scholarly journals Constitutive dynein activity inshe1mutants reveals differences in microtubule attachment at the yeast spindle pole body

2012 ◽  
Vol 23 (12) ◽  
pp. 2319-2326 ◽  
Author(s):  
Zane J. Bergman ◽  
Xue Xia ◽  
I. Alexandra Amaro ◽  
Tim C. Huffaker
Keyword(s):  
Spindle Pole Body ◽  
Spindle Pole ◽  
Microtubule Assembly ◽  
Microtubule Organizing Center ◽  
Microtubule Attachment ◽  
Pole Body ◽  
Organizing Center ◽  
Cytoplasmic Microtubules ◽  
G1 Cells

The organization of microtubules is determined in most cells by a microtubule-organizing center, which nucleates microtubule assembly and anchors their minus ends. In Saccharomyces cerevisiae cells lacking She1, cytoplasmic microtubules detach from the spindle pole body at high rates. Increased rates of detachment depend on dynein activity, supporting previous evidence that She1 inhibits dynein. Detachment rates are higher in G1 than in metaphase cells, and we show that this is primarily due to differences in the strengths of microtubule attachment to the spindle pole body during these stages of the cell cycle. The minus ends of detached microtubules are stabilized by the presence of γ-tubulin and Spc72, a protein that tethers the γ-tubulin complex to the spindle pole body. A Spc72–Kar1 fusion protein suppresses detachment in G1 cells, indicating that the interaction between these two proteins is critical to microtubule anchoring. Overexpression of She1 inhibits the loading of dynactin components, but not dynein, onto microtubule plus ends. In addition, She1 binds directly to microtubules in vitro, so it may compete with dynactin for access to microtubules. Overall, these results indicate that inhibition of dynein activity by She1 is important to prevent excessive detachment of cytoplasmic microtubules, particularly in G1 cells.

scholarly journals The Yeast Spindle Pole Body Component Spc72p Interacts with Stu2p and Is Required for Proper Microtubule Assembly

1998 ◽  
Vol 141 (5) ◽  
pp. 1169-1179 ◽  
Author(s):  
Xiaoyue Peter Chen ◽  
Hongwei Yin ◽  
Tim C. Huffaker
Keyword(s):  
Chromosome Segregation ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Microtubule Assembly ◽  
Temperature Sensitive ◽  
Pole Body ◽  
Cytoplasmic Microtubules ◽  
Spindle Elongation

We have previously shown that Stu2p is a microtubule-binding protein and a component of the Saccharomyces cerevisiae spindle pole body (SPB). Here we report the identification of Spc72p, a protein that interacts with Stu2p. Stu2p and Spc72p associate in the two-hybrid system and can be coimmunoprecipitated from yeast extracts. Stu2p and Spc72p also interact with themselves, suggesting the possibility of a multimeric Stu2p-Spc72p complex. Spc72p is an essential component of the SPB and is able to associate with a preexisting SPB, indicating that there is a dynamic exchange between soluble and SPB forms of Spc72p. Unlike Stu2p, Spc72p does not bind microtubules in vitro, and was not observed to localize along microtubules in vivo. A temperature-sensitive spc72 mutation causes defects in SPB morphology. In addition, most spc72 mutant cells lack cytoplasmic microtubules; the few cytoplasmic microtubules that are observed are excessively long, and some of these are unattached to the SPB. spc72 cells are able to duplicate and separate their SPBs to form a bipolar spindle, but spindle elongation and chromosome segregation rarely occur. The chromosome segregation block does not arrest the cell cycle; instead, spc72 cells undergo cytokinesis, producing aploid cells and polyploid cells that contain multiple SPBs.

scholarly journals Analysis of a Spindle Pole Body Mutant Reveals a Defect in Biorientation and Illuminates Spindle Forces

2005 ◽  
Vol 16 (1) ◽  
pp. 141-152 ◽  
Author(s):  
Tennessee J. Yoder ◽  
Mark A. McElwain ◽  
Susan E. Francis ◽  
Joy Bagley ◽  
Eric G.D. Muller ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Spindle Pole Body ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Spindle Pole ◽  
Microtubule Organizing Center ◽  
Bipolar Spindle ◽  
Pole Body ◽  
Cytoplasmic Microtubules ◽  
Mutant Cells

The spindle pole body (SPB) is the microtubule organizing center in Saccharomyces cerevisiae. An essential task of the SPB is to ensure assembly of the bipolar spindle, which requires a proper balancing of forces on the microtubules and chromosomes. The SPB component Spc110p connects the ends of the spindle microtubules to the core of the SPB. We previously reported the isolation of a mutant allele spc110-226 that causes broken spindles and SPB disintegration 30 min after spindle formation. By live cell imaging of mutant cells with green fluorescent protein (GFP)-Tub1p or Spc97p-GFP, we show that spc110-226 mutant cells have early defects in spindle assembly. Short spindles form but do not advance to the 1.5-μm stage and frequently collapse. Kinetochores are not arranged properly in the mutant cells. In 70% of the cells, no stable biorientation occurs and all kinetochores are associated with only one SPB. Examination of the SPB remnants by electron microscopy tomography and fluorescence microscopy revealed that the Spc110-226p/calmodulin complex is stripped off of the central plaque of the SPB and coalesces to from a nucleating structure in the nucleoplasm. The central plaque components Spc42p and Spc29p remain behind in the nuclear envelope. The delamination is likely due to a perturbed interaction between Spc42p and Spc110-226p as detected by fluorescence resonance energy transfer analysis. We suggest that the force exerted on the SPB by biorientation of the chromosomes pulls the Spc110-226p out of the SPB; removal of force exerted by coherence of the sister chromatids reduced fragmentation fourfold. Removal of the forces exerted by the cytoplasmic microtubules had no effect on fragmentation. Our results provide insights into the relative contributions of the kinetochore and cytoplasmic microtubules to the forces involved in formation of a bipolar spindle.

scholarly journals BIK1, a protein required for microtubule function during mating and mitosis in Saccharomyces cerevisiae, colocalizes with tubulin.

1990 ◽  
Vol 111 (6) ◽  
pp. 2573-2586 ◽  
Author(s):  
V Berlin ◽  
C A Styles ◽  
G R Fink
Keyword(s):  
Synthetic Lethality ◽  
Nuclear Fusion ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Microtubule Assembly ◽  
Chromosome Loss ◽  
Physical Interaction ◽  
Pole Body ◽  
Cytoplasmic Microtubules ◽  
And Function

BIK1 function is required for nuclear fusion, chromosome disjunction, and nuclear segregation during mitosis. The BIK1 protein colocalizes with tubulin to the spindle pole body and mitotic spindle. Synthetic lethality observed in double mutant strains containing a mutation in the BIK1 gene and in the gene for alpha- or beta-tubulin is consistent with a physical interaction between BIK1 and tubulin. Furthermore, over- or underexpression of BIK1 causes aberrant microtubule assembly and function, bik1 null mutants are viable but contain very short or undetectable cytoplasmic microtubules. Spindle formation often occurs strictly within the mother cell, probably accounting for the many multinucleate and anucleate bik1 cells. Elevated levels of chromosome loss in bik1 cells are indicative of defective spindle function. Nuclear fusion is blocked in bik1 x bik1 zygotes, which have truncated cytoplasmic microtubules. Cells overexpressing BIK1 initially have abnormally short or nonexistent spindle microtubules and long cytoplasmic microtubules. Subsequently, cells lose all microtubule structures, coincident with the arrest of division. Based on these results, we propose that BIK1 is required stoichiometrically for the formation or stabilization of microtubules during mitosis and for spindle pole body fusion during conjugation.

scholarly journals Brr6 drives the Schizosaccharomyces pombe spindle pole body nuclear envelope insertion/extrusion cycle

2011 ◽  
Vol 195 (3) ◽  
pp. 467-484 ◽  
Author(s):  
Tiina Tamm ◽  
Agnes Grallert ◽  
Emily P.S. Grossman ◽  
Isabel Alvarez-Tabares ◽  
Frances E. Stevens ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Mitotic Exit ◽  
Microtubule Organizing Center ◽  
Pole Body ◽  
Cytoplasmic Face ◽  
Organizing Center ◽  
Nuclear Component ◽  
Anaphase B

The fission yeast interphase spindle pole body (SPB) is a bipartite structure in which a bulky cytoplasmic domain is separated from a nuclear component by the nuclear envelope. During mitosis, the SPB is incorporated into a fenestra that forms within the envelope during mitotic commitment. Closure of this fenestra during anaphase B/mitotic exit returns the cytoplasmic component to the cytoplasmic face of an intact interphase nuclear envelope. Here we show that Brr6 is transiently recruited to SPBs at both SPB insertion and extrusion. Brr6 is required for both SPB insertion and nuclear envelope integrity during anaphase B/mitotic exit. Genetic interactions with apq12 and defective sterol assimilation suggest that Brr6 may alter envelope composition at SPBs to promote SPB insertion and extrusion. The restriction of the Brr6 domain to eukaryotes that use a polar fenestra in an otherwise closed mitosis suggests a conserved role in fenestration to enable a single microtubule organizing center to nucleate both cytoplasmic and nuclear microtubules on opposing sides of the nuclear envelope.

scholarly journals The microtubule polymerase Stu2 promotes oligomerization of the γ-TuSC for cytoplasmic microtubule nucleation

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Judith Gunzelmann ◽  
Diana Rüthnick ◽  
Tien-chen Lin ◽  
Wanlu Zhang ◽  
Annett Neuner ◽  
...  
Keyword(s):  
Budding Yeast ◽  
Spindle Pole Body ◽  
Family Members ◽  
Spindle Pole ◽  
Microtubule Nucleation ◽  
Cytoplasmic Microtubule ◽  
Pole Body ◽  
Cytoplasmic Microtubules

Stu2/XMAP215/ZYG-9/Dis1/Alp14/Msps/ch-TOG family members in association with with γ-tubulin complexes nucleate microtubules, but we know little about the interplay of these nucleation factors. Here, we show that the budding yeast Stu2 in complex with the γ-tubulin receptor Spc72 nucleates microtubules in vitro without the small γ-tubulin complex (γ-TuSC). Upon γ-TuSC addition, Stu2 facilitates Spc72–γ-TuSC interaction by binding to Spc72 and γ-TuSC. Stu2 together with Spc72–γ-TuSC increases microtubule nucleation in a process that is dependent on the TOG domains of Stu2. Importantly, these activities are also important for microtubule nucleation in vivo. Stu2 stabilizes Spc72–γ-TuSC at the minus end of cytoplasmic microtubules (cMTs) and an in vivo assay indicates that cMT nucleation requires the TOG domains of Stu2. Upon γ-tubulin depletion, we observed efficient cMT nucleation away from the spindle pole body (SPB), which was dependent on Stu2. Thus, γ-TuSC restricts cMT assembly to the SPB whereas Stu2 nucleates cMTs together with γ-TuSC and stabilizes γ-TuSC at the cMT minus end.

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.

scholarly journals The Organization of the Core Proteins of the Yeast Spindle Pole Body

2005 ◽  
Vol 16 (7) ◽  
pp. 3341-3352 ◽  
Author(s):  
Eric G.D. Muller ◽  
Brian E. Snydsman ◽  
Isabella Novik ◽  
Dale W. Hailey ◽  
Daniel R. Gestaut ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Mathematical Proof ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Central Component ◽  
Microtubule Organizing Center ◽  
The Core ◽  
Pole Body ◽  
Core Proteins

The spindle pole body (SPB) is the microtubule organizing center of Saccharomyces cerevisiae. Its core includes the proteins Spc42, Spc110 (kendrin/pericentrin ortholog), calmodulin (Cmd1), Spc29, and Cnm67. Each was tagged with CFP and YFP and their proximity to each other was determined by fluorescence resonance energy transfer (FRET). FRET was measured by a new metric that accurately reflected the relative extent of energy transfer. The FRET values established the topology of the core proteins within the architecture of SPB. The N-termini of Spc42 and Spc29, and the C-termini of all the core proteins face the gap between the IL2 layer and the central plaque. Spc110 traverses the central plaque and Cnm67 spans the IL2 layer. Spc42 is a central component of the central plaque where its N-terminus is closely associated with the C-termini of Spc29, Cmd1, and Spc110. When the donor-acceptor pairs were ordered into five broad categories of increasing FRET, the ranking of the pairs specified a unique geometry for the positions of the core proteins, as shown by a mathematical proof. The geometry was integrated with prior cryoelectron tomography to create a model of the interwoven network of proteins within the central plaque. One prediction of the model, the dimerization of the calmodulin-binding domains of Spc110, was confirmed by in vitro analysis.

scholarly journals Saccharomyces cerevisiaeCells with Defective Spindle Pole Body Outer Plaques Accomplish Nuclear Migration via Half-Bridge–organized Microtubules

1998 ◽  
Vol 9 (5) ◽  
pp. 977-991 ◽  
Author(s):  
Arndt Brachat ◽  
John V. Kilmartin ◽  
Achim Wach ◽  
Peter Philippsen
Keyword(s):  
Fluorescent Protein ◽  
Spindle Pole Body ◽  
Time Lapse ◽  
Nuclear Migration ◽  
Spindle Pole ◽  
Microtubule Organizing Center ◽  
Multinucleated Cells ◽  
Pole Body ◽  
Cytoplasmic Microtubules ◽  
Low Efficiency

Cnm67p, a novel yeast protein, localizes to the microtubule organizing center, the spindle pole body (SPB). Deletion ofCNM67 (YNL225c) frequently results in spindle misorientation and impaired nuclear migration, leading to the generation of bi- and multinucleated cells (40%). Electron microscopy indicated that CNM67 is required for proper formation of the SPB outer plaque, a structure that nucleates cytoplasmic (astral) microtubules. Interestingly, cytoplasmic microtubules that are essential for spindle orientation and nuclear migration are still present in cnm67Δ1 cells that lack a detectable outer plaque. These microtubules are attached to the SPB half- bridge throughout the cell cycle. This interaction presumably allows for low-efficiency nuclear migration and thus provides a rescue mechanism in the absence of a functional outer plaque. AlthoughCNM67 is not strictly required for mitosis, it is essential for sporulation. Time-lapse microscopy ofcnm67Δ1 cells with green fluorescent protein (GFP)-labeled nuclei indicated that CNM67 is dispensable for nuclear migration (congression) and nuclear fusion during conjugation. This is in agreement with previous data, indicating that cytoplasmic microtubules are organized by the half-bridge during mating.

scholarly journals Analysis of Tub4p, a yeast gamma-tubulin-like protein: implications for microtubule-organizing center function.

1996 ◽  
Vol 134 (2) ◽  
pp. 443-454 ◽  
Author(s):  
L G Marschall ◽  
R L Jeng ◽  
J Mulholland ◽  
T Stearns
Keyword(s):  
Fluorescent Protein ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Dependent Manner ◽  
Protein Fusion ◽  
Microtubule Organizing Center ◽  
Pole Body ◽  
Monopolar Spindle ◽  
Organizing Center ◽  
Gamma Tubulin

gamma-Tubulin is a conserved component of microtubule-organizing centers and is thought to be involved in microtubule nucleation. A recently discovered Saccharomyces cerevisiae gene (TUB4) encodes a tubulin that is related to, but divergent from, gamma-tubulins. TUB4 is essential for cell viability, and epitope-tagged Tub4 protein (Tub4p) is localized to the spindle pole body (Sobel, S.G., and M. Snyder. 1995.J. Cell Biol. 131:1775-1788). We have characterized the expression of TUB4, the association of Tub4p with the spindle pole body, and its role in microtubule organization. Tub4p is a minor protein in the cell, and expression of TUB4 is regulated in a cell cycle-dependent manner. Wild-type Tub4p is localized to the spindle pole body, and a Tub4p-green fluorescent protein fusion is able to associate with a preexisting spindle pole body, suggesting that there is dynamic exchange between cytoplasmic and spindle pole body forms of Tub4p. Perturbation of Tub4p function, either by conditional mutation or by depletion of the protein, results in spindle as well as spindle pole body defects, but does not eliminate the ability of microtubules to regrow from, or remain attached to, the spindle pole body. The spindle pole bodies in tub4 mutant cells duplicate but do not separate, resulting in a monopolar spindle. EM revealed that one spindle pole body of the duplicated pair appears to be defective for the nucleation of microtubules. These results offer insight into the role of gamma-tubulin in microtubule-organizing center function.

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