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 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 Mobility, Microtubule Nucleation and Structure of Microtubule-organizing Centers in Multinucleated Hyphae ofAshbya gossypii

2010 ◽  
Vol 21 (1) ◽  
pp. 18-28 ◽  
Author(s):  
Claudia Lang ◽  
Sandrine Grava ◽  
Tineke van den Hoorn ◽  
Rhonda Trimble ◽  
Peter Philippsen ◽  
...  
Keyword(s):  
Spindle Pole Body ◽  
Hyphal Growth ◽  
Spindle Pole ◽  
Microtubule Organizing Center ◽  
Microtubule Nucleation ◽  
Cytoplasmic Microtubule ◽  
Organizing Center ◽  
Cytoplasmic Side ◽  
Independent Mode

We investigated the migration of multiple nuclei in hyphae of the filamentous fungus Ashbya gossypii. Three types of cytoplasmic microtubule (cMT)-dependent nuclear movements were characterized using live cell imaging: short-range oscillations (up to 4.5 μm/min), rotations (up to 180° in 30 s), and long-range nuclear bypassing (up to 9 μm/min). These movements were superimposed on a cMT-independent mode of nuclear migration, cotransport with the cytoplasmic stream. This latter mode is sufficient to support wild-type-like hyphal growth speeds. cMT-dependent nuclear movements were led by a nuclear-associated microtubule-organizing center, the spindle pole body (SPB), which is the sole site of microtubule nucleation in A. gossypii. Analysis of A. gossypii SPBs by electron microscopy revealed an overall laminar structure similar to the budding yeast SPB but with distinct differences at the cytoplasmic side. Up to six perpendicular and tangential cMTs emanated from a more spherical outer plaque. The perpendicular and tangential cMTs most likely correspond to short, often cortex-associated cMTs and to long, hyphal growth-axis–oriented cMTs, respectively, seen by in vivo imaging. Each SPB nucleates its own array of cMTs, and the lack of overlapping cMT arrays between neighboring nuclei explains the autonomous nuclear oscillations and bypassing observed in A. gossypii hyphae.

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 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.

scholarly journals Nuclear Export Receptor Xpo1/Crm1 Is Physically and Functionally Linked to the Spindle Pole Body in Budding Yeast

2008 ◽  
Vol 28 (17) ◽  
pp. 5348-5358 ◽  
Author(s):  
Anja Neuber ◽  
Jacqueline Franke ◽  
Angelika Wittstruck ◽  
Gabriel Schlenstedt ◽  
Thomas Sommer ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Budding Yeast ◽  
Nuclear Export Signal ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Microtubule Organizing Center ◽  
Functional Link ◽  
Pole Body ◽  
Nuclear Export Receptor

ABSTRACT The spindle pole body (SPB) represents the microtubule organizing center in the budding yeast Saccharomyces cerevisiae. It is a highly structured organelle embedded in the nuclear membrane, which is required to anchor microtubules on both sides of the nuclear envelope. The protein Spc72, a component of the SPB, is located at the cytoplasmic face of this organelle and serves as a receptor for the γ-tubulin complex. In this paper we show that it is also a binding partner of the nuclear export receptor Xpo1/Crm1. Xpo1 binds its cargoes in a Ran-dependent fashion via a short leucine-rich nuclear export signal (NES). We show that binding of Spc72 to Xpo1 depends on Ran-GTP and a functional NES in Spc72. Mutations in this NES have severe consequences for mitotic spindle morphology in vivo. This is also the case for xpo1 mutants, which show a reduction in cytoplasmic microtubules. In addition, we find a subpopulation of Xpo1 localized at the SPB. Based on these data, we propose a functional link between Xpo1 and the SPB and discuss a role for this exportin in spindle biogenesis in budding yeast.

scholarly journals Novel Phosphorylation States of the Yeast Spindle Pole Body

2018 ◽  
Author(s):  
Kimberly K. Fong ◽  
Alex Zelter ◽  
Beth Graczyk ◽  
Jill M. Hoyt ◽  
Michael Riffle ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Spindle Pole Body ◽  
Mitotic Checkpoint ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Phosphorylation Sites ◽  
Microtubule Nucleation ◽  
Data Set ◽  
Pole Body

ABSTRACTPhosphorylation regulates yeast spindle pole body (SPB) duplication and separation and likely regulates microtubule nucleation. We report a phosphoproteomic analysis using tandem mass spectrometry of purifiedSaccharomyces cerevisiaeSPBs for two cell cycle arrests, G1/S and the mitotic checkpoint, expanding on previously reported phosphoproteomic data sets. We present a novel phosphoproteomic state of SPBs arrested in G1/S by acdc4-1temperature sensitive mutation, with particular interest in phosphorylation events on the γ-tubulin small complex (γ-TuSC). Thecdc4-1arrest is the earliest arrest at which microtubule nucleation has occurred at the newly duplicated SPB. Several novel phosphorylation sites were identified in G1/S and during mitosis on the microtubule nucleating γ-TuSC. These sites were analyzedin vivoby fluorescence microscopy and were shown to be required for proper regulation of spindle length. Additionally,in vivoanalysis of two mitotic sites in Spc97 found that phosphorylation of at least one of these sites is required for progression through the cell cycle. This phosphoproteomic data set not only broadens the scope of the phosphoproteome of SPBs, it also identifies several γ-TuSC phosphorylation sites influencing microtubule regulation.

scholarly journals Requirement for the Budding Yeast Polo Kinase Cdc5 in Proper Microtubule Growth and Dynamics

2008 ◽  
Vol 7 (3) ◽  
pp. 444-453 ◽  
Author(s):  
Chong J. Park ◽  
Jung-Eun Park ◽  
Tatiana S. Karpova ◽  
Nak-Kyun Soung ◽  
Li-Rong Yu ◽  
...  
Keyword(s):  
Budding Yeast ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Growth Defect ◽  
Temperature Sensitive ◽  
Independent Manner ◽  
Polo Kinase ◽  
Early Anaphase

ABSTRACT In many organisms, polo kinases appear to play multiple roles during M-phase progression. To provide new insights into the function of the budding yeast polo kinase Cdc5, we generated novel temperature-sensitive cdc5 mutants by mutagenizing the C-terminal noncatalytic polo box domain, a region that is critical for proper subcellular localization. One of these mutants, cdc5-11, exhibited a temperature-sensitive growth defect with an abnormal spindle morphology. Strikingly, provision of a moderate level of benomyl, a microtubule-depolymerizing drug, permitted cdc5-11 cells to grow significantly better than the isogenic CDC5 wild type in a FEAR (cdc Fourteen Early Anaphase Release)-independent manner. In addition, cdc5-11 required MAD2 for both cell growth and the benomyl-remedial phenotype. These results suggest that cdc5-11 is defective in proper spindle function. Consistent with this view, cdc5-11 exhibited abnormal spindle morphology, shorter spindle length, and delayed microtubule regrowth at the nonpermissive temperature. Overexpression of CDC5 moderately rescued the spc98-2 growth defect. Interestingly, both Cdc28 and Cdc5 were required for the proper modification of the spindle pole body components Nud1, Slk19, and Stu2 in vivo. They also phosphorylated these three proteins in vitro. Taken together, these observations suggest that concerted action of Cdc28 and Cdc5 on Nud1, Slk19, and Stu2 is important for proper spindle functions.

scholarly journals The Yeast Centrin, Cdc31p, and the Interacting Protein Kinase, Kic1p, Are Required for Cell Integrity

1998 ◽  
Vol 143 (3) ◽  
pp. 751-765 ◽  
Author(s):  
Donald S. Sullivan ◽  
Sue Biggins ◽  
Mark D. Rose
Keyword(s):  
Protein Kinase ◽  
Calcium Binding ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Permissive Temperature ◽  
Cell Integrity ◽  
Interacting Protein ◽  
Pole Body

Cdc31p is the yeast homologue of centrin, a highly conserved calcium-binding protein of the calmodulin superfamily. Previously centrins have been implicated only in microtubule-based processes. To elucidate the functions of yeast centrin, we carried out a two-hybrid screen for Cdc31p-interacting proteins and identified a novel essential protein kinase of 1,080 residues, Kic1p (kinase that interacts with Cdc31p). Kic1p is closely related to S. cerevisiae Ste20p and the p-21– activated kinases (PAKs) found in a wide variety of eukaryotic organisms. Cdc31p physically interacts with Kic1p by two criteria; Cdc31p coprecipitated with GST–Kic1p and it bound to GST–Kic1p in gel overlay assays. Furthermore, GST–Kic1p exhibited in vitro kinase activity that was CDC31-dependent. Although kic1 mutants were not defective for spindle pole body duplication, they exhibited a variety of mutant phenotypes demonstrating that Kic1p is required for cell integrity. We also found that cdc31 mutants, previously identified as defective for spindle pole body duplication, exhibited lysis and morphological defects. The cdc31 kic1 double mutants exhibited a drastic reduction in the range of permissive temperature, resulting in a severe lysis defect. We conclude that Kic1p function is dependent upon Cdc31p both in vivo and in vitro. We postulate that Cdc31p is required both for SPB duplication and for cell integrity/morphogenesis, and that the integrity/morphogenesis function is mediated through the Kic1p protein kinase.

scholarly journals Direct interaction between yeast spindle pole body components: Kar1p is required for Cdc31p localization to the spindle pole body.

1994 ◽  
Vol 125 (4) ◽  
pp. 843-852 ◽  
Author(s):  
S Biggins ◽  
M D Rose
Keyword(s):  
Calcium Binding ◽  
Spindle Pole Body ◽  
Direct Interaction ◽  
Spindle Pole ◽  
Pole Body ◽  
Blotting Technique ◽  
Essential Function ◽  
Body Components

The Saccharomyces cerevisiae genes KAR1 and CDC31 are required for the initial stages of spindle pole body (SPB) duplication in yeast. The Cdc31 protein is most related to caltractin/centrin, a calcium-binding protein present in microtubule organizing centers in many organisms. Because of a variety of genetic interactions between CDC31 and KAR1 (Vallen, E. A., W. Ho. M. Winey, and M. D. Rose. 1994. Genetics. In press), we wanted to determine whether Cdc31p and Kar1p physically interact. Cdc31p was expressed and purified from Escherichia coli and active for binding calcium. Using a protein blotting technique, Cdc31p bound to Kar1p in vitro via an essential domain in Kar1p required for SPB duplication (Vallen, E. A., M. A. Hiller, T. Y. Scherson, and M. D. Rose. 1992a. J. Cell Biol. 117:1277-1287). By immunofluorescence microscopy, we determined that the interaction also occurs in vivo. Cdc31p was localized to the SPB in wild-type cells but was mislocalized in a kar1 mutant strain. In a kar1 mutant containing a dominant CDC31 suppressor, Cdc31p was again localized to the SPB. Furthermore, the localization of Cdc31p to the SPB was affected by the overexpression of Kar1p-beta-galactosidase hybrids. Based on these data, we propose that the essential function of Kar1p is to localize Cdc31p to the SPB, and that this interaction is normally required for SPB duplication.

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.

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