scholarly journals Novel Roles for Saccharomyces cerevisiae Mitotic Spindle Motors

1999 ◽  
Vol 147 (2) ◽  
pp. 335-350 ◽  
Author(s):  
Frank R. Cottingham ◽  
Larisa Gheber ◽  
Dana L. Miller ◽  
M. Andrew Hoyt
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitotic Spindle ◽  
Structural Integrity ◽  
Cytoplasmic Dynein ◽  
Spindle Positioning ◽  
Bipolar Spindle ◽  
Accessory Factor ◽  
Spindle Function ◽  
Related Proteins ◽  
Spindle Structure

The single cytoplasmic dynein and five of the six kinesin-related proteins encoded by Saccharomyces cerevisiae participate in mitotic spindle function. Some of the motors operate within the nucleus to assemble and elongate the bipolar spindle. Others operate on the cytoplasmic microtubules to effect spindle and nuclear positioning within the cell. This study reveals that kinesin-related Kar3p and Kip3p are unique in that they perform roles both inside and outside the nucleus. Kar3p, like Kip3p, was found to be required for spindle positioning in the absence of dynein. The spindle positioning role of Kar3p is performed in concert with the Cik1p accessory factor, but not the homologous Vik1p. Kar3p and Kip3p were also found to overlap for a function essential for the structural integrity of the bipolar spindle. The cytoplasmic and nuclear roles of both these motors could be partially substituted for by the microtubule-destabilizing agent benomyl, suggesting that these motors perform an essential microtubule-destabilizing function. In addition, we found that yeast cell viability could be supported by as few as two microtubule-based motors: the BimC-type kinesin Cin8p, required for spindle structure, paired with either Kar3p or Kip3p, required for both spindle structure and positioning.

scholarly journals Saccharomyces cerevisiae kinesin- and dynein-related proteins required for anaphase chromosome segregation.

1995 ◽  
Vol 128 (4) ◽  
pp. 617-624 ◽  
Author(s):  
W S Saunders ◽  
D Koshland ◽  
D Eshel ◽  
I R Gibbons ◽  
M A Hoyt
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosome Segregation ◽  
Motor Proteins ◽  
Cytoplasmic Dynein ◽  
Gene Products ◽  
Loss Of Function ◽  
Direct Role ◽  
Double Deletion ◽  
Different Types ◽  
Related Proteins

The Saccharomyces cerevisiae kinesin-related gene products Cin8p and Kip1p function to assemble the bipolar mitotic spindle. The cytoplasmic dynein heavy chain homologue Dyn1p (also known as Dhc1p) participates in proper cellular positioning of the spindle. In this study, the roles of these motor proteins in anaphase chromosome segregation were examined. While no single motor was essential, loss of function of all three completely halted anaphase chromatin separation. As combined motor activity was diminished by mutation, both the velocity and extent of chromatin movement were reduced, suggesting a direct role for all three motors in generating a chromosome-separating force. Redundancy for function between different types of microtubule-based motor proteins was also indicated by the observation that cin8 dyn1 double-deletion mutants are inviable. Our findings indicate that the bulk of anaphase chromosome segregation in S. cerevisiae is accomplished by the combined actions of these three motors.

scholarly journals Stu1p Is Physically Associated with β-Tubulin and Is Required for Structural Integrity of the Mitotic Spindle

2002 ◽  
Vol 13 (6) ◽  
pp. 1881-1892 ◽  
Author(s):  
Hongwei Yin ◽  
Liru You ◽  
Danielle Pasqualone ◽  
Kristen M. Kopski ◽  
Tim C. Huffaker
Keyword(s):  
Mitotic Spindle ◽  
Structural Integrity ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Yeast Saccharomyces Cerevisiae ◽  
Spindle Poles ◽  
Balance Of Forces ◽  
Related Proteins ◽  
Β Tubulin

Formation of the bipolar mitotic spindle relies on a balance of forces acting on the spindle poles. The primary outward force is generated by the kinesin-related proteins of the BimC family that cross-link antiparallel interpolar microtubules and slide them past each other. Here, we provide evidence that Stu1p is also required for the production of this outward force in the yeast Saccharomyces cerevisiae. In the temperature-sensitive stu1–5mutant, spindle pole separation is inhibited, and preanaphase spindles collapse, with their previously separated poles being drawn together. The temperature sensitivity of stu1–5 can be suppressed by doubling the dosage of Cin8p, a yeast BimC kinesin–related protein. Stu1p was observed to be a component of the mitotic spindle localizing to the midregion of anaphase spindles. It also binds to microtubules in vitro, and we have examined the nature of this interaction. We show that Stu1p interacts specifically with β-tubulin and identify the domains required for this interaction on both Stu1p and β-tubulin. Taken together, these findings suggest that Stu1p binds to interpolar microtubules of the mitotic spindle and plays an essential role in their ability to provide an outward force on the spindle poles.

scholarly journals A Protein Interaction Map of the Mitotic Spindle

2007 ◽  
Vol 18 (10) ◽  
pp. 3800-3809 ◽  
Author(s):  
Jonathan Wong ◽  
Yuko Nakajima ◽  
Stefan Westermann ◽  
Ching Shang ◽  
Jung-seog Kang ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Interactions ◽  
Mitotic Spindle ◽  
Phosphorylation Site ◽  
Protein Functions ◽  
Regulation Mechanisms ◽  
Novel Interactions ◽  
Interaction Map ◽  
Spindle Function ◽  
Two Hybrid

The mitotic spindle consists of a complex network of proteins that segregates chromosomes in eukaryotes. To strengthen our understanding of the molecular composition, organization, and regulation of the mitotic spindle, we performed a system-wide two-hybrid screen on 94 proteins implicated in spindle function in Saccharomyces cerevisiae. We report 604 predominantly novel interactions that were detected in multiple screens, involving 303 distinct prey proteins. We uncovered a pattern of extensive interactions between spindle proteins reflecting the intricate organization of the spindle. Furthermore, we observed novel connections between kinetochore complexes and chromatin-modifying proteins and used phosphorylation site mutants of NDC80/TID3 to gain insights into possible phospho-regulation mechanisms. We also present analyses of She1p, a novel spindle protein that interacts with the Dam1 kinetochore/spindle complex. The wealth of protein interactions presented here highlights the extent to which mitotic spindle protein functions and regulation are integrated with each other and with other cellular activities.

Kinesin-related proteins required for structural integrity of the mitotic spindle

Cell ◽  
1992 ◽  
Vol 70 (3) ◽  
pp. 451-458 ◽  
Author(s):  
William S. Saunders ◽  
M.Andrew Hoyt
Keyword(s):  
Mitotic Spindle ◽  
Structural Integrity ◽  
Related Proteins

scholarly journals Cytoplasmic dynein is required for normal nuclear segregation in yeast

1993 ◽  
Vol 90 (23) ◽  
pp. 11172-11176 ◽  
Author(s):  
D Eshel ◽  
L A Urrestarazu ◽  
S Vissers ◽  
J C Jauniaux ◽  
J C van Vliet-Reedijk ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Division ◽  
Heavy Chain ◽  
Mitotic Spindle ◽  
Cytoplasmic Dynein ◽  
Predicted Amino Acid Sequence ◽  
Yeast Saccharomyces Cerevisiae ◽  
Abnormal Distribution ◽  
Bud Neck ◽  
Cytoplasmic Microtubules

We have identified the gene DYN1, which encodes the heavy chain of cytoplasmic dynein in the yeast Saccharomyces cerevisiae. The predicted amino acid sequence (M(r) 471,305) reveals the presence of four P-loop motifs, as in all dyneins known so far, and has 28% overall identity to the dynein heavy chain of Dictyostelium [Koonce, M. P., Grissom, P. M. & McIntosh, J. R. (1992) J. Cell Biol. 119, 1597-1604] with 40% identity in the putative motor domain. Disruption of DYN1 causes misalignment of the spindle relative to the bud neck during cell division and results in abnormal distribution of the dividing nuclei between the mother cell and the bud. Cytoplasmic dynein, by generating force along cytoplasmic microtubules, may play an important role in the proper alignment of the mitotic spindle in yeast.

scholarly journals RanGTP and CLASP1 cooperate to position the mitotic spindle

2013 ◽  
Vol 24 (16) ◽  
pp. 2506-2514 ◽  
Author(s):  
Stephen L. Bird ◽  
Rebecca Heald ◽  
Karsten Weis
Keyword(s):  
Mitotic Spindle ◽  
Nucleocytoplasmic Transport ◽  
Cytoplasmic Dynein ◽  
Small Gtpase ◽  
Mitotic Apparatus ◽  
Spindle Positioning ◽  
Molecule Inhibitor ◽  
Pulling Forces ◽  
Cortical Localization ◽  
Β Function

Accurate positioning of the mitotic spindle is critical to ensure proper distribution of chromosomes during cell division. The small GTPase Ran, which regulates a variety of processes throughout the cell cycle, including interphase nucleocytoplasmic transport and mitotic spindle assembly, was recently shown to also control spindle alignment. Ran is required for the correct cortical localization of LGN and nuclear-mitotic apparatus protein (NuMA), proteins that generate pulling forces on astral microtubules (MTs) through cytoplasmic dynein. Here we use importazole, a small-molecule inhibitor of RanGTP/importin-β function, to study the role of Ran in spindle positioning in human cells. We find that importazole treatment results in defects in astral MT dynamics, as well as in mislocalization of LGN and NuMA, leading to misoriented spindles. Of interest, importazole-induced spindle-centering defects can be rescued by nocodazole treatment, which depolymerizes astral MTs, or by overexpression of CLASP1, which does not restore proper LGN and NuMA localization but stabilizes astral MT interactions with the cortex. Together our data suggest a model for mitotic spindle positioning in which RanGTP and CLASP1 cooperate to align the spindle along the long axis of the dividing cell.

scholarly journals Cytoplasmic dynein plays a role in mammalian mitotic spindle formation.

1993 ◽  
Vol 123 (4) ◽  
pp. 849-858 ◽  
Author(s):  
E A Vaisberg ◽  
M P Koonce ◽  
J R McIntosh
Keyword(s):  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Cytoplasmic Dynein ◽  
Detectable Effect ◽  
Spindle Formation ◽  
Bipolar Spindle ◽  
Dynein Motor ◽  
Motor Enzymes ◽  
Chromosome Attachment

The formation and functioning of a mitotic spindle depends not only on the assembly/disassembly of microtubules but also on the action of motor enzymes. Cytoplasmic dynein has been localized to spindles, but whether or how it functions in mitotic processes is not yet known. We have cloned and expressed DNA fragments that encode the putative ATP-hydrolytic sites of the cytoplasmic dynein heavy chain from HeLa cells and from Dictyostelium. Monospecific antibodies have been raised to the resulting polypeptides, and these inhibit dynein motor activity in vitro. Their injection into mitotic mammalian cells blocks the formation of spindles in prophase or during recovery from nocodazole treatment at later stages of mitosis. Cells become arrested with unseparated centrosomes and form monopolar spindles. The injected antibodies have no detectable effect on chromosome attachment to a bipolar spindle or on motions during anaphase. These data suggest that cytoplasmic dynein plays a unique and important role in the initial events of bipolar spindle formation, while any later roles that it may play are redundant. Possible mechanisms of dynein's involvement in mitosis are discussed.

scholarly journals Mechanical design principles of a mitotic spindle

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Jonathan J Ward ◽  
Hélio Roque ◽  
Claude Antony ◽  
François Nédélec
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Structural Integrity ◽  
Mechanical Design ◽  
Structural Components ◽  
Cell Type ◽  
And Function ◽  
Anaphase B ◽  
The Relationship ◽  
Spindle Structure

An organised spindle is crucial to the fidelity of chromosome segregation, but the relationship between spindle structure and function is not well understood in any cell type. The anaphase B spindle in fission yeast has a slender morphology and must elongate against compressive forces. This ‘pushing’ mode of chromosome transport renders the spindle susceptible to breakage, as observed in cells with a variety of defects. Here we perform electron tomographic analyses of the spindle, which suggest that it organises a limited supply of structural components to increase its compressive strength. Structural integrity is maintained throughout the spindle's fourfold elongation by organising microtubules into a rigid transverse array, preserving correct microtubule number and dynamically rescaling microtubule length.

scholarly journals Co-translational protein targeting facilitates centrosomal recruitment of PCNT during centrosome maturation in vertebrates

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Guadalupe Sepulveda ◽  
Mark Antkowiak ◽  
Ingrid Brust-Mascher ◽  
Karan Mahe ◽  
Tingyoung Ou ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Protein Targeting ◽  
Mrna Localization ◽  
Cytoplasmic Dynein ◽  
Animal Cells ◽  
Bipolar Spindle ◽  
Spindle Poles ◽  
Cytoplasmic Proteins ◽  
Pericentriolar Material ◽  
Early Mitosis

As microtubule-organizing centers of animal cells, centrosomes guide the formation of the bipolar spindle that segregates chromosomes during mitosis. At mitosis onset, centrosomes maximize microtubule-organizing activity by rapidly expanding the pericentriolar material (PCM). This process is in part driven by the large PCM protein pericentrin (PCNT), as its level increases at the PCM and helps recruit additional PCM components. However, the mechanism underlying the timely centrosomal enrichment of PCNT remains unclear. Here, we show that PCNT is delivered co-translationally to centrosomes during early mitosis by cytoplasmic dynein, as evidenced by centrosomal enrichment of PCNT mRNA, its translation near centrosomes, and requirement of intact polysomes for PCNT mRNA localization. Additionally, the microtubule minus-end regulator, ASPM, is also targeted co-translationally to mitotic spindle poles. Together, these findings suggest that co-translational targeting of cytoplasmic proteins to specific subcellular destinations may be a generalized protein targeting mechanism.

scholarly journals Pericentromere tension is self-regulated by spindle structure in metaphase

2014 ◽  
Vol 205 (3) ◽  
pp. 313-324 ◽  
Author(s):  
Jeremy M. Chacón ◽  
Soumya Mukherjee ◽  
Breanna M. Schuster ◽  
Duncan J. Clarke ◽  
Melissa K. Gardner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Division ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Chromosome Structure ◽  
Wild Type ◽  
Checkpoint Signaling ◽  
Daughter Cells ◽  
Fluorescent Markers ◽  
Spindle Structure

During cell division, a mitotic spindle is built by the cell and acts to align and stretch duplicated sister chromosomes before their ultimate segregation into daughter cells. Stretching of the pericentromeric chromatin during metaphase is thought to generate a tension-based signal that promotes proper chromosome segregation. However, it is not known whether the mitotic spindle actively maintains a set point tension magnitude for properly attached sister chromosomes to facilitate robust mechanochemical checkpoint signaling. By imaging and tracking the thermal movements of pericentromeric fluorescent markers in Saccharomyces cerevisiae, we measured pericentromere stiffness and then used the stiffness measurements to quantitatively evaluate the tension generated by pericentromere stretch during metaphase in wild-type cells and in mutants with disrupted chromosome structure. We found that pericentromere tension in yeast is substantial (4–6 pN) and is tightly self-regulated by the mitotic spindle: through adjustments in spindle structure, the cell maintains wild-type tension magnitudes even when pericentromere stiffness is disrupted.

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