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 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 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 Spindle pole body-anchored Kar3 drives the nucleus along microtubules from another nucleus in preparation for nuclear fusion during yeast karyogamy

2013 ◽  
Vol 27 (3) ◽  
pp. 335-349 ◽  
Author(s):  
R. Gibeaux ◽  
A. Z. Politi ◽  
F. Nedelec ◽  
C. Antony ◽  
M. Knop
Keyword(s):  
Nuclear Fusion ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Pole Body

scholarly journals Bim1p/Yeb1p Mediates the Kar9p-dependent Cortical Attachment of Cytoplasmic Microtubules

2000 ◽  
Vol 11 (9) ◽  
pp. 2949-2959 ◽  
Author(s):  
Rita K. Miller ◽  
Soo-Chen Cheng ◽  
Mark D. Rose
Keyword(s):  
Fluorescent Protein ◽  
Binding Protein ◽  
Spindle Pole Body ◽  
Attachment Site ◽  
Spindle Pole ◽  
Cell Cortex ◽  
Microtubule Binding ◽  
Pole Body ◽  
Cytoplasmic Microtubules ◽  
Two Hybrid

In Saccharomyces cerevisiae, positioning of the mitotic spindle depends on the interaction of cytoplasmic microtubules with the cell cortex. In this process, cortical Kar9p in the bud acts as a link between the actin and microtubule cytoskeletons. To identify Kar9p-interacting proteins, a two-hybrid screen was conducted with the use of full-length Kar9p as bait, and three genes were identified: BIM1, STU2, andKAR9 itself. STU2 encodes a component of the spindle pole body. Bim1p is the yeast homologue of the human microtubule-binding protein EB1, which is a binding partner to the adenomatous polyposis coli protein involved in colon cancer. Eighty-nine amino acids within the third quarter of Bim1p was sufficient to confer interaction with Kar9p. The two-hybrid interactions were confirmed with the use of coimmunoprecipitation experiments. Genetic analysis placed Bim1p in the Kar9p pathway for nuclear migration. Bim1p was not required for Kar9p's cortical or spindle pole body localization. However, deletion ofBIM1 eliminated Kar9p localization along cytoplasmic microtubules. Furthermore, in the bim1 mutants, the cytoplasmic microtubules no longer intersected the cortical dot of Green Fluorescent Protein–Kar9p. These experiments demonstrate that the interaction of cytoplasmic microtubules with the Kar9p cortical attachment site requires the microtubule-binding protein Bim1p.

Ultrastructure of mitosis and the spindle pole body cycle in the smut fungus, Tilletia foetida

1985 ◽  
Vol 63 (1) ◽  
pp. 86-96 ◽  
Author(s):  
James A. Hoffmann ◽  
Blair J. Goates
Keyword(s):  
Nuclear Membrane ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Dense Layer ◽  
Double Structure ◽  
Pole Body ◽  
Cytoplasmic Microtubules ◽  
Spindle Microtubules ◽  
Dense Chromatin ◽  
Tilletia Foetida

The interphase nucleus in secondary sporidia of Tilletia foetida consists of mostly diffuse chromatin, one or two nucleoli, and an area of heterochromatin located opposite an electron-dense, extranuclear spindle pole body (SPB). The interphase SPB is an oval- to bar-shaped, double-structured disc that has a crystallinelike substructure. During nuclear migration into nascent sporidia, SPBs and nucleoli are randomly oriented. At the onset of division, chromatin begins to condense and the SPB becomes located on a nuclear protuberance. Cytoplasmic microtubules terminate at the SPBs and multivesicular bodies surround the SPBs from the early stages of SPB division to early postdivision. SPB discs become spheroid and each develops a medial, dense layer. Then, a basal, dense layer develops and elongates as the SPBs separate and become positioned on opposite sides of the nuclear protuberance. The nuclear membrane opens opposite the SPB during SPB division. The nucleolus is extruded into a nuclear bleb and degenerates. SPBs migrate to opposing sides of the nucleus and become diffuse as a microtubular spindle develops between them. Some spindle microtubules terminate at dense chromatin patches that are contiguous with the major mass of chromatin surrounding the spindle. During late division stages, spindle microtubules often appear to be closely juxtaposed. Except for polar openings adjacent to the SPBs, the nuclear membrane is entire until late division when it degenerates in the midregion of the nucleus. During early postdivision, the SPB condenses into a small, dense sphere as the chromatin and heterochromatin opposite the SPB become diffuse. The SPB then elongates into a dense bar and SPB material increases, except at the midportion, reforming the double structure of interphase.

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 Structural Mutants of the Spindle Pole Body Cause Distinct Alteration of Cytoplasmic Microtubules and Nuclear Dynamics in Multinucleated Hyphae

2010 ◽  
Vol 21 (5) ◽  
pp. 753-766 ◽  
Author(s):  
Claudia Lang ◽  
Sandrine Grava ◽  
Mark Finlayson ◽  
Rhonda Trimble ◽  
Peter Philippsen ◽  
...  
Keyword(s):  
Spindle Pole Body ◽  
Spindle Pole ◽  
Wild Type ◽  
Nuclear Dynamics ◽  
Pole Body ◽  
Nucleation Sites ◽  
Bridge Structures ◽  
Cytoplasmic Microtubules ◽  
Tangential Directions

In the multinucleate fungus Ashbya gossypii, cytoplasmic microtubules (cMTs) emerge from the spindle pole body outer plaque (OP) in perpendicular and tangential directions. To elucidate the role of cMTs in forward/backward movements (oscillations) and bypassing of nuclei, we constructed mutants potentially affecting cMT nucleation or stability. Hyphae lacking the OP components AgSpc72, AgNud1, AgCnm67, or the microtubule-stabilizing factor AgStu2 grew like wild- type but showed substantial alterations in the number, length, and/or nucleation sites of cMTs. These mutants differently influenced nuclear oscillation and bypassing. In Agspc72Δ, only long cMTs were observed, which emanate tangentially from reduced OPs; nuclei mainly moved with the cytoplasmic stream but some performed rapid bypassing. Agnud1Δ and Agcnm67Δ lack OPs; short and long cMTs emerged from the spindle pole body bridge/half-bridge structures, explaining nuclear oscillation and bypassing in these mutants. In Agstu2Δ only very short cMTs emanated from structurally intact OPs; all nuclei moved with the cytoplasmic stream. Therefore, long tangential cMTs promote nuclear bypassing and short cMTs are important for nuclear oscillation. Our electron microscopy ultrastructural analysis also indicated that assembly of the OP occurs in a stepwise manner, starting with AgCnm67, followed by AgNud1 and lastly AgSpc72.

Spindle pole body fusion in the smut fungus Tilletia foetida

1986 ◽  
Vol 64 (6) ◽  
pp. 1221-1223 ◽  
Author(s):  
Blair J. Goates ◽  
James A. Hoffmann
Keyword(s):  
Nuclear Fusion ◽  
Spindle Pole Body ◽  
Spindle Pole ◽  
Smut Fungus ◽  
Narrow Region ◽  
Pole Body ◽  
Spindle Pole Bodies ◽  
Tilletia Foetida

Fusion of double-structured, interphase spindle pole bodies (SPBs) occurred before nuclear fusion in heterokaryotic secondary sporidia. The SPBs of two separate nuclei were juxtaposed with their long axes perpendicular to each other. Also, SPBs were observed oriented with their long axes parallel and fused to each other at both ends. Fusion apparently continued toward the midportion of the SPBs. Nuclei were observed joined together in a narrow region. These nuclei appeared to share a single SPB that was located opposite to a protuberance on both nuclei. Following fusion, the SPB apparently returned to an interphase structure.

scholarly journals The Cyclin-dependent Kinase Cdc28p Regulates Multiple Aspects of Kar9p Function in Yeast

2007 ◽  
Vol 18 (4) ◽  
pp. 1187-1202 ◽  
Author(s):  
Jeffrey K. Moore ◽  
Rita K. Miller
Keyword(s):  
Spindle Pole Body ◽  
Spindle Pole ◽  
Cyclin Dependent Kinase ◽  
Hybrid Analysis ◽  
Yeast Saccharomyces Cerevisiae ◽  
Microtubule Associated Proteins ◽  
Pole Body ◽  
Associated Proteins ◽  
Cytoplasmic Microtubules ◽  
Two Hybrid

During mitosis in the yeast Saccharomyces cerevisiae, Kar9p directs one spindle pole body (SPB) toward the incipient daughter cell by linking the associated set of cytoplasmic microtubules (cMTs) to the polarized actin network on the bud cortex. The asymmetric localization of Kar9p to one SPB and attached cMTs is dependent on its interactions with microtubule-associated proteins and is regulated by the yeast Cdk1 Cdc28p. Two phosphorylation sites in Kar9p were previously identified. Here, we propose that the two sites are likely to govern Kar9p function through separate mechanisms, each involving a distinct cyclin. In the first mechanism, phosphorylation at serine 496 recruits Kar9p to one SPB. A phosphomimetic mutation at serine 496 bypasses the requirement of BIK1 and CLB5 in generating Kar9p asymmetry. In the second mechanism, Clb4p may target serine 197 of Kar9p for phosphorylation. This modification is required for Kar9p to direct cMTs to the bud. Two-hybrid analysis suggests that this phosphorylation may attenuate the interaction between Kar9p and the XMAP215-homologue Stu2p. We propose that phosphorylation at serine 197 regulates the release of Kar9p from Stu2p at the SPB, either to clear it from the mother-SPB or to allow it to travel to the plus end.

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.

Sign in / Sign up

Export Citation Format

Share Document