Components of the yeast spindle and spindle pole body

Yeast spindle pole bodies (SPBs) with attached nuclear microtubles were enriched approximately 600-fold from yeast cell extracts. 14 mAbs prepared against this enriched SPB fraction define at least three components of the SPB and spindle. Immunofluorescent staining of yeast cells showed that throughout the cell cycle two of the components (110 and 90 kD) were localized exclusively to the SPB region, and the other (80 kD) was localized both to the SPB region and to particulate dots in short spindles. Immunoelectron microscopy confirmed and extended most of these findings. Thus the 110-kD component was localized to a layer in the SPB just to the nuclear side of the plane of the inner nuclear membrane. The 90-kD component was localized in a layer across the cytoplasmic face of intact SPBs, and, in SPBs where nuclear microtubules were removed by extraction with DEAE-dextran, the 90-kD component was also found in an inner nuclear layer close to where spindle microtubules emerge. In intact SPBs with attached nuclear microtubules the anit-80-kD mAb labels microtubules, particularly those close to the SPB. These results begin to provide a preliminary molecular map of the SPB and should also enable the corresponding genes to be isolated.     

The spindle pole body of yeast

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, Saccharomyces cerevisiae, and the fission yeast, Schizosaccharomyces 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.     

Mutations in yeast calmodulin cause defects in spindle pole body functions and nuclear integrity

Yeast calmodulin (CaM) is required for the progression of nuclear division (Ohya, Y. and Y. Anraku. 1989. Curr. Genet. 15:113-120), although the precise mechanism and physiological role of CaM in this process are unclear. In this paper we have characterized the phenotype caused by a temperature-sensitive lethal mutation (cmdl-101) in the yeast CaM. The cmdl-101 mutation expresses a carboxyl-terminal half of the yeast CaM (Met72-Cys147) under the control of an inducible GAL1 promoter. Incubation of the cmdl-101 cells at a nonpermissive temperature causes a severe defect in chromosome segregation. The rate of chromosome loss in the cmdl-101 mutant is higher than wild-type cell even at permissive temperature. The primary visible defect observed by immunofluorescence and electron microscopic analyses is that the organization of spindle microtubules is abnormal in the cmdl-101 cells grown at nonpermissive temperature. Majority of budded cells arrested at the high temperature contain only one spindle pole body (SPB), which forms monopolar spindle, whereas the budded cells of the same strain incubated at permissive temperature all contain two SPBs. Using the freeze-substituted fixation method, we found that the integrity of the nuclear morphology of the cmdl-101 mutant cell is significantly disturbed. The nucleus in wild-type cells is round with smooth contours of nuclear envelope. However, the nuclear envelope in the mutant cells appears to be very flexible and forms irregular projections and invaginations that are never seen in wild-type cells. The deformation of the nuclear becomes much more severe as the incubation at nonpermissive temperature continues. The single SPB frequently localizes on the projections or the invaginations of the nuclear envelope. These observations suggest that CaM is required for the functions of SPB and spindle, and the integrity of nucleus.     

Microtubule organization by the budding yeast spindle pole body.

In budding yeast microtubule organizing functions are provided by the spindle pole body (SPB), a multi-layered structure that is embedded in the nuclear envelope throughout the cell cycle. The SPB organizes the nuclear and cytoplasmic microtubules which are spatially and functionally distinct. Microtubule formation in yeast requires the Tub4p-complex, containing the gamma-tubulin Tub4p, and two additional proteins, the SPB components Spc97p and Spc98p. The Tub4p complex assembles in the cytoplasm and is then anchored to the sides of the SPB which organize microtubules. This is achieved by the binding of Spc97p and Spc98p to so-called gamma-tubulin complex binding proteins (GTBPs) at the SPB. Spc72p is the yeast GTBP at the cytoplasmic side of the SPB, while Spc110p is the nuclear GTBP. Both GTBPs control the number of Tub4p complexes associated with the SPB and thereby the number of microtubules formed. In addition, the GTBPs may regulate the activity of the Tub4p complex. Homologues of Spc97p and Spc98p have been identified from yeast to mammalian cells and these are also part of gamma-tubulin complexes, suggesting that these related proteins may also interact with GTBPs at the centrosome. Candidates for GTBPs have been identified in mammalian and insect cells.     

Asymmetric mitotic segregation of the yeast spindle pole body.

The yeast KAR1 gene is required for spindle pole body (SPB) duplication and nuclear fusion. We determine here that KAR1-beta-galactosidase hybrid proteins localize to the outer face of the SPB. Remarkably, after SPB duplication, the hybrid protein was found associated with only one of the two SPBs, usually the one that enters the bud. Using an ndc1 mutant, which forms a defective SPB at the nonpermissive temperature, we found that the hybrid was exclusively associated with the "new" SPB. Two regions of KAR1 contribute to its localization; an internal 70 residue region was necessary and sufficient to localize hybrids to the SPB, and the hydrophobic carboxyl terminus localized proteins to the nuclear envelope. The localization domains correspond to two functional domains required for SPB duplication. We suggest that KAR1 is anchored to the nuclear envelope and interacts with at least one other SPB component during the cell cycle.     

Lessons from yeast: the spindle pole body and the centrosome

The yeast spindle pole body (SPB) is the functional equivalent of the centrosome. Most SPB components have been identified and their functions partly established. This involved a large variety of techniques which are described here, and the potential use of some of these in the centrosome field is highlighted. In particular, very useful structural information on the SPB was obtained from a reconstituted complex, the γ-tubulin complex, and also from a sub-particle, SPB cores, prepared by extraction of an enriched SPB preparation. The labelling of SPB proteins with GFP at the N or C termini, using GFP tags inserted into the genome, gave informative electron microscopy localization and fluorescence resonance energy transfer data. Examples are given of more precise functional data obtained by removing domains from one SPB protein, Spc110p, without affecting its essential function. Finally, a structural model for SPB duplication is described and the differences between SPB and centrosome duplication discussed.     

Constitutive dynein activity in She1 mutants reveals differences in microtubule attachment at the yeast spindle pole body

The organization of microtubules is determined in most cells by a microtubule-organizing center, which nucleates microtubule assembly and anchors their minus ends. In Saccharomyces cerevisiae cells lacking She1, cytoplasmic microtubules detach from the spindle pole body at high rates. Increased rates of detachment depend on dynein activity, supporting previous evidence that She1 inhibits dynein. Detachment rates are higher in G1 than in metaphase cells, and we show that this is primarily due to differences in the strengths of microtubule attachment to the spindle pole body during these stages of the cell cycle. The minus ends of detached microtubules are stabilized by the presence of γ-tubulin and Spc72, a protein that tethers the γ-tubulin complex to the spindle pole body. A Spc72-Kar1 fusion protein suppresses detachment in G1 cells, indicating that the interaction between these two proteins is critical to microtubule anchoring. Overexpression of She1 inhibits the loading of dynactin components, but not dynein, onto microtubule plus ends. In addition, She1 binds directly to microtubules in vitro, so it may compete with dynactin for access to microtubules. Overall, these results indicate that inhibition of dynein activity by She1 is important to prevent excessive detachment of cytoplasmic microtubules, particularly in G1 cells.     

Mutations in SPC110, encoding the yeast spindle pole body calmodulin-binding protein, cause defects in cell integrity as well as spindle formation

The 110 kDa spindle pole body component, Spc110p, is an essential target of calmodulin in budding yeast. Cells with mutations which reduce calmodulin binding to Spc110p are unable to form a mitotic spindle and die. Here we show that these effects can be overcome either directly by increasing extracellular calcium or calmodulin expression, which reverse the primary spindle defect, or indirectly through increased extracellular osmolarity or high dosage of MID2 or SLG1/HCS77/WSC1 which preserve viability. We propose that overcoming a cell integrity defect associated with the mitotic arrest enables the defective spindle pole bodies to provide sufficient function for proliferation of a large proportion of mutant cells. Our findings demonstrate a role for calcium in the Spc110p-calmodulin interaction in vivo and have important general implications for the interpretation of genetic interactions involving cell integrity genes.     

Intrinsic and cyclin-dependent kinase-dependent control of spindle pole body duplication in budding yeast

Centrosome duplication must be tightly controlled so that duplication occurs only once each cell cycle. Accumulation of multiple centrosomes can result in the assembly of a multipolar spindle and lead to chromosome mis-segregation and genomic instability. In metazoans, a centrosome-intrinsic mechanism prevents reduplication until centriole disengagement. Mitotic cyclin/cyclin-dependent kinases (CDKs) prevent reduplication of the budding yeast centrosome, called a spindle pole body (SPB), in late S-phase and G2/M, but the mechanism remains unclear. How SPB reduplication is prevented early in the cell cycle is also not understood. Here we show that, similar to metazoans, an SPB-intrinsic mechanism prevents reduplication early in the cell cycle. We also show that mitotic cyclins can inhibit SPB duplication when expressed before satellite assembly in early G1, but not later in G1, after the satellite had assembled. Moreover, electron microscopy revealed that SPBs do not assemble a satellite in cells expressing Clb2 in early G1. Finally, we demonstrate that Clb2 must localize to the cytoplasm in order to inhibit SPB duplication, suggesting the possibility for direct CDK inhibition of satellite components. These two mechanisms, intrinsic and extrinsic control by CDK, evoke two-step system that prevents SPB reduplication throughout the cell cycle.     

The organization of the core proteins of the yeast spindle pole body

The spindle pole body (SPB) is the microtubule organizing center of Saccharomyces cerevisiae. Its core includes the proteins Spc42, Spc110 (kendrin/pericentrin ortholog), calmodulin (Cmd1), Spc29, and Cnm67. Each was tagged with CFP and YFP and their proximity to each other was determined by fluorescence resonance energy transfer (FRET). FRET was measured by a new metric that accurately reflected the relative extent of energy transfer. The FRET values established the topology of the core proteins within the architecture of SPB. The N-termini of Spc42 and Spc29, and the C-termini of all the core proteins face the gap between the IL2 layer and the central plaque. Spc110 traverses the central plaque and Cnm67 spans the IL2 layer. Spc42 is a central component of the central plaque where its N-terminus is closely associated with the C-termini of Spc29, Cmd1, and Spc110. When the donor-acceptor pairs were ordered into five broad categories of increasing FRET, the ranking of the pairs specified a unique geometry for the positions of the core proteins, as shown by a mathematical proof. The geometry was integrated with prior cryoelectron tomography to create a model of the interwoven network of proteins within the central plaque. One prediction of the model, the dimerization of the calmodulin-binding domains of Spc110, was confirmed by in vitro analysis.     

Direct interaction between yeast spindle pole body components: Kar1p is required for Cdc31p localization to the spindle pole body

The Saccharomyces cerevisiae genes KAR1 and CDC31 are required for the initial stages of spindle pole body (SPB) duplication in yeast. The Cdc31 protein is most related to caltractin/centrin, a calcium-binding protein present in microtubule organizing centers in many organisms. Because of a variety of genetic interactions between CDC31 and KAR1 (Vallen, E. A., W. Ho. M. Winey, and M. D. Rose. 1994. Genetics. In press), we wanted to determine whether Cdc31p and Kar1p physically interact. Cdc31p was expressed and purified from Escherichia coli and active for binding calcium. Using a protein blotting technique, Cdc31p bound to Kar1p in vitro via an essential domain in Kar1p required for SPB duplication (Vallen, E. A., M. A. Hiller, T. Y. Scherson, and M. D. Rose. 1992a. J. Cell Biol. 117:1277-1287). By immunofluorescence microscopy, we determined that the interaction also occurs in vivo. Cdc31p was localized to the SPB in wild-type cells but was mislocalized in a kar1 mutant strain. In a kar1 mutant containing a dominant CDC31 suppressor, Cdc31p was again localized to the SPB. Furthermore, the localization of Cdc31p to the SPB was affected by the overexpression of Kar1p-beta-galactosidase hybrids. Based on these data, we propose that the essential function of Kar1p is to localize Cdc31p to the SPB, and that this interaction is normally required for SPB duplication.     

Mechanisms of genetic instability revealed by analysis of yeast spindle pole body duplication.

Aneuploidy and polyploidy are commonly observed in transformed cells. These states arise from failures during mitotic chromosome segregation, some of which can be traced to defects in the function or duplication of the centrosome. The centrosome is the organizing center for the mitotic spindle, and the equivalent organelle in the budding yeast, Saccharomyces cerevisiae, is the spindle pole body. We review how defects in spindle pole body duplication or function lead to genetic instability in yeast. There are several well documented instances of genetic instability in yeast that can be traced to the spindle pole body, all of which serve as models for genetic instability in transformed cells.     

NDC1: a nuclear periphery component required for yeast spindle pole body duplication

The spindle pole body (SPB) of Saccharomyces cerevisiae serves as the centrosome in this organism, undergoing duplication early in the cell cycle to generate the two poles of the mitotic spindle. The conditional lethal mutation ndc1-1 has previously been shown to cause asymmetric segregation, wherein all the chromosomes go to one pole of the mitotic spindle (Thomas, J. H., and D. Botstein. 1986. Cell. 44:65-76). Examination by electron microscopy of mutant cells subjected to the nonpermissive temperature reveals a defect in SPB duplication. Although duplication is seen to occur, the nascent SPB fails to undergo insertion into the nuclear envelope. The parental SPB remains functional, organizing a monopolar spindle to which all the chromosomes are presumably attached. Order-of-function experiments reveal that the NDC1 function is required in G1 after alpha-factor arrest but before the arrest caused by cdc34. Molecular analysis shows that the NDC1 gene is essential and that it encodes a 656 amino acid protein (74 kD) with six or seven putative transmembrane domains. This evidence for membrane association is further supported by immunofluorescent localization of the NDC1 product to the vicinity of the nuclear envelope. These findings suggest that the NDC1 protein acts within the nuclear envelope to mediate insertion of the nascent SPB.     

Structural role of Sfi1p-centrin filaments in budding yeast spindle pole body duplication

Centrins are calmodulin-like proteins present in centrosomes and yeast spindle pole bodies (SPBs) and have essential functions in their duplication. The Saccharomyces cerevisiae centrin, Cdc31p, binds Sfi1p on multiple conserved repeats; both proteins localize to the SPB half-bridge, where the new SPB is assembled. The crystal structures of Sfi1p-centrin complexes containing several repeats show Sfi1p as an alpha helix with centrins wrapped around each repeat and similar centrin-centrin contacts between each repeat. Electron microscopy (EM) shadowing of an Sfi1p-centrin complex with 15 Sfi1 repeats and 15 centrins bound showed filaments 60 nm long, compatible with all the Sfi1 repeats as a continuous alpha helix. Immuno-EM localization of the Sfi1p N and C termini showed Sfi1p-centrin filaments spanning the length of the half-bridge with the Sfi1p N terminus at the SPB. This suggests a model for SPB duplication where the half-bridge doubles in length by association of the Sfi1p C termini, thereby providing a new Sfi1p N terminus to initiate SPB assembly.     

Targeting Alp7/TACC to the spindle pole body is essential for mitotic spindle assembly in fission yeast

The conserved TACC protein family localises to the centrosome (the spindle pole body, SPB in fungi) and mitotic spindles, thereby playing a crucial role in bipolar spindle assembly. However, it remains elusive how TACC proteins are recruited to the centrosome/SPB. Here, using fission yeast Alp7/TACC, we have determined clustered five amino acid residues within the TACC domain required for SPB localisation. Critically, these sequences are essential for the functions of Alp7, including proper spindle formation and mitotic progression. Moreover, we have identified pericentrin-like Pcp1 as a loading factor to the mitotic SPB, although Pcp1 is not a sole platform.     

Spindle assembly without spindle pole body insertion into the nuclear envelope in fission yeast meiosis

Chromosoma - Centrosomes represent the major microtubule organizing center (MTOC) in eukaryotic cells and are responsible for nucleation of the spindle, the vehicle of chromosome segregation. In...     

Regulation of spindle pole body assembly and cytokinesis by the centrin-binding protein Sfi1 in fission yeast

Centrosomes play critical roles in the cell division cycle and ciliogenesis. Sfi1 is a centrin-binding protein conserved from yeast to humans. Budding yeast Sfi1 is essential for the initiation of spindle pole body (SPB; yeast centrosome) duplication. However, the recruitment and partitioning of Sfi1 to centrosomal structures have never been fully investigated in any organism, and the presumed importance of the conserved tryptophans in the internal repeats of Sfi1 remains untested. Here we report that in fission yeast, instead of doubling abruptly at the initiation of SPB duplication and remaining at a constant level thereafter, Sfi1 is gradually recruited to SPBs throughout the cell cycle. Like an sfi1Δ mutant, a Trp-to-Arg mutant (sfi1-M46) forms monopolar spindles and exhibits mitosis and cytokinesis defects. Sfi1-M46 protein associates preferentially with one of the two daughter SPBs during mitosis, resulting in a failure of new SPB assembly in the SPB receiving insufficient Sfi1. Although all five conserved tryptophans tested are involved in Sfi1 partitioning, the importance of the individual repeats in Sfi1 differs. In summary, our results reveal a link between the conserved tryptophans and Sfi1 partitioning and suggest a revision of the model for SPB assembly.     

MPS1 and MPS2: novel yeast genes defining distinct steps of spindle pole body duplication

It is crucial to the eucaryotic cell cycle that the centrosome undergo precise duplication to generate the two poles of the mitotic spindle. In the budding yeast Saccharomyces cerevisiae, centrosomal functions are provided by the spindle pole body (SPB), which is duplicated at the time of bud emergence in G1 of the cell cycle. Genetic control of this process has previously been revealed by the characterization of mutants in CDC31 and KAR1, which prevent SPB duplication and lead to formation of a monopolar spindle. Newly isolated mutations described here (mps1 and mps2, for monopolar spindle) similarly cause monopolar mitosis but their underlying effects on SPB duplication are unique. The MPS1 gene is found by electron microscopy to be essential for proper formation of the site at which the new SPB normally arises adjacent to the existing one. By contrast, a mutation in MPS2 permits duplication to proceed, but the newly formed SPB is structurally defective and unable to serve as a functional spindle pole. Distinct temporal requirements for the CDC31, MPS1, and MPS2 gene functions during the SPB duplication cycle further demonstrate the individual roles of these genes in the morphogenetic pathway.     

The role of the yeast spindle pole body and the mammalian centrosome in regulating late mitotic events

Centrosomes of vertebrate cells and spindle pole bodies (SPBs) of fungi were first recognized through their ability to organize microtubules. Recent studies suggest that centrosomes and SPBs also have a function in the regulation of cell cycle progression, in particular in controlling late mitotic events. Regulators of mitotic exit and cytokinesis are associated with the SPB of budding and fission yeast. Elucidation of the molecular roles played by these regulators is helping to clarify the function of the SPB in controlling progression though mitosis.     

An intrinsically disordered yeast prion arrests the cell cycle by sequestering a spindle pole body component

Intrinsically disordered proteins play causative roles in many human diseases. Their overexpression is toxic in many organisms, but the causes of toxicity are opaque. In this paper, we exploit yeast technologies to determine the root of toxicity for one such protein, the yeast prion Rnq1. This protein is profoundly toxic when overexpressed but only in cells carrying the endogenous Rnq1 protein in its [RNQ(+)] prion (amyloid) conformation. Surprisingly, toxicity was not caused by general proteotoxic stress. Rather, it involved a highly specific mitotic arrest mediated by the Mad2 cell cycle checkpoint. Monopolar spindles accumulated as a result of defective duplication of the yeast centrosome (spindle pole body [SPB]). This arose from selective Rnq1-mediated sequestration of the core SPB component Spc42 in the insoluble protein deposit (IPOD). Rnq1 does not normally participate in spindle pole dynamics, but it does assemble at the IPOD when aggregated. Our work illustrates how the promiscuous interactions of an intrinsically disordered protein can produce highly specific cellular toxicities through illicit, yet highly specific, interactions with the proteome.