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.     

Novel sfi1 alleles uncover additional functions for Sfi1p in bipolar spindle assembly and function

A variety of spindle and kinetochore defects have been shown to induce a mitotic delay through activation of the spindle checkpoint. With the aim of identifying novel mitotic defects we carried out a mad1 synthetic lethal screen in budding yeast. In this screen, four novel alleles of sfi1 were isolated. SFI1 is an essential gene, previously identified through its interaction with centrin/CDC31 and shown to be required for spindle pole body (SPB) duplication. The new mutations were all found in the C-terminal domain of Sfi1p, which has no known function, but it is well conserved among budding yeasts. Analysis of the novel sfi1 mutants, through a combination of light and electron microscopy, revealed duplicated SPBs <0.3 microm apart. Importantly, these SPBs have completed duplication, but they are not separated, suggesting a possible defect in splitting of the bridge. We discuss possible roles for Sfi1p in this step in bipolar spindle 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.     

Kar1 binding to Sfi1 C-terminal regions anchors the SPB bridge to the nuclear envelope

The yeast spindle pole body (SPB) is the functional equivalent of the mammalian centrosome. The half bridge is a SPB substructure on the nuclear envelope (NE), playing a key role in SPB duplication. Its cytoplasmic components are the membrane-anchored Kar1, the yeast centrin Cdc31, and the Cdc31-binding protein Sfi1. In G1, the half bridge expands into the bridge through Sfi1 C-terminal (Sfi1-CT) dimerization, the licensing step for SPB duplication. We exploited photo-activated localization microscopy (PALM) to show that Kar1 localizes in the bridge center. Binding assays revealed direct interaction between Kar1 and C-terminal Sfi1 fragments. kar1Δ cells whose viability was maintained by the dominant CDC31-16 showed an arched bridge, indicating Kar1’s function in tethering Sfi1 to the NE. Cdc31-16 enhanced Cdc31–Cdc31 interactions between Sfi1–Cdc31 layers, as suggested by binding free energy calculations. In our model, Kar1 binding is restricted to Sfi1-CT and Sfi1 C-terminal centrin-binding repeats, and centrin and Kar1 provide cross-links, while Sfi1-CT stabilizes the bridge and ensures timely SPB separation.     

Fission yeast cdc31p is a component of the half-bridge and controls SPB duplication

The fission yeast spindle pole body (SPB) is a nucleus-associated organelle that duplicates once each cell cycle during interphase. Duplicated SPBs serve as the poles of an intranuclear mitotic spindle after their insertion into the nuclear envelope in mitosis (Ding et al., Mol. Biol. Cell 8, 1461-1479). Here, we report the identification and characterization of Schizosaccharomyces pombe cdc31p, a member of the conserved calcium-binding centrin/CDC31 family. Immunofluorescence and immunoelectron microscopy show that cdc31p is a SPB component localized at the half-bridge structure of the SPB. cdc31 is an essential gene and Deltacdc31 cells and cdc31 conditional mutant cells arrest in mitosis with a monopolar mitotic spindle organized from a single SPB. EM analysis demonstrates that mutant cdc31 cells fail to duplicate the SPB. In addition, cdc31p exhibits genetic interactions with the SPB component sad1p and is required for sad1p localization. Finally, cdc31 mutant can undergo single or multiple rounds of septation before the exit from mitosis, suggesting that cdc31p activity or SPB duplication may be required for the proper coordination between the exit from mitosis and the initiation of septation.     

Spindle pole body duplication in fission yeast occurs at the G1/S boundary but maturation is blocked until exit from S by an event downstream of cdc10+

The regulation and timing of spindle pole body (SPB) duplication and maturation in fission yeast was examined by transmission electron microscopy. When cells are arrested at G1 by nitrogen starvation, the SPB is unduplicated. On release from G1, the SPBs were duplicated after 1–2 h. In cells arrested at S by hydroxyurea, SPBs are duplicated but not mature. In G1 arrest/release experiments with cdc2.33 cells at the restrictive temperature, SPBs remained single, whereas in cells at the permissive temperature, SPBs were duplicated. In cdc10 mutant cells, the SPBs seem not only to be duplicated but also to undergo partial maturation, including invagination of the nuclear envelope underneath the SPB. There may be an S-phase–specific inhibitor of SPB maturation whose expression is under control of cdc10⁺. This model was examined by induction of overreplication of the genome by overexpression of rum1p or cdc18p. In cdc18p-overexpressing cells, the SPBs are duplicated but not mature, suggesting that cdc18p is one component of this feedback mechanism. In contrast, cells overexpressing rum1p have large, deformed SPBs accompanied by other features of maturation and duplication. We propose a feedback mechanism for maturation of the SPB that is coupled with exit from S to trigger morphological changes.     

Saccharomyces cerevisiae Ndc1p Is a Shared Component of Nuclear Pore Complexes and Spindle Pole Bodies

We report a novel connection between nuclear pore complexes (NPCs) and spindle pole bodies (SPBs) revealed by our studies of the Saccharomyces cerevisiae NDC1 gene. Although both NPCs and SPBs are embedded in the nuclear envelope (NE) in yeast, their known functions are quite distinct. Previous work demonstrated that NDC1 function is required for proper SPB duplication (Winey, M., M.A. Hoyt, C. Chan, L. Goetsch, D. Botstein, and B. Byers. 1993. J. Cell Biol. 122:743–751). Here, we show that Ndc1p is a membrane protein of the NE that localizes to both NPCs and SPBs. Indirect immunofluorescence microscopy shows that Ndc1p displays punctate, nuclear peripheral localization that colocalizes with a known NPC component, Nup49p. Additionally, distinct spots of Ndc1p localization colocalize with a known SPB component, Spc42p. Immunoelectron microscopy shows that Ndc1p localizes to the regions of NPCs and SPBs that interact with the NE. The NPCs in ndc1-1 mutant cells appear to function normally at the nonpermissive temperature. Finally, we have found that a deletion of POM152, which encodes an abundant but nonessential nucleoporin, suppresses the SPB duplication defect associated with a mutation in the NDC1 gene. We show that Ndc1p is a shared component of NPCs and SPBs and propose a shared function in the assembly of these organelles into the NE.     

Sfi1p has conserved centrin-binding sites and an essential function in budding yeast spindle pole body duplication

Centrins are calmodulin-like proteins present in microtubule-organizing centers. The Saccharomyces cerevisiae centrin, Cdc31p, was functionally tagged with a single Z domain of protein A, and used in pull-down experiments to isolate Cdc31p-binding proteins. One of these, Sfi1p, localizes to the half-bridge of the spindle pole body (SPB), where Cdc31p is also localized. Temperature-sensitive mutants in SFI1 show a defect in SPB duplication and genetic interactions with cdc31-1. Sfi1p contains multiple internal repeats that are also present in a Schizosaccharomyces pombe protein, which also localizes to the SPB, and in several human proteins, one of which localizes close to the centriole region. Cdc31p binds directly to individual Sfi1 repeats in a 1:1 ratio, so a single molecule of Sfi1p binds multiple molecules of Cdc31p. The centrosomal human protein containing Sfi1 repeats also binds centrin in the repeat region, showing that this centrin-binding motif is conserved.     

The N-terminus of Sfi1 and yeast centrin Cdc31 provide the assembly site for a new spindle pole body

The spindle pole body (SPB) provides microtubule-organizing functions in yeast and duplicates exactly once per cell cycle. The first step in SPB duplication is the half-bridge to bridge conversion via the antiparallel dimerization of the centrin (Cdc31)-binding protein Sfi1 in anaphase. The bridge, which is anchored to the old SPB on the proximal end, exposes free Sfi1 N-termini (N-Sfi1) at its distal end. These free N-Sfi1 promote in G₁ the assembly of the daughter SPB (dSPB) in a yet unclear manner. This study shows that N-Sfi1 including the first three Cdc31 binding sites interacts with the SPB components Spc29 and Spc42, triggering the assembly of the dSPB. Cdc31 binding to N-Sfi1 promotes Spc29 recruitment and is essential for satellite formation. Furthermore, phosphorylation of N-Sfi1 has an inhibitory effect and delays dSPB biogenesis until G₁. Taking these data together, we provide an understanding of the initial steps in SPB assembly and describe a new function of Cdc31 in the recruitment of dSPB components.     

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.     

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.     

Spindle pole body duplication: a model for centrosome duplication?

The yeast spindle pole body (SPB) is the functional equivalent of the centrosome and forms the two poles of the mitotic spindle. Before mitosis, both SPBs and centrosomes are present as single copies and must be duplicated to form the bipolar spindle. SPB components have been identified using a combination of biochemistry and genetics, and their role during SPB duplication has been analysed using temperature-sensitive mutants. In this article, we describe structural aspects of SPB duplication and their possible relationship to centrosome duplication.     

Multi-step control of spindle pole body duplication by cyclin-dependent kinase

Organelles called centrosomes in metazoans or spindle pole bodies (SPBs) in yeast direct the assembly of a bipolar spindle that is essential for faithful segregation of chromosomes during mitosis. Abnormal accumulation of multiple centrosomes leads to genome instability, and has been observed in both tumour cells and cells with targeted mutations in tumour-suppressor genes. The defects that lead to centrosome amplification are not understood. We have recapitulated the multiple-centrosome phenotype in budding yeast by disrupting the activity of specific cyclin-dependent kinase (CDK) complexes. Our observations are reminiscent of mechanisms that govern DNA replication, and show that specific cyclin/CDK activities function both to promote SPB duplication and to prevent SPB reduplication.     

The Yeast CDC37 Gene Interacts with MPS1 and Is Required for Proper Execution of Spindle Pole Body Duplication

The MPS1 gene from Saccharomyces cerevisiae encodes an essential protein kinase required for spindle pole body (SPB) duplication and for the mitotic spindle assembly checkpoint. Cells with the mps1-1 mutation fail early in SPB duplication and proceed through monopolar mitosis with lethal consequences. We identified CDC37 as a multicopy suppressor of mps1-1 temperature-sensitive growth. Suppression is allele specific, and synthetic lethal interactions occur between mps1 and cdc37 alleles. We examined the cdc37-1 phenotype for defects related to the SPB cycle. The cdc37-1 temperature-sensitive allele causes unbudded, G1 arrest at Start (Reed, S.I. 1980. Genetics. 95: 561–577). Reciprocal shifts demonstrate that cdc37-1 arrest is interdependent with α-factor arrest but is not a normal Start arrest. Although the cells are responsive to α-factor at the arrest, SPB duplication is uncoupled from other aspects of G1 progression and proceeds past the satellite-bearing SPB stage normally seen at Start. Electron microscopy reveals side-by-side SPBs at cdc37-1 arrest. The outer plaque of one SPB is missing or reduced, while the other is normal. Using the mps2-1 mutation to distinguish between the SPBs, we find that the outer plaque defect is specific to the new SPB. This phenotype may arise in part from reduced Mps1p function: although Mps1p protein levels are unaffected by the cdc37-1 mutation, kinase activity is markedly reduced. These data demonstrate a requirement for CDC37 in SPB duplication and suggest a role for this gene in G1 control. CDC37 may provide a chaperone function that promotes the activity of protein kinases.     

New Alleles of the Yeast MPS1 Gene Reveal Multiple Requirements in Spindle Pole Body Duplication

In Saccharomyces cerevisiae, the Mps1p protein kinase is critical for both spindle pole body (SPB) duplication and the mitotic spindle assembly checkpoint. The mps1–1 mutation causes failure early in SPB duplication, and because the spindle assembly checkpoint is also compromised, mps1–1 cells proceed with a monopolar mitosis and rapidly lose viability. Here we report the genetic and molecular characterization of mps1–1 and five new temperature-sensitive alleles of MPS1. Each of the six alleles contains a single point mutation in the region of the gene encoding the protein kinase domain. The mutations affect several residues conserved among protein kinases, most notably the invariant glutamate in subdomain III. In vivo and in vitro kinase activity of the six epitope-tagged mutant proteins varies widely. Only two display appreciable in vitro activity, and interestingly, this activity is not thermolabile under the assay conditions used. While five of the six alleles cause SPB duplication to fail early, yielding cells with a single SPB, mps1–737 cells proceed into SPB duplication and assemble a second SPB that is structurally defective. This phenotype, together with the observation of intragenic complementation between this unique allele and two others, suggests that Mps1p is required for multiple events in SPB duplication.     

Ndj1, a telomere-associated protein,regulates centrosome separation in budding yeast meiosis

Yeast centrosomes (called spindle pole bodies [SPBs]) remain cohesive for hours during meiotic G2 when recombination takes place. In contrast, SPBs separate within minutes after duplication in vegetative cells. We report here that Ndj1, a previously known meiosis-specific telomere-associated protein, is required for protecting SPB cohesion. Ndj1 localizes to the SPB but dissociates from it ∼16 min before SPB separation. Without Ndj1, meiotic SPBs lost cohesion prematurely, whereas overproduction of Ndj1 delayed SPB separation. When produced ectopically in vegetative cells, Ndj1 caused SPB separation defects and cell lethality. Localization of Ndj1 to the SPB depended on the SUN domain protein Mps3, and removal of the N terminus of Mps3 allowed SPB separation and suppressed the lethality of NDJ1-expressing vegetative cells. Finally, we show that Ndj1 forms oligomeric complexes with Mps3, and that the Polo-like kinase Cdc5 regulates Ndj1 protein stability and SPB separation. These findings reveal the underlying mechanism that coordinates yeast centrosome dynamics with meiotic telomere movement and cell cycle progression.     

Redistribution of centrosomal proteins by centromeres and Polo kinase controls partial nuclear envelope breakdown in fission yeast

Proper mitotic progression in Schizosaccharomyces pombe requires partial nuclear envelope breakdown (NEBD) and insertion of the spindle pole body (SPB—yeast centrosome) to build the mitotic spindle. Linkage of the centromere to the SPB is vital to this process, but why that linkage is important is not well understood. Utilizing high-resolution structured illumination microscopy, we show that the conserved Sad1-UNC-84 homology-domain protein Sad1 and other SPB proteins redistribute during mitosis to form a ring complex around SPBs, which is a precursor for localized NEBD and spindle formation. Although the Polo kinase Plo1 is not necessary for Sad1 redistribution, it localizes to the SPB region connected to the centromere, and its activity is vital for redistribution of other SPB ring proteins and for complete NEBD at the SPB to allow for SPB insertion. Our results lead to a model in which centromere linkage to the SPB drives redistribution of Sad1 and Plo1 activation that in turn facilitate partial NEBD and spindle formation through building of a SPB ring structure.     

Spindle pole body components are reorganized during fission yeast meiosis

During meiosis, the centrosome/spindle pole body (SPB) must be regulated in a manner distinct from that of mitosis to achieve a specialized cell division that will produce gametes. In this paper, we demonstrate that several SPB components are localized to SPBs in a meiosis-specific manner in the fission yeast Schizosaccharomyces pombe. SPB components, such as Cut12, Pcp1, and Spo15, which stay on the SPB during the mitotic cell cycle, disassociate from the SPB during meiotic prophase and then return to the SPB immediately before the onset of meiosis I. Interestingly, the polo kinase Plo1, which normally localizes to the SPB during mitosis, is excluded from them in meiotic prophase, when meiosis-specific, horse-tail nuclear movement occurs. We found that exclusion of Plo1 during this period was essential to properly remodel SPBs, because artificial targeting of Plo1 to SPBs resulted in an overduplication of SPBs. We also found that the centrin Cdc31 was required for meiotic SPB remodeling. Thus Plo1 and a centrin play central roles in the meiotic SPB remodeling, which is essential for generating the proper number of meiotic SPBs and, thereby provide unique characteristics to meiotic divisions.     

Measuring Affinities of Fission Yeast Spindle Pole Body Proteins in Live Cells across the Cell Cycle

Characterizing protein-protein interactions is essential for understanding molecular mechanisms, although reproducing cellular conditions in vitro is challenging and some proteins are difficult to purify. We developed a method to measure binding to cellular structures using fission yeast cells as reaction vessels. We varied the concentrations of Sid2p and Mob1p (proteins of the septation initiation network) and measured their binding to spindle pole bodies (SPBs), the centrosome equivalent of yeast. From our measurements we infer that Sid2p and Mob1p both exist as monomeric, heterodimeric, and homodimeric species throughout the cell cycle. During interphase these species have widely different affinities for their common receptor Cdc11p on the SPB. The data support a model with a subset of Cdc11p binding the heterodimeric species with a K_d < 0.1 μM when Sid2p binds Mob1p-Cdc11p and K_d in the micromolar range when Mob1p binds Sid2p-Cdc11p. During mitosis an additional species presumed to be the phosphorylated Sid2p−Mob1p heterodimer binds SPBs with a lower affinity. Homodimers of Sid2p or Mob1p bind to the rest of Cdc11p at SPBs with lower affinity: K_ds > 10 μM during interphase and somewhat stronger during mitosis. These measurements allowed us to account for the fluctuations in Sid2p binding to SPBs throughout the cell cycle.     

Nuc2p, a subunit of the anaphase-promoting complex, inhibits septation initiation network following cytokinesis in fission yeast

In most cell types, mitosis and cytokinesis are tightly coupled such that cytokinesis occurs only once per cell cycle. The fission yeast Schizosaccharomyces pombe divides using an actomyosin-based contractile ring and is an attractive model for the study of the links between mitosis and cytokinesis. In fission yeast, the anaphase-promoting complex/cyclosome (APC/C) and the septation initiation network (SIN), a spindle pole body (SPB)–associated GTPase-driven signaling cascade, function sequentially to ensure proper coordination of mitosis and cytokinesis. Here, we find a novel interplay between the tetratricopeptide repeat (TPR) domain–containing subunit of the APC/C, Nuc2p, and the SIN, that appears to not involve other subunits of the APC/C. Overproduction of Nuc2p led to an increase in the presence of multinucleated cells, which correlated with a defect in actomyosin ring maintenance and localization of the SIN component protein kinases Cdc7p and Sid1p to the SPBs, indicative of defective SIN signaling. Conversely, loss of Nuc2p function led to increased SIN signaling, characterized by the persistent localization of Cdc7p and Sid1p on SPBs and assembly of multiple actomyosin rings and division septa. Nuc2p appears to function independently of the checkpoint with FHA and ring finger (CHFR)–related protein Dma1p, a known inhibitor of the SIN in fission yeast. Genetic and biochemical analyses established that Nuc2p might influence the nucleotide state of Spg1p GTPase, a key regulator of the SIN. We propose that Nuc2p, by inhibiting the SIN after cell division, prevents further deleterious cytokinetic events, thereby contributing to genome stability.