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 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.

Identification of an Spc110p-related protein in vertebrates

1997 ◽  
Vol 110 (20) ◽  
pp. 2533-2545 ◽  
Author(s):  
A.M. Tassin ◽  
C. Celati ◽  
M. Paintrand ◽  
M. Bornens
Keyword(s):  
Mammalian Cells ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Partial Purification ◽  
Related Protein ◽  
Microtubule Nucleation ◽  
Pole Body ◽  
Egg Extract ◽  
Ring Complex ◽  
Gamma Tubulin

Although varying in size and complexity, centrosomes have conserved functions throughout the evolutionary range of eukaryotes, and thus may display conserved components. In this work, we took advantage of the recent advances in the isolation of the budding yeast spindle pole body, the development of specific immunological probes and the molecular characterisation of genes involved in spindle pole body duplication or assembly. Screening a monoclonal antibody library against Saccharomyces cerevisiae spindle pole body components, we found that two monoclonal antibodies, directed against two different parts of the yeast Spc110p, decorate the centrosome from mammalian cells in an asymmetrical manner. Western blot experiments identified a 100 kDa protein specifically enriched in centrosome preparations from human cells. This protein is phosphorylated during mitosis and is tightly associated with the centrosome: only denaturing conditions such as 8 M urea were able to solubilise it. Purified immunoglobulins directed against Spc110p inhibit microtubule nucleation on isolated human centrosomes, using brain phosphocellulose-tubulin or Xenopus egg extract tubulin. This result suggested that the centrosomal 100 kDa protein could be involved in a microtubule nucleation complex. To test this hypothesis, we turned to Xenopus species, in which mAb anti-Spc110p decorated centrosomes from somatic cells and identified a 116 kDa protein in egg extract. We performed a partial purification of the gamma-tubulin-ring complex from egg extract. Sucrose gradient sedimentation, immunoprecipitation and native gels demonstrated that the Xenopus 116 kDa protein and gamma-tubulin were found in the same complex. Altogether, these results suggest the existence of an yeast Spc110-related protein in vertebrate centrosomes which is involved in microtubule nucleation.

scholarly journals The Sad1-UNC-84 homology domain in Mps3 interacts with Mps2 to connect the spindle pole body with the nuclear envelope

2006 ◽  
Vol 174 (5) ◽  
pp. 665-675 ◽  
Author(s):  
Sue L. Jaspersen ◽  
Adriana E. Martin ◽  
Galina Glazko ◽  
Thomas H. Giddings ◽  
Garry Morgan ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Integral Membrane Proteins ◽  
Microtubule Nucleation ◽  
The Core ◽  
Pole Body ◽  
C Terminus ◽  
Bridge Structures ◽  
Sun Domain

The spindle pole body (SPB) is the sole site of microtubule nucleation in Saccharomyces cerevisiae; yet, details of its assembly are poorly understood. Integral membrane proteins including Mps2 anchor the soluble core SPB in the nuclear envelope. Adjacent to the core SPB is a membrane-associated SPB substructure known as the half-bridge, where SPB duplication and microtubule nucleation during G1 occurs. We found that the half-bridge component Mps3 is the budding yeast member of the SUN protein family (Sad1-UNC-84 homology) and provide evidence that it interacts with the Mps2 C terminus to tether the half-bridge to the core SPB. Mutants in the Mps3 SUN domain or Mps2 C terminus have SPB duplication and karyogamy defects that are consistent with the aberrant half-bridge structures we observe cytologically. The interaction between the Mps3 SUN domain and Mps2 C terminus is the first biochemical link known to connect the half-bridge with the core SPB. Association with Mps3 also defines a novel function for Mps2 during SPB duplication.

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 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 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.

Role of gamma-tubulin in mitosis-specific microtubule nucleation from the Schizosaccharomyces pombe spindle pole body

1996 ◽  
Vol 109 (1) ◽  
pp. 165-177 ◽  
Author(s):  
H. Masuda ◽  
T. Shibata
Keyword(s):  
Protein Kinase ◽  
Mitotic Spindle ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Microtubule Nucleation ◽  
Amino Terminal ◽  
Pole Body ◽  
Gamma Tubulin ◽  
Mitotic Spindle Pole

The ability of the Schizosacchromyces pombe spindle pole body to nucleate microtubules is activated at the onset of mitosis for forming a mitotic spindle, but it is inactivated during interphase. We have previously developed an in vitro assay for studying the molecular mechanism of spindle pole body activation using permeabilized interphase S. pombe cells and Xenopus mitotic extracts. We have shown that the interphase spindle pole body is activated indirectly by p34cdc2 protein kinase in Xenopus mitotic extracts. In this study we examined the role of gamma-tubulin, a component of both interphase and mitotic spindle pole body, in formation of the microtubule nucleating complex at the mitotic spindle pole body. A polyclonal antibody specific to S. pombe gamma-tubulin inhibited both activation of the interphase spindle pole body and microtubule nucleation from the mitotic spindle pole body. Addition of bacterially expressed S. pombe gamma-tubulin or its amino-terminal fragments to Xenopus mitotic extracts inhibited spindle pole body activation. Affinity chromatography of partially fractionated Xenopus mitotic extracts with the amino-terminal fragment of S. pombe gamma-tubulin showed that fractions bound to the fragment supported the activation. The fractions did not contain Xenopus gamma-tubulin, showing that activation of the spindle pole body is not due to recruitment of Xenopus gamma-tubulin to the spindle pole body. The spindle pole body activation occurred in extracts depleted of p34cdc2 protein kinase or MAP kinase. The activity of the fractions bound to the fragment was inhibited by a protein kinase inhibitor, staurosporine. These results suggest that S. pombe gamma-tubulin is a component of the microtubule nucleating complex, and that the function of proteins that interact with gamma-tubulin is required for activation of the spindle pole body. We present possible models for the activation that convert the immature microtubule nucleating complex at interphase into the mature microtubule nucleating complex at mitosis.

scholarly journals Mps3p is a novel component of the yeast spindle pole body that interacts with the yeast centrin homologue Cdc31p

2002 ◽  
Vol 159 (6) ◽  
pp. 945-956 ◽  
Author(s):  
Sue L. Jaspersen ◽  
Thomas H. Giddings ◽  
Mark Winey
Keyword(s):  
Mitotic Spindle ◽  
Spindle Pole Body ◽  
Integral Membrane Protein ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Reading Frame ◽  
Pole Body ◽  
Monopolar Spindle

Accurate duplication of the Saccharomyces cerevisiae spindle pole body (SPB) is required for formation of a bipolar mitotic spindle. We identified mutants in SPB assembly by screening a temperature-sensitive collection of yeast for defects in SPB incorporation of a fluorescently marked integral SPB component, Spc42p. One SPB assembly mutant contained a mutation in a previously uncharacterized open reading frame that we call MPS3 (for monopolar spindle). mps3-1 mutants arrest in mitosis with monopolar spindles at the nonpermissive temperature, suggesting a defect in SPB duplication. Execution point experiments revealed that MPS3 function is required for the first step of SPB duplication in G1. Like cells containing mutations in two other genes required for this step of SPB duplication (CDC31 and KAR1), mps3-1 mutants arrest with a single unduplicated SPB that lacks an associated half-bridge. MPS3 encodes an essential integral membrane protein that localizes to the SPB half-bridge. Genetic interactions between MPS3 and CDC31 and binding of Cdc31p to Mps3p in vitro, as well as the fact that Cdc31p localization to the SPB is partially dependent on Mps3p function, suggest that one function for Mps3p during SPB duplication is to recruit Cdc31p, the yeast centrin homologue, to the half-bridge.

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