scholarly journals Key phosphorylation events in Spc29 and Spc42 guide multiple steps of yeast centrosome duplication

2018 ◽  
Vol 29 (19) ◽  
pp. 2280-2291 ◽  
Author(s):  
Michele Haltiner Jones ◽  
Eileen T. O’Toole ◽  
Amy S. Fabritius ◽  
Eric G. Muller ◽  
Janet B. Meehl ◽  
...  
Keyword(s):  
Cell Cycle Progression ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Centrosome Duplication ◽  
Phosphorylation Sites ◽  
Cycle Progression ◽  
Cellular Processes ◽  
Pole Body ◽  
Core Components ◽  
Spindle Elongation

Phosphorylation modulates many cellular processes during cell cycle progression. The yeast centrosome (called the spindle pole body, SPB) is regulated by the protein kinases Mps1 and Cdc28/Cdk1 as it nucleates microtubules to separate chromosomes during mitosis. Previously we completed an SPB phosphoproteome, identifying 297 sites on 17 of the 18 SPB components. Here we describe mutagenic analysis of phosphorylation events on Spc29 and Spc42, two SPB core components that were shown in the phosphoproteome to be heavily phosphorylated. Mutagenesis at multiple sites in Spc29 and Spc42 suggests that much of the phosphorylation on these two proteins is not essential but enhances several steps of mitosis. Of the 65 sites examined on both proteins, phosphorylation of the Mps1 sites Spc29-T18 and Spc29-T240 was shown to be critical for function. Interestingly, these two sites primarily influence distinct successive steps; Spc29-T240 is important for the interaction of Spc29 with Spc42, likely during satellite formation, and Spc29-T18 facilitates insertion of the new SPB into the nuclear envelope and promotes anaphase spindle elongation. Phosphorylation sites within Cdk1 motifs affect function to varying degrees, but mutations only have significant effects in the presence of an MPS1 mutation, supporting a theme of coregulation by these two kinases.

scholarly journals A microtubule polymerase cooperates with the kinesin-6 motor and a microtubule cross-linker to promote bipolar spindle assembly in the absence of kinesin-5 and kinesin-14 in fission yeast

2017 ◽  
Vol 28 (25) ◽  
pp. 3647-3659 ◽  
Author(s):  
Masashi Yukawa ◽  
Tomoki Kawakami ◽  
Masaki Okazaki ◽  
Kazunori Kume ◽  
Ngang Heok Tang ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Chromosome Segregation ◽  
Spindle Pole Body ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Bipolar Spindle ◽  
Pole Body ◽  
Cross Linker ◽  
Spindle Elongation

Accurate chromosome segregation relies on the bipolar mitotic spindle. In many eukaryotes, spindle formation is driven by the plus-end–directed motor kinesin-5 that generates outward force to establish spindle bipolarity. Its inhibition leads to the emergence of monopolar spindles with mitotic arrest. Intriguingly, simultaneous inactivation of the minus-end–directed motor kinesin-14 restores spindle bipolarity in many systems. Here we show that in fission yeast, three independent pathways contribute to spindle bipolarity in the absence of kinesin-5/Cut7 and kinesin-14/Pkl1. One is kinesin-6/Klp9 that engages with spindle elongation once short bipolar spindles assemble. Klp9 also ensures the medial positioning of anaphase spindles to prevent unequal chromosome segregation. Another is the Alp7/TACC-Alp14/TOG microtubule polymerase complex. Temperature-sensitive alp7cut7pkl1 mutants are arrested with either monopolar or very short spindles. Forced targeting of Alp14 to the spindle pole body is sufficient to render alp7cut7pkl1 triply deleted cells viable and promote spindle assembly, indicating that Alp14-mediated microtubule polymerization from the nuclear face of the spindle pole body could generate outward force in place of Cut7 during early mitosis. The third pathway involves the Ase1/PRC1 microtubule cross-linker that stabilizes antiparallel microtubules. Our study, therefore, unveils multifaceted interplay among kinesin-dependent and -independent pathways leading to mitotic bipolar spindle assembly.

scholarly journals Budding Yeast Bub2 Is Localized at Spindle Pole Bodies and Activates the Mitotic Checkpoint via a Different Pathway from Mad2

1999 ◽  
Vol 145 (5) ◽  
pp. 979-991 ◽  
Author(s):  
Roberta Fraschini ◽  
Elisa Formenti ◽  
Giovanna Lucchini ◽  
Simonetta Piatti
Keyword(s):  
Cell Cycle ◽  
Mitotic Spindle ◽  
Cell Cycle Progression ◽  
Budding Yeast ◽  
Spindle Pole Body ◽  
Mitotic Checkpoint ◽  
Spindle Pole ◽  
Cycle Progression ◽  
Spindle Pole Bodies

The mitotic checkpoint blocks cell cycle progression before anaphase in case of mistakes in the alignment of chromosomes on the mitotic spindle. In budding yeast, the Mad1, 2, 3, and Bub1, 2, 3 proteins mediate this arrest. Vertebrate homologues of Mad1, 2, 3, and Bub1, 3 bind to unattached kinetochores and prevent progression through mitosis by inhibiting Cdc20/APC-mediated proteolysis of anaphase inhibitors, like Pds1 and B-type cyclins. We investigated the role of Bub2 in budding yeast mitotic checkpoint. The following observations indicate that Bub2 and Mad1, 2 probably activate the checkpoint via different pathways: (a) unlike the other Mad and Bub proteins, Bub2 localizes at the spindle pole body (SPB) throughout the cell cycle; (b) the effect of concomitant lack of Mad1 or Mad2 and Bub2 is additive, since nocodazole-treated mad1 bub2 and mad2 bub2 double mutants rereplicate DNA more rapidly and efficiently than either single mutant; (c) cell cycle progression of bub2 cells in the presence of nocodazole requires the Cdc26 APC subunit, which, conversely, is not required for mad2 cells in the same conditions. Altogether, our data suggest that activation of the mitotic checkpoint blocks progression through mitosis by independent and partially redundant mechanisms.

scholarly journals Localization of the Kar3 kinesin heavy chain-related protein requires the Cik1 interacting protein

1994 ◽  
Vol 124 (4) ◽  
pp. 507-519 ◽  
Author(s):  
BD Page ◽  
LL Satterwhite ◽  
MD Rose ◽  
M Snyder
Keyword(s):  
Heavy Chain ◽  
Fusion Proteins ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Related Protein ◽  
Phenotypic Analysis ◽  
Interacting Protein ◽  
Cellular Processes ◽  
Pole Body ◽  
Kinesin Heavy Chain

The Kar3 protein (Kar3p), a protein related to kinesin heavy chain, and the Cik1 protein (Cik1p) appear to participate in the same cellular processes in S. cerevisiae. Phenotypic analysis of mutants indicates that both CIK1 and KAR3 participate in spindle formation and karyogamy. In addition, the expression of both genes is induced by pheromone treatment. In vegetatively growing cells, both Cik1::beta-gal and Kar3::beta-gal fusions localize to the spindle pole body (SPB), and after pheromone treatment both fusion proteins localize to the spindle pole body and cytoplasmic microtubules. The dependence of Cik1p and Kar3p localization upon one another was investigated by indirect immunofluorescence of fusion proteins in pheromone-treated cells. The Cik1p::beta-gal fusion does not localize to the SPB or microtubules in a kar3 delta strain, and the Kar3p::beta-gal fusion protein does not localize to microtubule-associated structures in a cik1 delta strain. Thus, these proteins appear to be interdependent for localization to the SPB and microtubules. Analysis by both the two-hybrid system and co-immunoprecipitation experiments indicates that Cik1p and kar3p interact, suggesting that they are part of the same protein complex. These data indicate that interaction between a putative kinesin heavy chain-related protein and another protein can determine the localization of motor activity and thereby affect the functional specificity of the motor complex.

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 Proper timing of cytokinesis is regulated by Schizosaccharomyces pombe Etd1

2009 ◽  
Vol 186 (5) ◽  
pp. 739-753 ◽  
Author(s):  
Juan Carlos García-Cortés ◽  
Dannel McCollum
Keyword(s):  
Fission Yeast ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Yeast Protein ◽  
Late Anaphase ◽  
Pole Body ◽  
Spindle Elongation ◽  
Cell Asymmetry ◽  
Guanosine Triphosphatase ◽  
Asymmetric Activation

Cytokinesis must be initiated only after chromosomes have been segregated in anaphase and must be terminated once cleavage is completed. We show that the fission yeast protein Etd1 plays a central role in both of these processes. Etd1 activates the guanosine triphosphatase (GTPase) Spg1 to trigger signaling through the septum initiation network (SIN) pathway and onset of cytokinesis. Spg1 is activated in late anaphase when spindle elongation brings spindle pole body (SPB)–localized Spg1 into proximity with its activator Etd1 at cell tips, ensuring that cytokinesis is only initiated when the spindle is fully elongated. Spg1 is active at just one of the two SPBs during cytokinesis. When the actomyosin ring finishes constriction, the SIN triggers disappearance of Etd1 from the half of the cell with active Spg1, which then triggers Spg1 inactivation. Asymmetric activation of Spg1 is crucial for timely inactivation of the SIN. Together, these results suggest a mechanism whereby cell asymmetry is used to monitor cytoplasmic partitioning to turn off cytokinesis signaling.

scholarly journals HIV-1 Vpr Induces Defects in Mitosis, Cytokinesis, Nuclear Structure, and Centrosomes

2004 ◽  
Vol 15 (4) ◽  
pp. 1793-1801 ◽  
Author(s):  
Fred Chang ◽  
Fabio Re ◽  
Sarah Sebastian ◽  
Shelley Sazer ◽  
Jeremy Luban
Keyword(s):  
Fission Yeast ◽  
Cell Cycle Progression ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Human Cells ◽  
Yeast Cells ◽  
M Cell ◽  
Pole Body ◽  
And Function ◽  
Hiv 1

Human immunodeficiency virus type 1 (HIV-1) Vpr is a 15-kDa accessory protein that contributes to several steps in the viral replication cycle and promotes virus-associated pathology. Previous studies demonstrated that Vpr inhibits G2/M cell cycle progression in both human cells and in the fission yeast Schizosaccharomyces pombe. Here, we report that, upon induction of vpr expression, fission yeast exhibited numerous defects in the assembly and function of the mitotic spindle. In particular, two spindle pole body proteins, sad1p and the polo kinase plo1p, were delocalized in vpr-expressing yeast cells, suggesting that spindle pole body integrity was perturbed. In addition, nuclear envelope structure, contractile actin ring formation, and cytokinesis were also disrupted. Similar Vpr-induced defects in mitosis and cytokinesis were observed in human cells, including aberrant mitotic spindles, multiple centrosomes, and multinucleate cells. These defects in cell division and centrosomes might account for some of the pathological effects associated with HIV-1 infection.

scholarly journals Direct experimental evidence for the existence, structural basis and function of astral forces during anaphase B in vivo

1991 ◽  
Vol 100 (2) ◽  
pp. 279-288 ◽  
Author(s):  
J.R. Aist ◽  
C.J. Bayles ◽  
W. Tao ◽  
M.W. Berns
Keyword(s):  
Critical Mass ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Structural Basis ◽  
Mitotic Apparatus ◽  
Pole Body ◽  
Spindle Microtubules ◽  
Spindle Elongation ◽  
And Function ◽  
Anaphase B

The existence, structural basis and function of astral forces that are active during anaphase B in the fungus, Nectria haematococca, were revealed by experiments performed on living cells. When one of the two asters of a mitotic apparatus was damaged, the entire mitotic apparatus migrated rapidly in the direction of the opposing astral forces, showing that the force that accelerated spindle pole body separation in earlier experiments is located in the asters. When a strong solution of the antimicrotubule drug, MBC, was applied at anaphase A, tubulin immunocytochemistry showed that both astral and spindle microtubules were destroyed completely in less than a minute. As a result, separation of the spindle pole bodies during anaphase B almost stopped. By contrast, disrupting only the spindle microtubules with a laser microbeam increased the rate of spindle pole body separation more than fourfold. Taken together, these two experiments show that the astral forces are microtubule-dependent. When only one of the two or three bundles of spindle microtubules was broken at very early anaphase B, most such diminished spindles elongated at a normal rate, whereas others elongated at an increased rate. This result suggests that only a critical mass or number of spindle microtubules needs be present for the rate of spindle elongation to be fully governed, and that astral forces can accelerate the elongation of a weakened or diminished spindle.

scholarly journals Fission yeast MOZART1/Mzt1 is an essential γ-tubulin complex component required for complex recruitment to the microtubule organizing center, but not its assembly

2013 ◽  
Vol 24 (18) ◽  
pp. 2894-2906 ◽  
Author(s):  
Hirohisa Masuda ◽  
Risa Mori ◽  
Masashi Yukawa ◽  
Takashi Toda
Keyword(s):  
Fission Yeast ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Microtubule Organizing Center ◽  
Microtubule Organization ◽  
Unique Role ◽  
Pole Body ◽  
Organizing Center ◽  
Core Components

γ-Tubulin plays a universal role in microtubule nucleation from microtubule organizing centers (MTOCs) such as the animal centrosome and fungal spindle pole body (SPB). γ-Tubulin functions as a multiprotein complex called the γ-tubulin complex (γ-TuC), consisting of GCP1–6 (GCP1 is γ-tubulin). In fungi and flies, it has been shown that GCP1–3 are core components, as they are indispensable for γ-TuC complex assembly and cell division, whereas the other three GCPs are not. Recently a novel conserved component, MOZART1, was identified in humans and plants, but its precise functions remain to be determined. In this paper, we characterize the fission yeast homologue Mzt1, showing that it is essential for cell viability. Mzt1 is present in approximately equal stoichiometry with Alp4/GCP2 and localizes to all the MTOCs, including the SPB and interphase and equatorial MTOCs. Temperature-sensitive mzt1 mutants display varying degrees of compromised microtubule organization, exhibiting multiple defects during both interphase and mitosis. Mzt1 is required for γ-TuC recruitment, but not sufficient to localize to the SPB, which depends on γ-TuC integrity. Intriguingly, the core γ-TuC assembles in the absence of Mzt1. Mzt1 therefore plays a unique role within the γ-TuC components in attachment of this complex to the major MTOC site.

Mitosis and septum formation in the basidiomycete Helicobasidium mompa

1986 ◽  
Vol 64 (1) ◽  
pp. 130-145 ◽  
Author(s):  
Timothy M. Bourett ◽  
David J. McLaughlin
Keyword(s):  
Endoplasmic Reticulum ◽  
Functional Significance ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Multivesicular Bodies ◽  
Spindle Poles ◽  
Astral Microtubule ◽  
Pole Body ◽  
Helicobasidium Mompa ◽  
Spindle Elongation

Mitosis in clampless, dikaryotic hyphae of Helicobasidium mompa (Basidiomycota, Auriculariales sensu lato) was studied in apical and penultimate cells by correlating light microscopic and ultrastructural observations. Mitosis lasts about 10.5 min. In penultimate cells, mitosis occurs in the base of a branch whose initiation involves rupture of the wall. The extranuclear interphase spindle pole body contains two three-layered discs. Prophase is discerned by the polarization of the nucleus into a karyokinetic and a nucleolar region. During prometaphase, the spindle pole body discs move into the plane of the nuclear envelope where they occupy gaps. The spindle pole is enclosed by a cap of endoplasmic reticulum. At metaphase, nuclei lie side by side, the nucleolus resides in a nuclear evagination, and the spindle pole body discs are five layered. At anaphase, both chromatin to pole movement and extensive spindle elongation occur, astral microtubule populations reach a maximum, and multivesicular bodies aggregate at the spindle poles. Septa contain simple pores and form at the site previously occupied by the dividing nuclei. The results are compared with mitotic cycles in higher fungi and their evolutionary, phylogenetic, and functional significance is discussed.

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