The molecular architecture of the yeast spindle pole body core determined by Bayesian integrative modeling

Microtubule-organizing centers (MTOCs) form, anchor, and stabilize the polarized network of microtubules in a cell. The central MTOC is the centrosome that duplicates during the cell cycle and assembles a bipolar spindle during mitosis to capture and segregate sister chromatids. Yet, despite their importance in cell biology, the physical structure of MTOCs is poorly understood. Here we determine the molecular architecture of the core of the yeast spindle pole body (SPB) by Bayesian integrative structure modeling based on in vivo fluorescence resonance energy transfer (FRET), small-angle x-ray scattering (SAXS), x-ray crystallography, electron microscopy, and two-hybrid analysis. The model is validated by several methods that include a genetic analysis of the conserved PACT domain that recruits Spc110, a protein related to pericentrin, to the SPB. The model suggests that calmodulin can act as a protein cross-linker and Spc29 is an extended, flexible protein. The model led to the identification of a single, essential heptad in the coiled-coil of Spc110 and a minimal PACT domain. It also led to a proposed pathway for the integration of Spc110 into the SPB.

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Vesicle Docking to the Spindle Pole Body Is Necessary to Recruit the Exocyst During Membrane Formation inSaccharomyces cerevisiae

Molecular Biology of the Cell ◽

10.1091/mbc.e10-07-0563 ◽

2010 ◽

Vol 21 (21) ◽

pp. 3693-3707 ◽

Cited By ~ 11

Author(s):

Erin M. Mathieson ◽

Yasuyuki Suda ◽

Mark Nickas ◽

Brian Snydsman ◽

Trisha N. Davis ◽

...

Keyword(s):

De Novo ◽

Spindle Pole Body ◽

Coiled Coil ◽

Resonance Energy ◽

Initiation Site ◽

Spindle Pole ◽

Rab Gtpase ◽

Membrane Formation ◽

Vesicle Docking ◽

Pole Body

During meiosis II in Saccharomyces cerevisiae, the cytoplasmic face of the spindle pole body, referred to as the meiosis II outer plaque (MOP), is modified in both composition and structure to become the initiation site for de novo formation of a membrane called the prospore membrane. The MOP serves as a docking complex for precursor vesicles that are targeted to its surface. Using fluorescence resonance energy transfer analysis, the orientation of coiled-coil proteins within the MOP has been determined. The N-termini of two proteins, Mpc54p and Spo21p, were oriented toward the outer surface of the structure. Mutations in the N-terminus of Mpc54p resulted in a unique phenotype: precursor vesicles loosely tethered to the MOP but did not contact its surface. Thus, these mpc54 mutants separate the steps of vesicle association and docking. Using these mpc54 mutants, we determined that recruitment of the Rab GTPase Sec4p, as well as the exocyst components Sec3p and Sec8p, to the precursor vesicles requires vesicle docking to the MOP. This suggests that the MOP promotes membrane formation both by localization of precursor vesicles to a particular site and by recruitment of a second tethering complex, the exocyst, that stimulates downstream events of fusion.

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

Molecular Biology of the Cell ◽

10.1091/mbc.e04-08-0703 ◽

2005 ◽

Vol 16 (1) ◽

pp. 141-152 ◽

Cited By ~ 22

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.

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Nuf2, a spindle pole body-associated protein required for nuclear division in yeast.

The Journal of Cell Biology ◽

10.1083/jcb.125.4.853 ◽

1994 ◽

Vol 125 (4) ◽

pp. 853-866 ◽

Cited By ~ 43

Author(s):

M A Osborne ◽

G Schlenstedt ◽

T Jinks ◽

P A Silver

Keyword(s):

Nuclear Division ◽

Spindle Pole Body ◽

Coiled Coil ◽

Temperature Shift ◽

Spindle Pole ◽

Temperature Sensitive ◽

Yeast Saccharomyces Cerevisiae ◽

Culture Cells ◽

Pole Body ◽

Associated Proteins

The NUF2 gene of the yeast Saccharomyces cerevisiae encodes an essential 53-kd protein with a high content of potential coiled-coil structure similar to myosin. Nuf2 is associated with the spindle pole body (SPB) as determined by coimmunofluorescence with known SPB proteins. Nuf2 appears to be localized to the intranuclear region and is a candidate for a protein involved in SPB separation. The nuclear association of Nuf2 can be disrupted, in part, by 1 M salt but not by the detergent Triton X-100. All Nuf2 can be removed from nuclei by 8 M urea extraction. In this regard, Nuf2 is similar to other SPB-associated proteins including Nufl/SPC110, also a coiled-coil protein. Temperature-sensitive alleles of NUF2 were generated within the coiled-coil region of Nuf2 and such NUF2 mutant cells rapidly arrest after temperature shift with a single undivided or partially divided nucleus in the bud neck, a shortened mitotic spindle and their DNA fully replicated. In sum, Nuf2 is a protein associated with the SPB that is critical for nuclear division. Anti-Nuf2 antibodies also recognize a mammalian 73-kd protein and display centrosome staining of mammalian tissue culture cells suggesting the presence of a protein with similar function.

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Microtubules in the fungal pathogen Ustilago maydis are highly dynamic and determine cell polarity

Journal of Cell Science ◽

10.1242/jcs.114.3.609 ◽

2001 ◽

Vol 114 (3) ◽

pp. 609-622 ◽

Cited By ~ 6

Author(s):

G. Steinberg ◽

R. Wedlich-Soldner ◽

M. Brill ◽

I. Schulz

Keyword(s):

Cell Polarity ◽

Fungal Pathogens ◽

Spindle Pole Body ◽

Ustilago Maydis ◽

Spindle Pole ◽

Microtubule Organization ◽

Growth Region ◽

Alpha Tubulin ◽

Pole Body

Many fungal pathogens undergo a yeast-hyphal transition during their pathogenic development that requires rearrangement of the cytoskeleton, followed by directed membrane traffic towards the growth region. The role of microtubules and their dynamic behavior during this process is not well understood. Here we set out to elucidate the organization, cellular role and in vivo dynamics of microtubules in the dimorphic phytopathogen Ustilago maydis. Hyphae and unbudded yeast-like cells of U. maydis contain bundles of spindle pole body-independent microtubules. At the onset of bud formation two spherical tubulin structures focus microtubules towards the growth region, suggesting that they support polar growth in G(2), while spindle pole body-nucleated astral microtubules participate in nuclear migration in M and early G(1). Conditional mutants of an essential alpha-tubulin gene from U. maydis, tub1, confirmed a role for interphase microtubules in determination of cell polarity and growth. Observation of GFP-Tub1 fusion protein revealed that spindle pole body-independent and astral microtubules are dynamic, with elongation and shrinkage rates comparable to those found in vertebrate systems. In addition, very fast depolymerization was measured within microtubule bundles. Unexpectedly, interphase microtubules underwent bending and rapid translocations within the cell, suggesting that unknown motor activities participate in microtubule organization in U. maydis. Movies available on-line: http://www.biologists.com/JCS/movies/jcs1792.html

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S. pombe sporulation-specific coiled-coil protein Spo15p is localized to the spindle pole body and essential for its modification

Journal of Cell Science ◽

10.1242/jcs.113.3.545 ◽

2000 ◽

Vol 113 (3) ◽

pp. 545-554 ◽

Cited By ~ 8

Author(s):

S. Ikemoto ◽

T. Nakamura ◽

M. Kubo ◽

C. Shimoda

Keyword(s):

Life Cycle ◽

Fusion Gene ◽

Spindle Pole Body ◽

Coiled Coil ◽

Spindle Pole ◽

Wild Type ◽

Membrane Formation ◽

Pole Body ◽

Spindle Pole Bodies ◽

Meiotic Spindles

Spindle pole bodies in the fission yeast Schizosaccharomyces pombe are required during meiosis, not only for spindle formation but also for the assembly of forespore membranes. The spo15 mutant is defective in the formation of forespore membranes, which develop into spore envelopes. The spo15(+)gene encodes a protein with a predicted molecular mass of 223 kDa, containing potential coiled-coil regions. The spo15 gene disruptant was not lethal, but was defective in spore formation. Northern and western analyses indicated that spo15(+) was expressed not only in meiotic cells but also in vegetative cells. When the spo15-GFP fusion gene was expressed by the authentic spo15 promoter during vegetative growth and sporulation, the fusion protein colocalized with Sad1p, which is a component of spindle pole bodies. Meiotic divisions proceeded in spo15delta cells with kinetics similar to those in wild-type cells. In addition, the morphology of the mitotic and meiotic spindles and the nuclear segregation were normal in spo15delta. Intriguingly, transformation of spindle pole bodies from a punctate to a crescent form prior to forespore membrane formation was not observed in spo15delta cells. We conclude that Spo15p is associated with spindle pole bodies throughout the life cycle and plays an indispensable role in the initiation of spore membrane formation.

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Specific coiled-coil interactions contribute to a global model of the structure of the spindle pole body

Journal of Structural Biology ◽

10.1016/j.jsb.2010.01.022 ◽

2010 ◽

Vol 170 (2) ◽

pp. 246-256 ◽

Cited By ~ 6

Author(s):

Nora Zizlsperger ◽

Amy E. Keating

Keyword(s):

Spindle Pole Body ◽

Coiled Coil ◽

Spindle Pole ◽

Global Model ◽

Pole Body

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Sister Kinetochore Recapture in Fission Yeast Occurs by Two Distinct Mechanisms, Both Requiring Dam1 and Klp2

Molecular Biology of the Cell ◽

10.1091/mbc.e07-09-0910 ◽

2008 ◽

Vol 19 (4) ◽

pp. 1646-1662 ◽

Cited By ~ 36

Author(s):

Yannick Gachet ◽

Céline Reyes ◽

Thibault Courthéoux ◽

Sherilyn Goldstone ◽

Guillaume Gay ◽

...

Keyword(s):

Fission Yeast ◽

Spindle Pole Body ◽

Spindle Pole ◽

Cell Analysis ◽

Microtubule Depolymerization ◽

Directed Movement ◽

Pole Body ◽

Sister Kinetochore ◽

Intranuclear Microtubules

In eukaryotic cells, proper formation of the spindle is necessary for successful cell division. We have studied chromosome recapture in the fission yeast Schizosaccharomyces pombe. We show by live cell analysis that lost kinetochores interact laterally with intranuclear microtubules (INMs) and that both microtubule depolymerization (end-on pulling) and minus-end–directed movement (microtubule sliding) contribute to chromosome retrieval to the spindle pole body (SPB). We find that the minus-end–directed motor Klp2 colocalizes with the kinetochore during its transport to the SPB and contributes to the effectiveness of retrieval by affecting both end-on pulling and lateral sliding. Furthermore, we provide in vivo evidence that Dam1, a component of the DASH complex, also colocalizes with the kinetochore during its transport and is essential for its retrieval by either of these mechanisms. Finally, we find that the position of the unattached kinetochore correlates with the size and orientation of the INMs, suggesting that chromosome recapture may not be a random process.

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

The Journal of Cell Biology ◽

10.1083/jcb.200111025 ◽

2002 ◽

Vol 156 (3) ◽

pp. 453-465 ◽

Cited By ~ 52

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

eLife ◽

10.7554/elife.39932 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 24

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.

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