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.     

Licensing of Yeast Centrosome Duplication Requires Phosphoregulation of Sfi1

Duplication of centrosomes once per cell cycle is essential for bipolar spindle formation and genome maintenance and is controlled in part by cyclin-dependent kinases (Cdks). Our study identifies Sfi1, a conserved component of centrosomes, as the first Cdk substrate required to restrict centrosome duplication to once per cell cycle. We found that reducing Cdk1 phosphorylation by changing Sfi1 phosphorylation sites to nonphosphorylatable residues leads to defects in separation of duplicated spindle pole bodies (SPBs, yeast centrosomes) and to inappropriate SPB reduplication during mitosis. These cells also display defects in bipolar spindle assembly, chromosome segregation, and growth. Our findings lead to a model whereby phosphoregulation of Sfi1 by Cdk1 has the dual function of promoting SPB separation for spindle formation and preventing premature SPB duplication. In addition, we provide evidence that the protein phosphatase Cdc14 has the converse role of activating licensing, likely via dephosphorylation of Sfi1.     

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.     

IBD2 encodes a novel component of the Bub2p-dependent spindle checkpoint in the budding yeast Saccharomyces cerevisiae

During mitosis, genomic integrity is maintained by the proper coordination of mitotic events through the spindle checkpoint. The bifurcated spindle checkpoint blocks cell cycle progression at metaphase by monitoring unattached kinetochores and inhibits mitotic exit in response to the incorrect orientation of the mitotic spindle. Bfa1p is a spindle checkpoint regulator of budding yeast in the Bub2p checkpoint pathway for proper mitotic exit. We have isolated a novel Bfa1p interacting protein named Ibd2p in the budding yeast Saccharomyces cerevisiae. We found that IBD2 (Inhibition of Bud Division 2) is not an essential gene but its deletion mutant proceeded through the cell cycle in the presence of microtubule-destabilizing drugs, thereby inducing a sharp decrease in viability. In addition, overexpression of Mps1p caused partial mitotic arrest in ibd2Delta as well as in bub2Delta, suggesting that IBD2 encodes a novel component of the spindle checkpoint downstream of MPS1. Overexpression of Ibd2p induced mitotic arrest with increased levels of Clb2p in wild type and mad2Delta, but not in deletion mutants of BUB2 and BFA1. Pds1p was also stabilized by the overexpression of Ibd2p in wild-type cells. The mitotic arrest defects observed in ibd2Delta in the presence of nocodazole were restored by additional copies of BUB2, BFA1, and CDC5, whereas an extra copy of IBD2 could not rescue the mitotic arrest defects of bub2Delta and bfa1Delta. The mitotic arrest defects of ibd2Delta were not recovered by MAD2, or vice versa. Analysis of the double mutant combinations ibd2Deltamad2Delta, ibd2Deltabub2Delta, and ibd2Deltadyn1Delta showed that IBD2 belongs to the BUB2 epistasis group. Taken together, these data demonstrate that IBD2 encodes a novel component of the BUB2-dependent spindle checkpoint pathway that functions upstream of BUB2 and BFA1.     

cut11+: A Gene Required for Cell Cycle-dependent Spindle Pole Body Anchoring in the Nuclear Envelope and Bipolar Spindle Formation in Schizosaccharomyces pombe

The “cut” mutants of Schizosaccharomyces pombe are defective in spindle formation and/or chromosome segregation, but they proceed through the cell cycle, resulting in lethality. Analysis of temperature-sensitive alleles of cut11⁺ suggests that this gene is required for the formation of a functional bipolar spindle. Defective spindle structure was revealed with fluorescent probes for tubulin and DNA. Three-dimensional reconstruction of mutant spindles by serial sectioning and electron microscopy showed that the spindle pole bodies (SPBs) either failed to complete normal duplication or were free floating in the nucleoplasm. Localization of Cut11p tagged with the green fluorescent protein showed punctate nuclear envelope staining throughout the cell cycle and SPBs staining from early prophase to mid anaphase. This SPB localization correlates with the time in the cell cycle when SPBs are inserted into the nuclear envelope. Immunoelectron microscopy confirmed the localization of Cut11p to mitotic SPBs and nuclear pore complexes. Cloning and sequencing showed that cut11⁺ encodes a novel protein with seven putative membrane-spanning domains and homology to the Saccharomyces cerevisiae gene NDC1. These data suggest that Cut11p associates with nuclear pore complexes and mitotic SPBs as an anchor in the nuclear envelope; this role is essential for mitosis.     

The mitotic arrest in response to hypoxia and of polar bodies during early embryogenesis requires Drosophila Mps1

Mps1 kinase plays an evolutionary conserved role in the mitotic spindle checkpoint. This system precludes anaphase onset until all chromosomes have successfully attached to spindle microtubules via their kinetochores. Mps1 overexpression in budding yeast is sufficient to trigger a mitotic arrest, which is dependent on the other mitotic checkpoint components, Bub1, Bub3, Mad1, Mad2, and Mad3. Therefore, Mps1 might act at the top of the mitotic checkpoint cascade. Moreover, in contrast to the other mitotic checkpoint components, Mps1 is essential for spindle pole body duplication in budding yeast. Centrosome duplication in mammalian cells might also be controlled by Mps1 , but the fission yeast homolog is not required for spindle pole body duplication. Our phenotypic characterizations of Mps1 mutant embryos in Drosophila do not reveal an involvement in centrosome duplication, while the mitotic spindle checkpoint is defective in these mutants. In addition, our analyses reveal novel functions. We demonstrate that Mps1 is also required for the arrest of cell cycle progression in response to hypoxia. Finally, we show that Mps1 and the mitotic spindle checkpoint are responsible for the developmental cell cycle arrest of the three haploid products of female meiosis that are not used as the female pronucleus.     

A field guide to the Mps1 family of protein kinases

Cell cycle events must be faithfully executed and properly integrated to ensure genetic stability. The Mps1 family of protein kinases has recently emerged as a critical regulator of genetic stability, because they regulate several processes central to mitotic fidelity. The spindle checkpoint monitors alignment of mitotic chromosomes, and centrosomes control cell cycle entry, mitotic spindle assembly, and cytokinesis. Several studies have shown that vertebrate orthologues of budding yeast Mps1p regulate the spindle checkpoint. More recently it has been demonstrated that human Mps1 is also required for centrosome duplication, normal mitotic progression, and cytokinesis.     

A Field Guide to the Mps1 Family of Protein Kinases

Cell cycle events must be faithfully executed and properly integrated to ensure genetic stability. The Mps1 family of protein kinases has recently emerged as a critical regulator of genetic stability, because they regulate several processes central to mitotic fidelity. The spindle checkpoint monitors alignment of mitotic chromosomes, and centrosomes control cell cycle entry, mitotic spindle assembly, and cytokinesis. Several studies have shown that vertebrate orthologues of budding yeast Mps1p regulate the spindle checkpoint. More recently it has been demonstrated that human Mps1 is also required for centrosome duplication, normal mitotic progression, and cytokinesis.     

Requirement for Bbp1p in the proper mitotic functions of Cdc5p in Saccharomyces cerevisiae

The polo-box domain of the budding yeast polo kinase Cdc5p plays an essential role for targeting the catalytic activity of Cdc5p to spindle pole bodies (SPBs) and cytokinetic neck-filaments. Here, we report the isolation of Bbp1p as a polo-box interacting protein by a yeast two-hybrid screen. Bbp1p localizes to the periphery of the central plaque of the SPB and plays an important role in SPB duplication. Similarly, Cdc5p localized to the cytoplasmic periphery of the SPB. In vitro binding studies showed that Cdc5p interacted with the N-terminal domain of Bbp1p (Bbp1pDeltaC), but apparently not with Mps2p, a component shown to form a stable complex with Bbp1p. In addition, Bbp1p, but likely not Mps2p, was required for proper localization of Cdc5p to the SPB. The C-terminal coiled-coil domain of Bbp1p (Bbp1p(243-385)), which is crucial for both the homodimerization and the SPB localization, could target the localization-defective Cdc5pDeltaC to the SPB and induce the release of Cdc14p from the nucleolus. Consistent with this observation, expression of CDC5DeltaC-BBP1(243-385) under CDC5 promoter control partially complemented the cdc5Delta defect. These data suggest that Bbp1pDeltaC interacts with the polo-box domain of Cdc5p, and this interaction is critical for the subcellular localization and mitotic functions of Cdc5p.     

A novel allele of Saccharomyces cerevisiae NDC1 reveals a potential role for the spindle pole body component Ndc1p in nuclear pore assembly

Both the spindle pole body (SPB) and the nuclear pore complex (NPC) are essential organelles embedded in the nuclear envelope throughout the life cycle of the budding yeast Saccharomyces cerevisiae. However, the mechanism by which these two multisubunit structures are inserted into the nuclear envelope during their biogenesis is not well understood. We have previously shown that Ndc1p is the only known integral membrane protein that localizes to both the SPBs and the NPCs and is required for SPB duplication. For this study, we generated a novel temperature-sensitive (ts) allele of NDC1 to investigate the role of Ndc1p at the NPCs. Yeast cells carrying this allele (ndc1-39) failed to insert the SPB into the nuclear envelope at the restrictive temperature. Importantly, the double mutation of ndc1-39 and NPC assembly mutant nic96-1 resulted in cells with enhanced growth defects. While nuclear protein import and NPC distribution in the nuclear envelope were unaffected, ndc1-39 mutants failed to properly incorporate the nucleoporin Nup49p into NPCs. These results provide evidence that Ndc1p is required for NPC assembly in addition to its role in SPB duplication. We postulate that Ndc1p is crucial for the biogenesis of both the SPBs and the NPCs at the step of insertion into the nuclear envelope.     

Three-dimensional electron microscopy analysis of ndc10-1 mutant reveals an aberrant organization of the mitotic spindle and spindle pole body defects in Saccharomyces cerevisiae

Kinetochore components play a major role in regulating the transmission of genetic information during cell division. Ndc10p, a kinetochore component of the essential CBF3 complex in budding yeast is required for chromosome attachment to the mitotic spindle. ndc10-1 mutant was shown to display chromosome mis-segregation as well as an aberrant mitotic spindle (Goh and Kilmartin, 1993). In addition, Ndc10p localizes along the spindle microtubules (Muller-Reichert et al., 2003). To further understand the role of Ndc10p in the mitotic apparatus, we performed a three-dimensional electron microscopy (EM) reconstruction of mitotic spindles from serial sections of cryo-immobilized ndc10-1 mutant cells. This analysis reveals a dramatic reduction in the number of microtubules present in the half-spindle, which is connected to the newly formed spindle pole body (SPB) in ndc10-1 cells. Moreover, in contrast to wild-type (WT) cells, ndc10-1 cells showed a significantly lower signal intensity of the SPB components Spc42p and Spc110p fused with GFP, in mother cell bodies compared with buds. A subsequent EM analysis also showed clear defects in the newly formed SPB, which remains in the mother cell during anaphase. These results suggest that Ndc10p is required for maturation of the newly formed SPB. Intriguingly, mutations in other kinetochore components, ndc80-1 and spc24-1, showed kinetochore detachment from the spindle, similar to ndc10-1, but did not display defects in SPBs. This suggests that unattached kinetochores are not sufficient to cause SPB defects in ndc10-1 cells. We propose that Ndc10p, alongside its role in kinetochore–microtubule interaction, is also essential for SPB maturation and mitotic spindle integrity.     

Complex formation between Mad1p, Bub1p and Bub3p is crucial for spindle checkpoint function

The spindle checkpoint delays the metaphase to anaphase transition in response to defects in kinetochore-microtubule interactions in the mitotic apparatus (see [1] [2] [3] [4] for reviews). The Mad and Bub proteins were identified as key components of the spindle checkpoint through budding yeast genetics [5] [6] and are highly conserved [3]. Most of the spindle checkpoint proteins have been localised to kinetochores, yet almost nothing is known about the molecular events which take place there. Mad1p forms a tight complex with Mad2p [7], and has been shown to recruit Mad2p to kinetochores [8]. Similarly, Bub3p binds to Bub1p [9] and may target it to kinetochores [10]. Here, we show that budding yeast Mad1p has a regulated association with Bub1p and Bub3p during a normal cell cycle and that this complex is found at significantly higher levels once the spindle checkpoint is activated. We find that formation of this complex requires Mad2p and Mps1p but not Mad3p or Bub2p. In addition, we identify a conserved motif within Mad1p that is essential for Mad1p-Bub1p-Bub3p complex formation. Mutation of this motif abolishes checkpoint function, indicating that formation of the Mad1p-Bub1p-Bub3p complex is a crucial step in the spindle checkpoint mechanism.     

酿酒酵母Sfi1p的结构及生物学功能

纺锤体极体(spindle pole body,SPB)是酵母细胞的微管组织中心,它在细胞分裂及细胞遗传稳定性的维持过程中起着极其重要的作用,是细胞生物学领域热门的研究方向.Sfi1p是酿酒酵母SPB的必需蛋白并且横跨整个半桥,该蛋白与SPB的复制有关,它的缺失或突变会导致SPB复制失败,在哺乳动物的中心体也存在酵母Sfi1p的同源蛋白.本文系统的介绍了酵母Sfi1p及其在人类中心体中的同源蛋白hSfi1p的结构特征,并且阐明了Sfi1p在SPB复制与分离、核配及生孢等细胞周期过程中的作用.对Sfi1p的功能研究,将有助于解决SPB研究过程中重要的科学问题,同时为中心体中Sfi1p同源蛋白的功能研究提供良好的借鉴.     

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.     

The yeast protein kinase Mps1p is required for assembly of the integral spindle pole body component Spc42p

Saccharomyces cerevisiae MPS1 encodes an essential protein kinase that has roles in spindle pole body (SPB) duplication and the spindle checkpoint. Previously characterized MPS1 mutants fail in both functions, leading to aberrant DNA segregation with lethal consequences. Here, we report the identification of a unique conditional allele, mps1-8, that is defective in SPB duplication but not the spindle checkpoint. The mutations in mps1-8 are in the noncatalytic region of MPS1, and analysis of the mutant protein indicates that Mps1-8p has wild-type kinase activity in vitro. A screen for dosage suppressors of the mps1-8 conditional growth phenotype identified the gene encoding the integral SPB component SPC42. Additional analysis revealed that mps1-8 exhibits synthetic growth defects when combined with certain mutant alleles of SPC42. An epitope-tagged version of Mps1p (Mps1p-myc) localizes to SPBs and kinetochores by immunofluorescence microscopy and immuno-EM analysis. This is consistent with the physical interaction we detect between Mps1p and Spc42p by coimmunoprecipitation. Spc42p is a substrate for Mps1p phosphorylation in vitro, and Spc42p phosphorylation is dependent on Mps1p in vivo. Finally, Spc42p assembly is abnormal in a mps1-1 mutant strain. We conclude that Mps1p regulates assembly of the integral SPB component Spc42p during SPB duplication.     

The budding yeast Polo-like kinase Cdc5 is released from the nucleus during anaphase for timely mitotic exit

Polo-like kinases are important regulators of multiple mitotic events; however, how Polo-like kinases are spatially and temporally regulated to perform their many tasks is not well understood. Here, we examined the subcellular localization of the budding yeast Polo-like kinase Cdc5 using a functional Cdc5-GFP protein expressed from the endogenous locus. In addition to the well-described localization of Cdc5 at the spindle pole bodies (SPBs) and the bud neck, we found that Cdc5-GFP accumulates in the nucleus in early mitosis but is released to the cytoplasm in late mitosis in a manner dependent on the Cdc14 phosphatase. This Cdc5 release from the nucleus is important for mitotic exit because artificial sequestration of Cdc5 in the nucleus by addition of a strong nuclear localization signal (NLS) resulted in mitotic exit defects. We identified a key cytoplasmic target of Cdc5 as Bfa1, an inhibitor of mitotic exit. Our study revealed a novel layer of Cdc5 regulation and suggests the existence of a possible coordination between Cdc5 and Cdc14 activity.     

Yeast Dam1p Is Required to Maintain Spindle Integrity during Mitosis and Interacts with the Mps1p Kinase

We have identified a mutant allele of the DAM1 gene in a screen for mutations that are lethal in combination with the mps1-1 mutation. MPS1 encodes an essential protein kinase that is required for duplication of the spindle pole body and for the spindle assembly checkpoint. Mutations in six different genes were found to be lethal in combination with mps1-1, of which only DAM1 was novel. The remaining genes encode a checkpoint protein, Bub1p, and four chaperone proteins, Sti1p, Hsc82p, Cdc37p, and Ydj1p. DAM1 is an essential gene that encodes a protein recently described as a member of a microtubule binding complex. We report here that cells harboring the dam1-1 mutation fail to maintain spindle integrity during anaphase at the restrictive temperature. Consistent with this phenotype, DAM1 displays genetic interactions with STU1, CIN8, and KAR3, genes encoding proteins involved in spindle function. We have observed that a Dam1p-Myc fusion protein expressed at endogenous levels and localized by immunofluorescence microscopy, appears to be evenly distributed along short mitotic spindles but is found at the spindle poles at later times in mitosis.     

Chemical genetics reveals a role for Mps1 kinase in kinetochore attachment during mitosis

Accurate chromosome segregation depends on proper assembly and function of the kinetochore and the mitotic spindle. In the budding yeast, Saccharomyces cerevisiae, the highly conserved protein kinase Mps1 has well-characterized roles in spindle pole body (SPB, yeast centrosome equivalent) duplication and the mitotic checkpoint. However, an additional role for Mps1 is suggested by phenotypes of MPS1 mutations that include genetic interactions with kinetochore mutations and meiotic chromosome segregation defects and also by the localization of Mps1 at the kinetochore, the latter being independent of checkpoint activation. We have developed a new MPS1 allele, mps1-as1, that renders the kinase specifically sensitive to a cell-permeable ATP analog inhibitor, allowing us to perform high-resolution execution point experiments that identify a novel role for Mps1 subsequent to SPB duplication. We demonstrate, by using both fixed- and live-cell fluoresence techniques, that cells lacking Mps1 function show severe defects in mitotic spindle formation, sister kinetochore positioning at metaphase, and chromosome segregation during anaphase. Taken together, our experiments are consistent with an important role for Mps1 at the kinetochore in mitotic spindle assembly and function.     

Saccharomyces cerevisiae Duo1p and Dam1p,Novel Proteins Involved in Mitotic Spindle Function

In this paper, we describe the identification and characterization of two novel and essential mitotic spindle proteins, Duo1p and Dam1p. Duo1p was isolated because its overexpression caused defects in mitosis and a mitotic arrest. Duo1p was localized by immunofluorescence, by immunoelectron microscopy, and by tagging with green fluorescent protein (GFP), to intranuclear spindle microtubules and spindle pole bodies. Temperature-sensitive duo1 mutants arrest with short spindles. This arrest is dependent on the mitotic checkpoint. Dam1p was identified by two-hybrid analysis as a protein that binds to Duo1p. By expressing a GFP–Dam1p fusion protein in yeast, Dam1p was also shown to be associated with intranuclear spindle microtubules and spindle pole bodies in vivo. As with Duo1p, overproduction of Dam1p caused mitotic defects. Biochemical experiments demonstrated that Dam1p binds directly to microtubules with micromolar affinity. We suggest that Dam1p might localize Duo1p to intranuclear microtubules and spindle pole bodies to provide a previously unrecognized function (or functions) required for mitosis.     

Regulation of the Bfa1p-Bub2p complex at spindle pole bodies by the cell cycle phosphatase Cdc14p

The budding yeast mitotic exit network (MEN) is a GTPase-driven signal transduction cascade that controls the release of the phosphatase Cdc14p from the nucleolus in anaphase and thereby drives mitotic exit. We show that Cdc14p is partially released from the nucleolus in early anaphase independent of the action of the MEN components Cdc15p, Dbf2p, and Tem1p. Upon release, Cdc14p binds to the spindle pole body (SPB) via association with the Bfa1p-Bub2p GTPase activating protein complex, which is known to regulate the activity of the G protein Tem1p. Cdc14p also interacts with this GTPase. The association of the MEN component Mob1p with the SPB acts as a marker of MEN activation. The simultaneous binding of Cdc14p and Mob1p to the SPB in early anaphase suggests that Cdc14p initially activates the MEN. In a second, later step, which coincides with mitotic exit, Cdc14p reactivates the Bfa1p-Bub2p complex by dephosphorylating Bfa1p. This inactivates the MEN and displaces Mob1p from SPBs. These data indicate that Cdc14p activates the MEN in early anaphase but later inactivates it through Bfa1p dephosphorylation and so restricts MEN activity to a short period in anaphase.