) and in vivo (using the two hybrid system and genetic experiments) (Geiser et al. 1993; M. Stark, personal communication). Cal- modulin is normally detected throughout the cell and is especially concentrated at the bud tip (Brockerhoff and Davis 1992; Sun et al. 1992b). However, using mild fix- ation conditions, calmodulin staining can be observed at the SPB region in many lysed cells (Geiser et al. 1993). Interestingly, calmodulin has been localized to the centrosomal region of mammalian ceils, although it role during mitosis is unknown (Welsh et al. 1978, 1979; Sweet and Welsh 1988), Cells containing particular temperature-sensitive mutants of the cahnodulin gene (cmdl-1 and cmdl-101 mutants) exhibit mitotic defects (Davis 1992; Sun et al. 1992a). One of these mutations, cmdl-1, has been shown to be suppressible by deletion of the Nuflp calmodulin binding region. Thus, calmodulin may either serve as a negative regulator of some cellular event (Geiser et al. 1993), or as a positive regulator that inactivates an inhibitory function of the carboxy-terminus of Nuflp. Nuclear microtubules are often detached from the SPBs of temperature-sensitive cmdl-lO1 mutants incubated at the restrictive temperature (Sun et al. 1992a). Perhaps calmodulin participates in the attachment of either the inner plaque and/or its associated microtubules to the SPB. A possible role for calmodulin in microtubule disassembly is described below.

Genetic screens have identified two additional SPB components, Cdc31 and Karl (Conde and Fink 1976; Hartwell et al. 1973; Rose and Fink 1987; Spang et al. 1993; Biggin and Rose, personal communication). Im- munoelectron microscopic analysis has been used to localize Cdc31 to the half bridge in cells containing a sin-

gle SPB, and to the bridge in cells containing duplicated, but unseparated SPBs (Spang et al. 1993). Cdc31 is a putative calcium-binding protein (Baum et al. 1986) that exhibits 50% sequence identity to centrin/caltractin, a highly conserved component associated with the Chlamydomonas MTOC (the basal body) (Huang et al. 1988) and the human centrosome (Lee and Huang 1993).

Karl-fl-galactosidase (fl-gal) fusions have been localized to the vicinity of the SPB (Vallen et al. 1992), but the precise location of authentic Karl within the SPB has not been determined. However, labeled Karl binds to Cdc31 in gel blot overlay assays indicating that these proteins interact in vitro (Biggin and Rose, personal communication). Furthermore, karP s mutants and karl deletion mutants are suppressible by certain dominant CDC31 mutations, suggesting possible interactions in vivo (Vallen and Rose, personal communication) Cdc31 fails to localize to the SPB in a temperature-sensitive karl-A 13 mutant, whereas a Cdc31 dominant suppressor protein localizes properly in this strain (Biggin and Rose, personal communication). Taken together, these data indicate that the essential function of Karl may be to tether the Cdc31 protein at the SPB.

Prote ins associated with the SPB

Several proteins associated with the SPB have been identified (Table 1). A putative coiled-coil protein called Nuf2 has been localized to the nuclear side of the SPB by immunofluorescence experiments (Osborne etal. 1994). nuf2 temperature-sensitive mutants arrest with replicated DNA but fail to set up a mitotic spindle apparatus. The yeast Spal protein has also been found to reside in the vicinity of the SPB by indirect immunofluorescence experiments (Snyder and Davis 1988; M. Snyd- er, unpublished), spal-A mutants exhibit defects in chromosome segregation and nuclear fusion during conjugation (karyogamy; see below) (Snyder i989; Snyder and Davis 1988). Finally, an 80 kDa polypeptide that associates with microtubules on the nuclear side of the SPB has been identified (Rout and Kilmartin 1990); the role of this protein is not known.

Microtubule motor proteins have also been found to be associated with the SPB. The cut7 kinesin-related protein of S. pombe localizes to the SPB and is predicted to contain a motor domain at its amino-terminus (Hagan and Yanagida 1992). Conditional cut7 mutants fail to set up a proper spindle apparatus at the restrictive temperature and form two unconnected half spindles. Hagan and Yanagida (1992) proposed that the cut7 protein may function in the proper separation of the SPBs (see below).

The Kar3 motor complex is associated with the SPB of S. cerevisiae. Kar3 is a minus end-directed microtubule motor protein with a kinesin-related motor domain at its carboxy-terminus (Meluh and Rose 1990; M. Rose and S. Endow, personal communication).The non-motor domain at the amino-terminus interacts with another protein, Cikl, which is a potential Kar3 light chain (Page et al.

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1994). The Cikl/Kar3(non-motor domain) complex associates with microtubules in vivo. The authentic Cikl protein has been localized to the SPB region in pheromone- treated cells (Page and Snyder 1992), and both Cikl-fl-gal and Kar3-fl-gal fusion proteins have been localized to the SPB and along nuclear microtubules in vegetative cells (Page and Snyder 1992; Page et al. 1994). Given its two micrombule binding domains, the Kar3 motor complex is expected to crosslink microtubules and slide them past one another (Meluh and Rose 1990).

Strains deleted for CIK1 and/or KAR3 exhibit severe defects in chromosome segregation and fail to undergo karyogamy. Although a role for a minus end-directed motor protein that slides microtubules is apparent in karyogamy (see below), the role of this type of motor for sliding microtubules in vegetative cells is less clear. kar3-A and cikl-A cells have enhanced microtubule arrays both in pheromone-treated and vegetative cells (Meluh and Rose 1990; Page and Snyder 1992). Before the directionality of the Kar3 motor was known, Rose (1991) proposed that Kar3, as a plus end motor, might be useful for mediating disassembly at the pole, as has been suggested to occur at mammalian MTOCs (Mitchi- son and Salmon 1992; see below). Failure to disassemble microtubules at the SPB could explain the prominent arrays. A similar role in microtubule disassembly can be proposed for Kar3 as a minus end-directed motor now that directionality has been established (S. Endow, M. Rose and E. Salmon, personal communication). The Kar3 complex might bind to two microtubules from the same pole; movement of Kar3 toward the SPB would drive one microtubule into the polar region where it might become disassembled. Alternatively, the motor it- self might disassemble microtubules during its movement.

The association of proteins with the MTOC of any organism must be interpreted cautiously. Minus end microtubule motor proteins such as Kar3 might function along the length of the microtubule, but accumulate at the pole (Page and Snyder 1993; Page et al. 1994). Furthermore, if microtubule disassembly does occur at the pole, then proteins statically associated with microtubules might also preferentially accumulate at the pole. Another con- cern is that apparent SPB-associated proteins might cor- respond to other structures that reside near the SPB. For example, yeast kinetochore proteins appear to be associated with the SPB throughout much of the cell cycle (Goh and Kilmartin 1993; see below). It is conceivable that some proteins, for example Nuf2, Kar3 or Spc80, are components of yeast kinetochores or some other SPB-associated structure.

Microtubule assembly at the yeast SPB

One of the most interesting aspects of MTOCs is their ability to nucleate microtubule assembly. In S. cerevisiae both nuclear and cytoplasmic microtubules are visible throughout the cell cycle (Byers and Goetsch 1973, 1975; Kilmartin and Adams 1984; Adams and Pringle 1984). The microtubule arrays are comparatively simple

in both budding yeast and fission yeast as compared with the arrays in other organisms. On the order of tens of microtubules are visible from a single pole (Peterson and Ris 1976), as opposed to hundreds or thousands that are estimated to be present at a mammalian MTOC (Brink- ley 1985).

Electron microscopic studies of serial sections of S. cerevisiae mitotic cells have revealed that there are two types of microtubules in the mitotic spindle apparatus: a limited number of "pole to pole" microtubules (5-12), which extend from one SPB to, or close to, the other SPB, and "discontinuous" microtubules, which extend from the SPB and terminate in the nucleoplasm (Peter- son and Ris 1976). These discontinuous microtubules are presumed to interact with chromosomes, although this has not been directly demonstrated. However, consistent with the possibility that discontinuous microtubules interact with chromosomes, their number near one pole approximates the number of chromosomes within the cell. Diploid cells, which have 32 chromosomes, contain approximately 30-40 discontinuous microtubules while haploids have -15. Interestingly, isolated SPBs of diploid cells nucleate twice as many microtubules as those of haploid cells (Byers and Goetsch 1975; Hyams and Borisy 1978; Byers et al. 1978) and the SPBs of diploids often appear larger than those of haploids. Thus, nuclear microtubule assembly capacity cor- relates with ploidy.

Nucleation of microtubule assembly is a highly regulated event in S. pombe. The SPB of interphase cells does not contain microtubules (Hagan and Hyams 1988). After the G2/M transition the SPB is tightly associated with the nuclear envelope and microtubules are now evident (Hagan and Hyams 1988). Using permeabilized cells Matsuda et al. (1992) demonstrated that SPBs of interphase cells were not competent to nucleate microtubule assembly, whereas mitotic cells were able to nucleate assembly. Incubation of interphase S. pombe cells with mitotic extracts of Xenopus oocytes, which contain an activated cdc2 kinase, allowed nucleation of assembly at the SPB; treatment with interphase Xenopus extracts, which do not have an active cdc2 kinase, did not allow nucleation. These results suggest that a cdc2 kinase pathway might be responsible for activating SPBs, thereby rendering them competent for microtubule assembly.

The mechanism by which the nucleation of microtubule assembly occurs is unknown. T-tubulin, which is found at the MTOCs of many organisms including the centrosomes of humans and Drosophila, and the SPBs of Aspergillus and S. pombe (Horio et al. 1991; Oakley and Oakley 1989; Stearns et al. 1991; Zheng et al. 1991), has been shown to be directly or indirectly involved in nucleating microtubule assembly in human cells and As- pergillus (Joshi et al. 1992; Oakley et al. 1990). In S. pombe, T-tubulin is found at the SPB throughout the cell cycle, even when it is not competent for nucleating microtubule assembly, indicating that its presence alone does not ensure microtubule assembly (Horio etal. 1991). Presumably modification of this protein or inter- action with another protein is required for generating nucleating activity in this organism. To date, ),-tubulin has

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Model 1 disassembly (perhaps upon binding calcium). For example, calmodulin could be involved in detachment of microtubules from their nucleating site, and thereby make them accessible for a disassembly mechanism. It will be of interest to know the orientation of the Nuflp/Spc110 polypeptide within the spacer region and whether calmodulin is associated with the inner or central plaque. If calmodulin plays a role in either microtubule assembly or disassembly, then it would be expected to reside in or near the inner plaque.

Model 2

i:iiii~iii:U m

::i)i::iiii:)i:ii:iii:ii - ~

Fig. 2. Models for microtubule disassembly at the pole. In Model 1, microtubules detach from a nucleating site (black region). Mi- crotubule disassembly occurs at the microtubule end adjacent to the pole. Attachment to the pole is maintained by lateral connec- tions, perhaps via microtubule motors. In the version depicted, the microtubule nucleating sites are still present; a similar scheme can be envisioned in which the nucleating region is removed from the end. In Model 2 microtubule disassembly occurs from within the protofilaments themselves. This type of model was originally proposed by Inoue and Ritter (1975) and more recently by M. Seme- nov (personal communication)

not been found in S. cerevisiae indicating that it is either not present or is highly diverged from that of other organisms.

Photolabeling experiments from other organisms have suggested that microtubule disassembly occurs at the MTOCs (e.g. Mitchinson and Salmon 1992), although the data do not rigorously exclude the possibility that microtubule disassembly occurs along the length of the microtubule (lnoue 1975). Given that nucleation of microtubule assembly occurs at the poles, presumably on defined structural material, it is difficult to envision how disassembly occurs at the nucleating region (Fig. 2). Ei- ther the microtubule must detach from its initial nucleating site (or disassemble the site) and remain attached via lateral attachments (Model 1; see Sawin et al. 1992), or alternatively, disassembly occurs from the sides of microtubules that are adjacent to the nucleating regions (Model 2) or from along the length of the entire microtubule (see Inoue and Ritter 1975). Experiments to determine whether microtubule disassembly occurs at the yeast SPB have not been performed.

Calmodulin may play a role in the regulation of nuclear microtubule disassembly, which would explain the long nuclear microtubules evident in many cells of a temperature-sensitive calmodulin mutant, cmdl-lO1 (Sun et al. 1992a). Microtubule disassembly could be mediated in one of several ways. Calmodulin might acti- vate a kinase or phosphatase involved in regulating this process. Alternatively, calmodulin might induce conformational changes in proteins involved in microtubule

Duplication of the yeast SPB

Another intriguing aspect of MTOCs is the curious man- ner in which they duplicate. Typically the new MTOC forms adjacent to the old MTOC. Presumably components at the old MTOC nucleate assembly of new components at an adjacent site.

In S. cerevisiae SPB duplication begins sometime during G1, when an electron-dense satellite appears on the cytoplasmic face of the nuclear envelope adjacent to the half bridge (Byers and Goetsch 1973). The satellite is present in temperature-sensitive cdc28 cells shifted to the restrictive temperature and in cells treated with c~ factor (Byers 1981b; Byers and Goetsch 1975), indicating that this step occurs independently of the Cdc28 G1 kinase. At the G1/S transition two SPBs separated by a bridge are observed. Thus, post-satellite formation events are presumably regulated by the Cdc28 kinase. Since the satellite forms adjacent to the half bridge, it is likely that the nucleating component that initiates SPB duplication is directly or indirectly associated with the half bridge.

Two components that participate in the SPB duplication process are Cdc3l and Karl. As noted above, Cdc31 is a component of the half bridge, and Karl is likely to be associated with this structure. Temperature- sensitive cdc31 and karl mutants fail to form satellites at the restrictive temperature and arrest with enlarged SPBs (Byers 1981b; Rose et al. 1987). Based on functions proposed for calmodulin, Cdc31 might function in the duplication process in one of several ways. Perhaps Cdc31 regulates the activities of a kinase or phosphatase. Alter- natively, Cdc31 might regulate conformational changes in an interacting protein, perhaps by a calcium-regulated event. Regulated conformational changes might initiate assembly of a new SPB by allowing SPB components to insert into the nuclear envelope, or by some other means.

Analysis of other yeast temperature-sensitive mutants had identified potential intermediates in the SPB duplication process (Fig. 3). mpsl cells arrest with a single large SPB that lacks a satellite but contains an enlarged half bridge (Winey etal . 1991). raps2 and ndcl cells shifted to the restrictive temperature contain one normal SPB and an additional defective SPB on the cytoplasmic face of the nuclear envelope (Thomas and Botstein 1986; Winey et al. 1991, 1993). The defective SPB is not in- serted in the nuclear envelope, lacks nuclear microtubules and contains only two of the three plaques. There- fore, the raps1 and ndcl mutants are thought to be defec-

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~-factor

/

-CDC31 / -MP -NPS2 / -KARl -NDC1

~ ~ Slm ~ +

Fig. 3. Duplication of the yeast spindle pole body. Adapted from Winey and Byers (Winey et al. 1991). The upper row indicates a normal SPB duplication. A possible intermediate is indicated in brackets. The lower row depicts the arrested SPB morphology in the absence of the indicated proteins. MPS1 and MPS2 are required after the a -factor arrest point. Presumably the satellite is lost in mpsI-1 cells shifted to the restrictive temperature

tive in the insertion or proper assembly of the newly formed SPB into the nuclear membrane.

DNA sequence analysis indicates that MPS1 encodes a protein kinase homolog (M. Winey, personal communication). Presumably phosphorylation by the Mpsl protein is critical for an essential SPB duplication step. The Ndcl protein has been localized throughout the nuclear envelope, and the predicted sequence of Ndcl contains either six or seven transmembrane segments (Winey et al. 1993). One likely possibility for the role of Ndc2 in SPB duplication is that Ndcl is directly involved in the assembly of a critical SPB component into the nuclear envelope. Alternatively, the effect of Ndcl might be more indirect; for example, ndcl mutants might fail to import an endoplasmic reticulum component that is important for SPB assembly. (As in other organisms, the yeast endoplasmic reticulum is continuous with the nuclear envelope.) The predicted sequence of Mps2 does not share significant homology with other proteins in the sequence databases (M. Winey, personal communication).

By comparsion with S. cerevisiae, very little is known about components involved in SPB duplication in S. pombe. SPB duplication in S. pombe occurs at the G2/M transition (Hagan and Hyams 1988) and is presumably regulated by the cdc2 G2 kinase.

Even more enigmatic than the mechanisms of SPB duplication is the process by which the SPB reduces in size during meiosis. In S. cerevisiae the number of microtubules nucleated by the haploid SPB are each approximately one-half that of a diploid cell and the size of haploid SPBs often appears smaller; therefore the SPB must decrease both its size and microtubule nucleating capacity during meiosis. Genetic data suggests that this occurs during meiosis I. Two mutants defective in SPB duplication, cdc31 and ndcl, still progress through meiosis I and arrest at meiosis II, indicating that duplication is not necessary for meiosis I (Byers 1981b; Thom- as and Botstein 1986). Since two poles presumably are formed during meiosis I in cdc31 and ndcl mutants, it is reasonable to speculate that SPB reduction forms them. SPB reduction might normally occur at this stage.

Separation of the SPB

Once SPBs have replicated, they separate during S-phase to form a short spindle (Byers and Goetsch 1973).

Temperature-sensitive cdc4 and cdc34 mutants arrest at the restrictive temperature with duplicated side-by-side SPBs (Byers 1981b; Byers and Goetsch 1973), but con- tinue to undergo budding. It is not clear whether the SPBs in these mutants are defective, and it is therefore not known whether the defects reside in the duplication or SPB separation processes, or, more likely in the cell cycle regulatory machinery. The Cdc4 gene product is predicted to contain a large domain with motifs similar to those found in the fl subunits of GTP-binding proteins, such as transducin (Fong et al. 1986; Yochem and Byers 1987); the predicted sequence of Cdc34 is homologous to that of ubiquitin-conjugating enzymes (Goebl et al. 1988), which are involved in protein degradation (Jentsch 1922). The Cdc34 protein might participate in degradation of either a SPB component whose cleavage is necessary for SPB separation, or a regulatory component that controls this event (e.g., a Cdc28 regulator whose loss is required for SPB separation).

Two genes important for SPB separation are CIN8 and KIP1, which encode functionally redundant kinesin- related proteins (Hoyt et al. 1992; Roof et al. 1992; Saunders and Hoyt 1992). Deletion of KIP1 causes no detectable defects and deletion of CIN8 causes only mi- nor defects in chromosome segregation; however, deletion of both genes results in lethality (Roof et al. 1992; Saunders and Hoyt 1992). Temperature-sensitive cin8-3 kipl-A strains fail to form a spindle when shifted to the restrictive temperature and arrest with side-by-side SPBs (Hoyt et al. 1992). In addition to their role in SPB separation and establishment of the mitotic spindle apparatus, Cin8 and Kip1 are also required for the maintenance of the spindle apparatus (Hoyt et al. 1992). When cin8-3 kipl-A are first arrested with a short spindle by hydroxy- urea treatment, and then shifted to the restrictive temperature, the spindle apparatus collapses, and side-by- side SPBs are again observed. It has been suggested that the Cin8 and Kipl proteins function as plus end-directed motors that slide microtubules emanating from different SPBs past one another, thereby separating the poles (Fig. 4A; Hoyt et al. 1992). The cut7 protein of S. pombe has been proposed to separate SPBs in a similar fashion; the protein might reside at one SPB and act on microtubules emanating from the opposing SPB (Fig. 4B; Hagan and Yanagida 1992). MTOC separating kinesin-related proteins have also been identified in Aspergillus (Enos and Morris 1990), suggestive of a possible universal role for

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A u ~ i n

B I m m ~ m l m m

Jli ll Fig. 4A, B. Separation of the SPBs. A Cin8 and Kipl as plus end directed- microtubule motor proteins might directly or indirectly crosslink microtubules and slide them past each other to estab- lish the spindle apparatus. B The Cut7 plus end-directed motor protein is associated with the SPB and could also poten- tially participate in separation of the poles. B is adapted from Hagan and Ya- nigida (1992) and Page and Snyder (1993)

these proteins in establishment of the mitotic spindle apparatus.

Positioning and segregation of the yeast SPB

The location of the SPB within the cell is nonrandom. During vegetative growth, the SPB lies on the opposite side of the nucleus from the nucleolus (Yang et al. 1989). It has been suggested that short intranuclear microtubules that emanate from the SPB are attached to centromeres throughout the cell cycle and cluster centromere-proximal regions of the chromosomes near the SPB (Yang et al. 1989). Regions closer to the telo- meres, such as the rDNA, would preferentially lie on the opposite side of the nucleus. Consistent with this hy- pothesis, Gob and Kilmartin (1993) have. found that a centromere protein Cbf2/Ndcl0 localizes in the vicinity of the SPB. It is not clear what biological functions are conferred by the nonrandom positioning of SPBs relative to chromosomes or the nucleolus within the cell. It has been suggested that attachment of chromosomes to centromeres throughout the cell cycle might be important for the non-random segregation of chromatids at mitosis (Murray and Szostak 1985). However, this mechanism seems unlikely since newly replicated chromatids are segregated randomly (Neff and Burke 1991).

Another curious phenomenon is the possible relationship between the SPB and nuclear pore complexes. Two temperature-sensitive mutants of yeast, rat2 and rat3, exhibit defects in mRNA export from the nucleus (D. Amberg, C. Copeland, M. Snyder and C. Cole, unpublished). The mutants contain clustered nuclear pore complexes and in many cases the cluster appears to be associated with the SPB. Perhaps assembly of nuclear pore complexes and the SPB occur within a distinct domain of the nucleus. In mammalian cells the nuclear lamina is thought to play an important role in the organization and/or distribution of components of the nuclear envelope (Newport and Forbes 1987; Whytock et al. 1990); in yeast a well-defined nuclear lamina has not been described (Hurt et al. 1992), so its role in this process is not known.

The SPB is also positioned with respect to the bud and site of bud formation (Byers and Goetsch 1975; Snyder et al. 1991). During the unbudded phase the SPB orients toward the incipient bud site (Snyder et al. 1991). After bud formation and growth the SPB remains orient-

ed toward the bud, and cytoplasmic microtubules extend into the bud (Byers and Goetsch 1975). It has been suggested that the SPB and/or its associated microtubules participates in bud site selection (Byers and Goetsch 1975; Byers 1981a). However, in normal haploid and diploid mother cells, which form buds adjacent to the previous site of cytokinesis (Friefelder 1960; Snyder 1989), this clearly cannot be the case. At the end of cytokinesis the SPBs in these cells reside on the side of the nucleus distal to the site where the next bud will form. In haploid cells, it has been shown that components involved in budding often assemble at the incipient bud site while the SPB still resides on the distal side of the nucleus (Snyder et al. 1991); thus the SPB and/or its associated microtubules cannot normally be responsible for directing the assembly of components at the new bud site. Cortical "tags", in which new components assemble adjacent to cortical markers, appear to be responsible for bud site selection in these cells (Snyder etal. 1991; Madden et al. 1992; Flescher et al. 1993). It is still possible however, that SPBs and/or their associated microtubules participate in bud site selection in cells emerging from stationary phase and/or in diploid daughter cells, in which the new bud forms opposite the SPB (see Madden and Snyder 1992).

SPB orientation toward the incipient bud might be mediated by microtubule capture sites at the incipient bud and at or near the emerging bud tip (Snyder et al. 1991). A candidate protein involved in capture and/or orientation of the SPB is cytoplasmic dynein, which is important for maintaining the orientation of the mitotic spindle apparatus (Eshel et al. 1993; Li et al. 1993).

In S. pombe, the position of the SPB is also not random. Like S. cerevisiae, the SPB lies preferentially opposite the nucleolus; it also resides near a mitochondrian (McCully and Robinow 1971; Tanaka and Kanabe 1986). For S. pombe, the positioning of the SPB relative to the nucleolus may be a consequence of the mitotic behavior of chromosomes. During mitosis centromeres are expected to be pulled to the poles and telomeric regions trail (RaN 1885). In S. pombe, the nucleolus (which is more telomeric than the bulk of the genomic DNA) is al- ways trailing during mitosis (Granot and Snyder 1991; Hirano et al. 1989); persistance of the pattern in interphase cells in a "Rabl-like" configuration would account for the SPB residing opposite the nucleolus. In S. cerevisiae, the nucleolus does not trail during mitosis and can-

377

Vegetative Conjugation Meiosis Growth

Chromosome Karyogamy Chromosome Segregation Segregation

Prospore Wall Budding (?) Formation

Fig. 5. Roles of microtubules and the SPB in yeast. Not shown is a role for microtubules in positioning of the nucleus (and spindle apparatus) during vegetative growth

not account for the SPB/nucleolus orientation in those cells (Yang et al. 1989; Granot and Snyder 1991).

Following SPB duplication, the daughter SPBs will segregate into the progeny cells. In S. cerevisiae, segregation of SPBs is not random. Indirect immunofluorescence experiments indicate that the Karl::fl-gal fusion protein associates with only one SPB; this SPB segregates primarily into the bud (Vallen et al. 1992). Analy- sis of ndcl mutants indicates that the Karl::fl-gal fusion is associated with the defective (and presumably) new SPB. Thus, segregation of the SPBs is a nonrandom process; the old SPB remains in the mother cell and the new SPB segregates to the daughter. Models to explain the nonrandom segregation of the yeast SPB are presented in Hyman and Stearns (1992) and Page and Snyder (1993).

Roles of the yeast SPB microtubules during vegetative growth, karyogamy and meiosis

The SPB and microtubules are involved in a variety of diverse functions (summarized in Fig. 5 for S. cerevisiae). During vegetative growth of S. cerevisiae, the SPB organizes the microtubules of the mitotic spindle apparatus and the cytoplasmic microtubule array. Some of the cytoplasmic microtubules extend to the nascent bud site of unbudded cells and to or near the tip of the bud in budded cells. Cytoplasmic microtubules play a role in orienting the spindle apparatus (Sullivan and Huffaker 1992; Palmer et al. 1992) and they also appear to play a nonessential function in bud formation and/or growth (Lillie and Brown 1992). Nocodazole-treated cells and tubulin mutants form buds normally, indicating that microtubules are not essential for bud formation and growth (Jacobs et al. 1988; Huffaker et al. 1988). How- ever, temperature-sensitive mutations in the yeast gene encoding a myosin V protein, MY02, fail to form buds at the restrictive temperature (Johnston et al. 1991); this defect is rescued by overexpression of SMY1, a gene encoding a kinesin-related protein (Lillie and Brown 1992). Thus, microtubule-based motors can contribute to bud formation and growth, and presumably play an an- cillary role in these processes.

Cytoplasmic microtubules also appear to function in nuclear fusion during mating. In S. cerevisiae, when haploid yeast cells of opposite mating types are mixed together, the cells first contact at the tips of their mating projections (reviewed in Sprague and Thorner 1992). The cell walls break down at the contact site to form a heterokaryon containing two separated nuclei. The SPBs from these nuclei face one another in the heterokaryon, and an interdigitating microtubule array extends between them. The nuclei are drawn together using microtubules, and electron microscopic studies suggest that nuclear fusion initiates at the SPBs (Byers and Goetsch 1975). Mi- crotubules are essential for mediating the nuclear fusion; nocodazole-treated cells and tub2 tubulin mutants are defective in karyogamy (Delgado and Conde 1984; Huf- faker et al. 1988).

The Kar3 motor complex is important for nuclear fusion; both Kar3 and Cikl are required for karyogamy (Po-liana and Conde 1982; Meluh and Rose 1990; Page and Snyder 1992). A likely mechanism for describing how the Kar3/Cikl complex functions is that the motor domain of Kar3 interacts with the microtubules of one SPB; the nonmotor domain in association with Cikl functions to bind microtubules from the other SPB. As the Kar3 complex moves in the minus end direction, the microtubules slide past one another and the two SPBs are drawn toward one another (in a fashion opposite that shown for Fig. 4A). The Kar3 complex could function either in the interzone region of overlapping microtubules and/or near the SPB.

Studies of nuclear fusion in the fission yeast Schizo- saccharomyces octosporus, have revealed a number of similarities between fission yeast and budding yeast (Ashton and Moens 1982). Cell wall breakdown, orientation of the two SPBs toward one another, and migra- tion of the nuclei toward one another leading to fusion at the SPB are generally similar between the two types of yeast. However, there appear to be some interesting differences (Ashton and Moens 1982). In S. octosporus, the SPB of only one nucleus contains microtubules (suggesting that it is now competent for assembly); two additional cytoplasmic microtubule bundles, each emanating near a haploid nucleus, extend past the SPB of the other nucleus. It seems likely that interdigitating microtubules, and motors analogous to those of the Kar3/Cikl complex will be important for nuclear fusion in fission yeast.

In addition to its roles in chromosome segregation and karyogamy, the SPB of S, cerevisiae also appears to play a unique role during meiosis. During meiosis II the outer plaque of the SPB is significantly enlarged (Moens and Rapport 1971). The prospore wall begins forming adjacent to the modified outer plaque, while the nuclei are still interconnected. This observation suggests that the SPB participates in, and perhaps directs, the initiation of prospore wall formation.

Conclusion and future issues

Our understanding of the SPB is still extremely limited. It is expected that the handful of SPB proteins now iden-

378

tiffed wil l const i tu te on ly a minor f ract ion o f the total SPB components . The prote ins that par t ic ipa te in nucle- a t ing mic ro tubu le a s sembly have not been d i scovered in S. cerevisiae nor is it k n o w n how their act ivi ty is regula t - ed. Whe the r mic ro tubu le d i s a s sembly occurs at the poles is not known. The pa thway by which SPB dupl ica t ion occurs and how this pa thway is regu la ted is only begin- ning to be resolved. The conse rved proper t ies o f the SPB (dup l ica t ion and micro tubu le a s sembly ) and the high de- gree o f conserva t ion of pro te ins such as Cdc31/cen t r in sugges t that many of these p rocesses wil l be h igh ly conserved with other eukaryotes at both a ce l lu lar and molecular level. Thus, fur ther ana lys is o f SPB dupl ica t ion and funct ion in yeas t m a y reveal universa l p r inc ip les ap- p l i cab le to other organisms.

Acknowledgements. I thank members of my laboratory for stimu- lating discussions, and M. Winey and M. Rose for sharing unpublished information. Jennifer Barrett, Christine Costigan, Scott Erdman, Haya Friedman, Marc Freeman, Kevin Madden, Suzanne Sobel, and Misha Semenov provided comments on the manu- script. Research from our laboratory was supported by NIH grant GM36494.

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the spindle pole body of yeast

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