scholarly journals Sid2p, a Spindle Pole Body Kinase That Regulates the Onset of Cytokinesis

1999 ◽  
Vol 146 (4) ◽  
pp. 777-790 ◽  
Author(s):  
Cynthia A. Sparks ◽  
Mary Morphew ◽  
Dannel McCollum
Keyword(s):  
Cell Division ◽  
Kinase Activity ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Vitro Assay ◽  
Daughter Cells ◽  
Division Site ◽  
Pole Body ◽  
Ring Constriction

The fission yeast Schizosaccharomyces pombe divides by medial fission through the use of an actomyosin contractile ring. Precisely at the end of anaphase, the ring begins to constrict and the septum forms. Proper coordination of cell division with mitosis is crucial to ensure proper segregation of chromosomes to daughter cells. The Sid2p kinase is one of several proteins that function as part of a novel signaling pathway required for initiation of medial ring constriction and septation. Here, we show that Sid2p is a component of the spindle pole body at all stages of the cell cycle and localizes transiently to the cell division site during medial ring constriction and septation. A medial ring and an intact microtubule cytoskeleton are required for the localization of Sid2p to the division site. We have established an in vitro assay for measuring Sid2p kinase activity, and found that Sid2p kinase activity peaks during medial ring constriction and septation. Both Sid2p localization to the division site and activity depend on the function of all of the other septation initiation genes: cdc7, cdc11, cdc14, sid1, spg1, and sid4. Thus, Sid2p, a component of the spindle pole body, by virtue of its transient localization to the division site, appears to determine the timing of ring constriction and septum delivery in response to activating signals from other Sid gene products.

scholarly journals The Cdc31p-binding protein Kar1p is a component of the half bridge of the yeast spindle pole body.

1995 ◽  
Vol 128 (5) ◽  
pp. 863-877 ◽  
Author(s):  
A Spang ◽  
I Courtney ◽  
K Grein ◽  
M Matzner ◽  
E Schiebel
Keyword(s):  
Cell Cycle ◽  
Essential Gene ◽  
Binding Protein ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Yeast Cells ◽  
Vitro Assay ◽  
Pole Body ◽  
Cell Biol

KAR1 has been identified as an essential gene which is involved in karyogamy of mating yeast cells and in spindle pole body duplication of mitotic cells (Rose, M. D., and G. R. Fink. 1987. Cell. 48:1047-1060). We investigated the cell cycle-dependent localization of the Kar1 protein (Kar1p) and its interaction with other SPB components. Kar1p is associated with the spindle pole body during the entire cell cycle of yeast. Immunoelectron microscopic studies with anti-Kar1p antibodies or with the monoclonal antibody 12CA5 using an epitope-tagged, functional Kar1p revealed that Kar1p is associated with the half bridge or the bridge of the spindle pole body. Cdc31p, a Ca(2+)-binding protein, was previously identified as the first component of the half bridge of the spindle pole body (Spang, A., I. Courtney, U. Fackler, M. Matzner, and E. Schiebel. 1993. J. Cell Biol. 123:405-416). Using an in vitro assay we demonstrate that Cdc31p specifically interacts with a short sequence within the carboxyl terminal half of Kar1p. The potential Cdc31p-binding sequence of Kar1p contains three acidic amino acids which are not found in calmodulin-binding peptides, explaining the different substrate specificities of Cdc31p and calmodulin. Cdc31p was also able to bind to the carboxy terminus of Nuflp/Spc110p, another component of the SPB (Kilmartin, J. V., S. L. Dyos, D. Kershaw, and J. T. Finch. 1993. J. Cell Biol. 123:1175-1184). The association of Kar1p with the spindle pole body was independent of Cdc31p. Cdc31p, on the other hand, was not associated with SPBs of kar1 cells.

scholarly journals Stu2p: A Microtubule-Binding Protein that Is an Essential Component of the Yeast Spindle Pole Body

1997 ◽  
Vol 139 (5) ◽  
pp. 1271-1280 ◽  
Author(s):  
Peijing Jeremy Wang ◽  
Tim C. Huffaker
Keyword(s):  
Essential Gene ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Cold Sensitivity ◽  
Gene Encoding ◽  
Microtubule Binding ◽  
Amino Acid Region ◽  
Pole Body ◽  
Spindle Function

Previously we isolated tub2-423, a cold-sensitive allele of the Saccharomyces cerevisiae gene encoding β-tubulin that confers a defect in mitotic spindle function. In an attempt to identify additional proteins that are important for spindle function, we screened for suppressors of the cold sensitivity of tub2-423 and obtained two alleles of a novel gene, STU2. STU2 is an essential gene and encodes a protein whose sequence is similar to proteins identified in a variety of organisms. Stu2p localizes primarily to the spindle pole body (SPB) and to a lesser extent along spindle microtubules. Localization to the SPB is not dependent on the presence of microtubules, indicating that Stu2p is an integral component of the SPB. Stu2p also binds microtubules in vitro. We have localized the microtubule-binding domain of Stu2p to a highly basic 100-amino acid region. This region contains two imperfect repeats; both repeats appear to contribute to microtubule binding to similar extents. These results suggest that Stu2p may play a role in the attachment, organization, and/or dynamics of microtubule ends at the SPB.

scholarly journals Identification of Ribonucleotide Reductase Protein R1 as an Activator of Microtubule Nucleation in Xenopus Egg Mitotic Extracts

2000 ◽  
Vol 11 (12) ◽  
pp. 4173-4187 ◽  
Author(s):  
Saeko Takada ◽  
Takehiko Shibata ◽  
Yasushi Hiraoka ◽  
Hirohisa Masuda
Keyword(s):  
Ribonucleotide Reductase ◽  
Large Subunit ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Microtubule Nucleation ◽  
Pole Body ◽  
Recombinant Mouse ◽  
Xenopus Egg ◽  
Essential Subunit

Microtubule nucleation on the centrosome and the fungal equivalent, the spindle pole body (SPB), is activated at the onset of mitosis. We previously reported that mitotic extracts prepared fromXenopus unfertilized eggs convert the interphase SPB of fission yeast into a competent state for microtubule nucleation. In this study, we have purified an 85-kDa SPB activator from the extracts and identified it as the ribonucleotide reductase large subunit R1. We further confirmed that recombinant mouse R1 protein was also effective for SPB activation. On the other hand, another essential subunit of ribonucleotide reductase, R2 protein, was not required for SPB activation. SPB activation by R1 protein was suppressed in the presence of anti-R1 antibodies or a partial oligopeptide of R1; the oligopeptide also inhibited aster formation on Xenopussperm centrosomes. In accordance, R1 was detected in animal centrosomes by immunofluorescence and immunoblotting with anti-R1 antibodies. In addition, recombinant mouse R1 protein bound to γ- and α/β-tubulin in vitro. These results suggest that R1 is a bifunctional protein that acts on both ribonucleotide reduction and centrosome/SPB activation.

scholarly journals The cortical protein Lte1 promotes mitotic exit by inhibiting the spindle position checkpoint kinase Kin4

2011 ◽  
Vol 193 (6) ◽  
pp. 1033-1048 ◽  
Author(s):  
Daniela Trinca Bertazzi ◽  
Bahtiyar Kurtulmus ◽  
Gislene Pereira
Keyword(s):  
Kinase Activity ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Mitotic Exit ◽  
Cell Protein ◽  
Cell Axis ◽  
Pole Body ◽  
Catalytically Active ◽  
Surveillance Mechanism ◽  
Spindle Position

The spindle position checkpoint (SPOC) is an essential surveillance mechanism that allows mitotic exit only when the spindle is correctly oriented along the cell axis. Key SPOC components are the kinase Kin4 and the Bub2–Bfa1 GAP complex that inhibit the mitotic exit–promoting GTPase Tem1. During an unperturbed cell cycle, Kin4 associates with the mother spindle pole body (mSPB), whereas Bub2–Bfa1 is at the daughter SPB (dSPB). When the spindle is mispositioned, Bub2–Bfa1 and Kin4 bind to both SPBs, which enables Kin4 to phosphorylate Bfa1 and thereby block mitotic exit. Here, we show that the daughter cell protein Lte1 physically interacts with Kin4 and inhibits Kin4 kinase activity. Specifically, Lte1 binds to catalytically active Kin4 and promotes Kin4 hyperphosphorylation, which restricts Kin4 binding to the mSPB. This Lte1-mediated exclusion of Kin4 from the dSPB is essential for proper mitotic exit of cells with a correctly aligned spindle. Therefore, Lte1 promotes mitotic exit by inhibiting Kin4 activity at the dSPB.

scholarly journals Role of Septins in the Orientation of Forespore Membrane Extension during Sporulation in Fission Yeast

2010 ◽  
Vol 30 (8) ◽  
pp. 2057-2074 ◽  
Author(s):  
Masayuki Onishi ◽  
Takako Koga ◽  
Aiko Hirata ◽  
Taro Nakamura ◽  
Haruhiko Asakawa ◽  
...  
Keyword(s):  
Spindle Pole Body ◽  
Leading Edge ◽  
Spindle Pole ◽  
Pole Body ◽  
Membrane Extension ◽  
Fusion Site ◽  
Phosphatidylinositol 4 ◽  
Ring Shaped Structure

ABSTRACT During yeast sporulation, a forespore membrane (FSM) initiates at each spindle-pole body and extends to form the spore envelope. We used Schizosaccharomyces pombe to investigate the role of septins during this process. During the prior conjugation of haploid cells, the four vegetatively expressed septins (Spn1, Spn2, Spn3, and Spn4) coassemble at the fusion site and are necessary for its normal morphogenesis. Sporulation involves a different set of four septins (Spn2, Spn5, Spn6, and the atypical Spn7) that does not include the core subunits of the vegetative septin complex. The four sporulation septins form a complex in vitro and colocalize interdependently to a ring-shaped structure along each FSM, and septin mutations result in disoriented FSM extension. The septins and the leading-edge proteins appear to function in parallel to orient FSM extension. Spn2 and Spn7 bind to phosphatidylinositol 4-phosphate [PtdIns(4)P] in vitro, and PtdIns(4)P is enriched in the FSMs, suggesting that septins bind to the FSMs via this lipid. Cells expressing a mutant Spn2 protein unable to bind PtdIns(4)P still form extended septin structures, but these structures fail to associate with the FSMs, which are frequently disoriented. Thus, septins appear to form a scaffold that helps to guide the oriented extension of the FSM.

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

2014 ◽  
Vol 25 (18) ◽  
pp. 2735-2749 ◽  
Author(s):  
I-Ju Lee ◽  
Ning Wang ◽  
Wen Hu ◽  
Kersey Schott ◽  
Jürg Bähler ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Fission Yeast ◽  
Budding Yeast ◽  
Binding Protein ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Centrosome Duplication ◽  
Pole Body ◽  
The Individual

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.

scholarly journals Pachytene arrest and other meiotic effects of the start mutations in Saccharomyces cerevisiae.

Genetics ◽  
1989 ◽  
Vol 123 (1) ◽  
pp. 29-43 ◽  
Author(s):  
E O Shuster ◽  
B Byers
Keyword(s):  
Cell Division ◽  
Nuclear Division ◽  
Spindle Pole Body ◽  
Mitotic Cell ◽  
Spindle Pole ◽  
Mitotic Cell Cycle ◽  
Chromosomal Dna ◽  
Pole Body ◽  
Vegetative Cycle

Abstract Mutations in the Start class of cell division cycle genes (CDC28, CDC36 and CDC39) define the point in the G1 phase of the vegetative cycle at which the cell becomes committed to completing another round of cell division. Genetic, cytological and biochemical data demonstrate that these mutations cause meiotic cells to become arrested at pachytene following completion of both chromosomal DNA replication and spindle pole body (SPB) duplication. In contrast these mutations have previously been found to cause arrest of the mitotic cell cycle prior to either of these landmark events, so the role of the Start genes in these events during vegetative growth must be indirect. Our observations are consistent with the hypothesis that CDC28, CDC36 and CDC39 are required for irreversible commitment to nuclear division in both the mitotic and meiotic pathways. CDC28 was additionally found to be required for the SPB separation that precedes spindle formation in preparation for the second meiotic division. Cytological and genetic analyses of this requirement revealed both that such separation may fail independently at either SPB and that ascospore formation can proceed independently of SPB separation.

scholarly journals Relief of the Dma1-mediated checkpoint requires Dma1 autoubiquitination and dynamic localization

2018 ◽  
Vol 29 (18) ◽  
pp. 2176-2189 ◽  
Author(s):  
Christine M. Jones ◽  
Jun-Song Chen ◽  
Alyssa E. Johnson ◽  
Zachary C. Elmore ◽  
Sierra N. Cullati ◽  
...  
Keyword(s):  
Cell Division ◽  
Chromosome Segregation ◽  
Ubiquitin Ligase ◽  
Spindle Pole Body ◽  
Mitotic Checkpoint ◽  
Spindle Pole ◽  
Checkpoint Activation ◽  
Pole Body ◽  
Septation Initiation Network ◽  
Anaphase B

Chromosome segregation and cell division are coupled to prevent aneuploidy and cell death. In the fission yeast Schizosaccharomyces pombe, the septation initiation network (SIN) promotes cytokinesis, but upon mitotic checkpoint activation, the SIN is actively inhibited to prevent cytokinesis from occurring before chromosomes have safely segregated. SIN inhibition during the mitotic checkpoint is mediated by the E3 ubiquitin ligase Dma1. Dma1 binds to the CK1-phosphorylated SIN scaffold protein Sid4 at the spindle pole body (SPB), and ubiquitinates it. Sid4 ubiquitination antagonizes the SPB localization of the Pololike kinase Plo1, the major SIN activator, so that SIN signaling is delayed. How this checkpoint is silenced once spindle defects are resolved has not been clear. Here we establish that Dma1 transiently leaves SPBs during anaphase B due to extensive autoubiquitination. The SIN is required for Dma1 to return to SPBs later in anaphase. Blocking Dma1 removal from SPBs by permanently tethering it to Sid4 prevents SIN activation and cytokinesis. Therefore, controlling Dma1’s SPB dynamics in anaphase is an essential step in S. pombe cell division and the silencing of the Dma1-dependent mitotic checkpoint.

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

2002 ◽  
Vol 156 (3) ◽  
pp. 453-465 ◽  
Author(s):  
Andrea R. Castillo ◽  
Janet B. Meehl ◽  
Garry Morgan ◽  
Amy Schutz-Geschwender ◽  
Mark Winey
Keyword(s):  
Protein Kinase ◽  
Spindle Pole Body ◽  
Spindle Checkpoint ◽  
Spindle Pole ◽  
Physical Interaction ◽  
Gene Encoding ◽  
Pole Body ◽  
Conditional Allele

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.

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