Pcp1p, an Spc110p-related calmodulin target at the centrosome of the fission yeast Schizosaccharomyces pombe.

In the budding yeast Saccharomyces cerevisiae, the calmodulin-binding protein Spc110p/Nuf1p facilitates mitotic spindle formation from the fungal centrosome or spindle pole body (SPB). The human Spc110p orthologue kendrin is a centrosomal, calmodulin-binding pericentrin isoform that is specifically overexpressed in carcinoma cells. Here we establish an evolutionary and functional link between Spc110p and kendrin through identification and analysis of similar calmodulin-binding proteins in the fission yeast Schizosaccharomyces pombe (Pcp1p, pole target of calmodulin in S. pombe) and the filamentous fungus Aspergillus nidulans. Like Spc110p and kendrin, Pcp1p and the A. nidulans protein contain predicted coiled-coil secondary structure and a COOH-terminal calmodulin-binding region. Green fluorescent protein fusions of Pcp1p localize to the SPB as analyzed by fluorescence and immunoelectron microscopy. Pcp1p overexpression causes chromosome missegregation, multiple mitotic spindle fragments, and multiple abnormal SPB-like structures, a phenotype remarkably similar to that of many human carcinoma lines, which exhibit chromosome and spindle defects, and supernumerary centrosomes.     

Dissociation of the Nuf2-Ndc80 complex releases centromeres from the spindle-pole body during meiotic prophase in fission yeast

In the fission yeast Schizosaccharomyces pombe, centromeres remain clustered at the spindle-pole body (SPB) during mitotic interphase. In contrast, during meiotic prophase centromeres dissociate from the SPB. Here we examined the behavior of centromere proteins in living meiotic cells of S. pombe. We show that the Nuf2-Ndc80 complex proteins (Nuf2, Ndc80, Spc24, and Spc25) disappear from the centromere in meiotic prophase when the centromeres are separated from the SPB. The centromere protein Mis12 also dissociates during meiotic prophase; however, Mis6 remains throughout meiosis. When cells are induced to meiosis by inactivation of Pat1 kinase (a key negative regulator of meiosis), centromeres remain associated with the SPB during meiotic prophase. However, inactivation of Nuf2 by a mutation causes the release of centromeres from the SPB in pat1 mutant cells, suggesting that the Nuf2-Ndc80 complex connects centromeres to the SPB. We further found that removal of the Nuf2-Ndc80 complex from the centromere and centromere-SPB dissociation are caused by mating pheromone signaling. Because pat1 mutant cells also show aberrant chromosome segregation in the first meiotic division and this aberration is compensated by mating pheromone signaling, dissociation of the Nuf2-Ndc80 complex may be associated with remodeling of the kinetochore for meiotic chromosome segregation.     

Spc24 interacts with Mps2 and is required for chromosome segregation, but is not implicated in spindle pole body duplication

Mps2 (monopolar spindle protein) is a coiled-coil protein found at the spindle pole body (SPB) and at the nuclear envelope that is required for insertion of the SPB into the nuclear envelope. We identified three proteins that interact with Mps2 in a two-hybrid screen: Bbp1, Ynl107w and Spc24. All three proteins contain coiled-coil motifs that appear to be required for their interaction with Mps2. In this work, we verified the Mps2-Spc24 interaction by co-immunoprecipitation in vivo and by the in vitro interaction of recombinant proteins. Previous two-hybrid screens with Spc24 as bait had identified Spc25 and Ndc80 as putative interacting partners, and we verified these interactions in vivo by purification of TAP-tagged derivatives of Spc24 and Ndc80. Finally, we found that spc24 thermosensitive mutants had a chromosome segregation defect, but no apparent defect in SPB duplication. These results are consistent with recently published data showing that Spc24, Spc25 and Ndc80 are peripheral kinetochore com-ponents required for chromosome segregation. The Mps2-Spc24 interaction may contribute to the localization of Spc24 and other kinetochore components to the inner plaque of the SPB.     

Interaction with calmodulin is required for the function of Spc110p, an essential component of the yeast spindle pole body.

NUF1/SPC110, encoding a nuclear filament-related protein which is a component of the yeast spindle pole body (SPB), has been identified in a screen designed to isolate genes encoding targets of yeast calmodulin. Spc110p interacts with calmodulin by two different criteria and the calmodulin interacting region has been localized within the C-terminus of the protein. Point mutations between residues 898 and 917 further define the calmodulin binding site within this region. Mutations in this domain which abolish calmodulin binding in vitro prevent Spc110p function in vivo, demonstrating that calmodulin binding by Spc110p has important functional consequences. In keeping with a role for calmodulin in Spc110p function, we show that calmodulin localizes to the yeast SPB when cells are prepared under appropriate conditions. Non-functional mutant Spc110 proteins which cannot bind calmodulin are present at lowered steady-state levels in the cell; when their level is increased by elevated gene dosage, partial recovery of Spc110p function is seen. Overexpression of calmodulin suppresses the defect(s) associated with the mutant Spc110 proteins, supporting the notion that Spc110p stability is a consequence of its ability to bind calmodulin and pointing to a direct role for calmodulin in Spc110p function.     

Spc42p: a phosphorylated component of the S. cerevisiae spindle pole body (SPD) with an essential function during SPB duplication

The 42-kD component of the S. cerevisiae spindle pole body (SPB) localizes to the electron-dense central plaque of the SPB. We have cloned the corresponding gene SPC42 (spindle pole component) and show that it is essential. Seven temperature-sensitive (ts) mutants in SPC42 were prepared by error-prone PCR. We found that a change to a proline residue in a potential coiled-coil region of Spc42p was responsible for the ts phenotype in at least three alleles, suggesting that formation of the coiled-coil is essential in normal function. The mutant cells showed a phenotype of predominantly single or bilobed SPBs often with an accumulation of unstructured electron-dense material associated with the bridge structure adjacent to the SPB. This phenotype suggests a defect in SPB duplication. This was confirmed by examining synchronized mutant cells that lose viability when SPB duplication is attempted. Spc42p is a phosphoprotein which shows some cell cycle-regulated phosphorylation. Overexpression of Spc42p causes the formation of a disc- or dome-shaped polymer composed of phosphorylated Spc42p, which is attached to the central plaque and associated with the outer nuclear membrane. Taken together, these data suggest that Spc42p forms a polymeric layer at the periphery of the SPB central plaque which has an essential function during SPB duplication and may facilitate attachment of the SPB to the nuclear membrane.     

Cnm67p is a spacer protein of the Saccharomyces cerevisiae spindle pole body outer plaque

In Saccharomyces cerevisiae, the spindle pole body (SPB) is the functional homolog of the mammalian centrosome, responsible for the organization of the tubulin cytoskeleton. Cytoplasmic (astral) microtubules essential for the proper segregation of the nucleus into the daughter cell are attached at the outer plaque on the SPB cytoplasmic face. Previously, it has been shown that Cnm67p is an integral component of this structure; cells deleted for CNM67 are lacking the SPB outer plaque and thus experience severe nuclear migration defects. With the use of partial deletion mutants of CNM67, we show that the N- and C-terminal domains of the protein are important for nuclear migration. The C terminus, not the N terminus, is essential for Cnm67p localization to the SPB. On the other hand, only the N terminus is subject to protein phosphorylation of a yet unknown function. Electron microscopy of SPB serial thin sections reveals that deletion of the N- or C-terminal domains disturbs outer plaque formation, whereas mutations in the central coiled-coil domain of Cnm67p change the distance between the SPB core and the outer plaque. We conclude that Cnm67p is the protein that connects the outer plaque to the central plaque embedded in the nuclear envelope, adjusting the space between them by the length of its coiled-coil.     

A novel mechanism of intragenic complementation between Phe to Ala calmodulin mutations

Calmodulin (CaM) performs essential functions in cell proliferation in Saccharomyces cerevisiae. Previously, we isolated fourteen temperature-sensitive Phe-to-Ala mutations of the CaM-encoding gene CMD1. These mutations were classified into four intragenic complementation groups, suggesting that each group represents a loss of CaM interaction with its specific essential target protein. Nuf1p/Spc110p, one of the essential targets, is a spindle pole body component that is required for proper mitosis. We investigated which intragenic complementation group of CaM represents the malfunction of Nuf1p. Immunoprecipitation analysis showed that two cmd1 mutations belonging to two distinct intragenic complementation groups had the most severely impaired complex formation with Nuf1p at the restrictive temperature. The temperature-sensitive growth of these cmd1 mutants was suppressed by a CaM-independent dominant allele of NUF1. Additionally, these mutants displayed characteristic mitotic defects: an increased ratio of artificial chromosome loss, which could be suppressed by the CaM-independent dominant allele of NUF1, and aberrant microtubule structures. These results indicate that these cmd1 mutants display the temperature-sensitive growth due to the compromised interaction with Nuf1p. However, the interaction was restored in a heterozygous diploid of the two cmd1 alleles, suggesting that intragenic complementation between these cmd1 alleles occurs by a novel mechanism, whereby co-presence of both mutant proteins rescues the interaction with Nuf1p.     

The essential mitotic target of calmodulin is the 110-kilodalton component of the spindle pole body in Saccharomyces cerevisiae.

Two independent methods identified the spindle pole body component Nuf1p/Spc110p as the essential mitotic target of calmodulin. Extragenic suppressors of cmd1-1 were isolated and found to define three loci, XCM1, XCM2, and XCM3 (extragenic suppressor of cmd1-1). The gene encoding a dominant suppressor allele of XCM1 was cloned. On the basis of DNA sequence analysis, genetic cosegregation, and mutational analysis, XCM1 was identified as NUF1/SPC110. Independently, a C-terminal portion of Nuf1p/Spc110p, amino acid residues 828 to 944, was isolated as a calmodulin-binding protein by the two-hybrid system. As assayed by the two-hybrid system, Nuf1p/Spc110p interacts with wild-type calmodulin and triple-mutant calmodulins defective in binding Ca2+ but not with two mutant calmodulins that confer a temperature-sensitive phenotype. Deletion analysis by the two-hybrid system mapped the calmodulin-binding site of Nuf1p/Spc110p to amino acid residues 900 to 927. Direct binding between calmodulin and Nuf1p/Spc110p was demonstrated by a modified gel overlay assay. Furthermore, indirect immunofluorescence with fixation procedures known to aid visualization of spindle pole body components localized calmodulin to the spindle pole body. Sequence analysis of five suppressor alleles of NUF1/SPC110 indicated that suppression of cmd1-1 occurs by C-terminal truncation of Nuf1p/Spc110p at amino acid residues 856, 863, or 881, thereby removing the calmodulin-binding site.     

Saccharomyces cerevisiae MPS2 encodes a membrane protein localized at the spindle pole body and the nuclear envelope.

The MPS2 (monopolar spindle two) gene is one of several genes required for the proper execution of spindle pole body (SPB) duplication in the budding yeast Saccharomyces cerevisiae (). We report here that the MPS2 gene encodes an essential 44-kDa protein with two putative coiled-coil regions and a hydrophobic sequence. Although MPS2 is required for normal mitotic growth, some null strains can survive; these survivors exhibit slow growth and abnormal ploidy. The MPS2 protein was tagged with nine copies of the myc epitope, and biochemical fractionation experiments show that it is an integral membrane protein. Visualization of a green fluorescent protein (GFP) Mps2p fusion protein in living cells and indirect immunofluorescence microscopy of 9xmyc-Mps2p revealed a perinuclear localization with one or two brighter foci of staining corresponding to the SPB. Additionally, immunoelectron microscopy shows that GFP-Mps2p localizes to the SPB. Our analysis suggests that Mps2p is required as a component of the SPB for insertion of the nascent SPB into the nuclear envelope.     

Spc110p: assembly properties and role in the connection of nuclear microtubules to the yeast spindle pole body.

Spc110p is an essential component of the budding yeast spindle pole body (SPB). It binds calmodulin and contains a long central coiled-coil rod which acts as a spacer element between the central plaque of the SPB and the ends of the nuclear or spindle microtubules. This suggests that the essential function of Spc110p is to connect the nuclear microtubules to the SPB. To confirm this, we examined the phenotype of ts alleles of SPC110, one of which contains a mutation in the calmodulin binding site and was suppressed by overexpression of calmodulin. The alleles fail to form a functional mitotic spindle because spindle microtubules are not properly connected to the SPB. We also examined the phenotype of the toxic overexpression of either the wild-type or a truncated version of Spc110p containing a deletion of most of the coiled-coil domain. Both of these proteins form large ordered spheroidal polymers in the nucleus. The polymerization of the truncated Spc110p appears to be initiated inside the SPB from the position where Spc110p is normally located, and as the polymer grows in size it severs the connection between the nuclear microtubules and the SPB. The polymers were purified and are composed of Spc110p and calmodulin. A model for the structure of the polymer is proposed.     

Spatiotemporal regulations of Wee1 at the G2/M transition

Wee1 is a protein kinase that negatively regulates mitotic entry in G2 phase by suppressing cyclin B-Cdc2 activity, but its spatiotemporal regulations remain to be elucidated. We observe the dynamic behavior of Wee1 in Schizosaccharomyces pombe cells and manipulate its localization and kinase activity to study its function. At late G2, nuclear Wee1 efficiently suppresses cyclin B-Cdc2 around the spindle pole body (SPB). During the G2/M transition when cyclin B-Cdc2 is highly enriched at the SPB, Wee1 temporally accumulates at the nuclear face of the SPB in a cyclin B-Cdc2-dependent manner and locally suppresses both cyclin B-Cdc2 activity and spindle assembly to counteract a Polo kinase-dependent positive feedback loop. Then Wee1 disappears from the SPB during spindle assembly. We propose that regulation of Wee1 localization around the SPB during the G2/M transition is important for proper mitotic entry and progression.     

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.     

Sli15 associates with the ipl1 protein kinase to promote proper chromosome segregation in Saccharomyces cerevisiae.

The conserved Ipl1 protein kinase is essential for proper chromosome segregation and thus cell viability in the budding yeast Saccharomyces cerevisiae. Its human homologue has been implicated in the tumorigenesis of diverse forms of cancer. We show here that sister chromatids that have separated from each other are not properly segregated to opposite poles of ipl1-2 cells. Failures in chromosome segregation are often associated with abnormal distribution of the spindle pole-associated Nuf2-GFP protein, thus suggesting a link between potential spindle pole defects and chromosome missegregation in ipl1 mutant cells. A small fraction of ipl1-2 cells also appears to be defective in nuclear migration or bipolar spindle formation. Ipl1 associates, probably directly, with the novel and essential Sli15 protein in vivo, and both proteins are localized to the mitotic spindle. Conditional sli15 mutant cells have cytological phenotypes very similar to those of ipl1 cells, and the ipl1-2 mutation exhibits synthetic lethal genetic interaction with sli15 mutations. sli15 mutant phenotype, like ipl1 mutant phenotype, is partially suppressed by perturbations that reduce protein phosphatase 1 function. These genetic and biochemical studies indicate that Sli15 associates with Ipl1 to promote its function in chromosome segregation.     

.用酵母双杂交系统筛选CENP-E相互作用蛋白

纺锤体检验点(spindle checkpoint)是一个重要的细胞分裂生化调节通路, 可监督染色体正确分离和传代．着丝粒相关蛋白E (centromere-associated protein E, CENP-E)是一个分子量为312 kD的微管马达驱动蛋白,可以衔接纺锤体微管与动点并参与纺锤体检验点调控．为研究CENP-E的作用机理,以其动点结合区域为诱饵蛋白,用酵母双杂交技术从人HeLa细胞 cDNA 文库中筛选出了Nuf2蛋白．体外的pull-down实验和体内的免疫共沉淀实验表明, Nuf2蛋白通过其卷曲螺旋(coiled-coil) 功能域特异结合CENP-E的 C 末端区域,间接免疫荧光显示Nuf2与CENP-E共定位于细胞有丝分裂期染色体的动点．由此推论, CENP-E 通过Nuf2的直接作用参与构筑动点-微管界面,进而参与细胞有丝分裂纺锤体检验点信号转导通路,为染色体正确分离发挥调控作用．     

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.     

Separate domains of KAR1 mediate distinct functions in mitosis and nuclear fusion

The yeast KAR1 gene is essential for mitotic growth and important for nuclear fusion. Mutations in KAR1 prevent duplication of the spindle pole body (SPB), and affect functions associated with both the nuclear and cytoplasmic microtubules. The localization of hybrid Kar1-lacZ proteins, described elsewhere (Vallen, E. A., T. Y. Scherson, T. Roberts, K. van Zee, and M. D. Rose. 1992. Cell. In press), suggest that the protein is associated with the SPB. In this paper, we report a deletion analysis demonstrating that the mitotic and karyogamy functions of KAR1 are separate and independent, residing in discrete functional domains. One region, here shown to be essential for mitosis, coincided with a part of the protein that is both necessary and sufficient to target Karl-lacZ hybrid proteins to the SPB (Vallen, E. A., T. Y. Scherson, T. Roberts, K. van Zee, and M. D. Rose. 1992. Cell. In press). Complementation testing demonstrated that deletions in this interval did not affect nuclear fusion. A second region, required only for karyogamy, was necessary for the localization of a Kar3-lacZ hybrid protein to the SPB. These data suggest a model for the roles of Kar1p and Kar3p, a kinesin-like protein, in nuclear fusion. Finally, a third region of KAR1 was found to be important for both mitosis and karyogamy. This domain included the hydrophobic carboxy terminus and is sufficient to target a lacZ-Kar1 hybrid protein to the nuclear envelope (Vallen E. A., T. Y. Scherson, T. Roberts, K. van Zee, and M. D. Rose. 1992. Cell. In press). Altogether, the essential mitotic regions of KAR1 comprised 20% of the coding sequence. We propose a model for Kar1p in which the protein is composed of several protein-binding domains tethered to the nuclear envelope via its hydrophobic tail.     

Nud1p links astral microtubule organization and the control of exit from mitosis

The budding yeast spindle pole body (SPB) not only organizes the astral and nuclear microtubules but is also associated with a number of cell-cycle regulators that control mitotic exit. Here, we describe that the core SPB component Nud1p is a key protein that functions in both processes. The astral microtubule organizing function of Nud1p is mediated by its interaction with the gamma-tubulin complex binding protein Spc72p. This function of Nud1p is distinct from its role in cell-cycle control: Nud1p binds the spindle checkpoint control proteins Bfa1p and Bub2p to the SPB, and is part of the mitotic exit network (MEN) in which it functions upstream of CDC15 but downstream of LTE1. In conditional lethal nud1-2 cells, the MEN component Tem1p, a GTPase, is mislocalized, whereas the kinase Cdc15p is still associated with the SPB. Thus, in nud1-2 cells the failure of Tem1p to interact with Cdc15p at the SPB probably prevents mitotic exit.     

Time-lapse video microscopy analysis reveals astral microtubule detachment in the yeast spindle pole mutant cnm67

Saccharomyces cerevisiae cnm67Delta cells lack the spindle pole body (SPB) outer plaque, the main attachment site for astral (cytoplasmic) microtubules, leading to frequent nuclear segregation failure. We monitored dynamics of green fluorescent protein-labeled nuclei and microtubules over several cell cycles. Early nuclear migration steps such as nuclear positioning and spindle orientation were slightly affected, but late phases such as rapid oscillations and insertion of the anaphase nucleus into the bud neck were mostly absent. Analyzes of microtubule dynamics revealed normal behavior of the nuclear spindle but frequent detachment of astral microtubules after SPB separation. Concomitantly, Spc72 protein, the cytoplasmic anchor for the gamma-tubulin complex, was partially lost from the SPB region with dynamics similar to those observed for microtubules. We postulate that in cnm67Delta cells Spc72-gamma-tubulin complex-capped astral microtubules are released from the half-bridge upon SPB separation but fail to be anchored to the cytoplasmic side of the SPB because of the absence of an outer plaque. However, successful nuclear segregation in cnm67Delta cells can still be achieved by elongation forces of spindles that were correctly oriented before astral microtubule detachment by action of Kip3/Kar3 motors. Interestingly, the first nuclear segregation in newborn diploid cells never fails, even though astral microtubule detachment occurs.