Kinesin-6 Klp9 plays motor-dependent and -independent roles in collaboration with Kinesin-5 Cut7 and the microtubule crosslinker Ase1 in fission yeast

ABSTRACTBipolar mitotic spindles play a critical part in accurate chromosome segregation. During late mitosis, spindle microtubules undergo drastic elongation towards the cell cortex in a process called anaphase B. Two kinesin motors, Kinesin-5 and Kinesin-6, are thought to generate outward forces to drive spindle elongation, and the microtubule crosslinker Ase1/PRC1 maintains structural integrity of antiparallel microtubules. However, how these three proteins orchestrate this process remains unknown. Here we explore the functional interplay among fission yeast Kinesin-5/Cut7, Kinesin-6/Klp9 and Ase1. Using total internal reflection fluorescence microscopy, we show that Klp9 is a processive plus end-directed motor. klp9Δase1Δ is synthetically lethal. Surprisingly, this lethality is not ascribable to the defective motor activity of Klp9; instead, it is dependent upon an NLS and coiled coil domains within the non-motor region. We isolated a cut7 mutant (cut7-122) that displays temperature sensitivity only in the absence of Klp9. Interestingly, cut7-122 is impaired specifically in late mitotic stages. cut7-122klp9Δ double mutant cells exhibit additive defects in spindle elongation. Together, we propose that Klp9 plays dual roles during anaphase B; one is motor-dependent that collaborates with Cut7 in force generation, while the other is motor-independent and ensures structural integrity of spindle microtubules together with Ase1.

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Mcp5, a meiotic cell cortex protein, is required for nuclear movement mediated by dynein and microtubules in fission yeast

The Journal of Cell Biology ◽

10.1083/jcb.200512129 ◽

2006 ◽

Vol 173 (1) ◽

pp. 27-33 ◽

Cited By ~ 34

Author(s):

Takamune T. Saito ◽

Daisuke Okuzaki ◽

Hiroshi Nojima

Keyword(s):

Fission Yeast ◽

Meiotic Prophase ◽

Coiled Coil ◽

Cell Cortex ◽

Ph Domain ◽

Nuclear Movement ◽

Recombination Rates ◽

Homologous Chromosome Pairing ◽

Cortical Localization ◽

Pulling Force

During meiotic prophase I of the fission yeast Schizosaccharomyces pombe, oscillatory nuclear movement occurs. This promotes homologous chromosome pairing and recombination and involves cortical dynein, which plays a pivotal role by generating a pulling force with the help of an unknown dynein anchor. We show that Mcp5, the homologue of the budding yeast dynein anchor Num1, may be this putative dynein anchor. mcp5+ is predominantly expressed during meiotic prophase, and GFP-Mcp5 localizes at the cell cortex. Moreover, the mcp5Δ strain lacks the oscillatory nuclear movement. Accordingly, homologous pairing and recombination rates of the mcp5Δ strain are significantly reduced. Furthermore, the cortical localization of dynein heavy chain 1 appears to be reduced in mcp5Δ cells. Finally, the full function of Mcp5 requires its coiled-coil and pleckstrin homology (PH) domains. Our results suggest that Mcp5 localizes at the cell cortex through its PH domain and functions as a dynein anchor, thereby facilitating nuclear oscillation.

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Spindle dynamics and cell cycle regulation of dynein in the budding yeast, Saccharomyces cerevisiae.

The Journal of Cell Biology ◽

10.1083/jcb.130.3.687 ◽

1995 ◽

Vol 130 (3) ◽

pp. 687-700 ◽

Cited By ~ 272

Author(s):

E Yeh ◽

R V Skibbens ◽

J W Cheng ◽

E D Salmon ◽

K Bloom

Keyword(s):

Saccharomyces Cerevisiae ◽

Nuclear Translocation ◽

Spindle Pole ◽

Cell Cortex ◽

Wild Type ◽

Immunofluorescent Localization ◽

Yeast Saccharomyces Cerevisiae ◽

Spindle Dynamics ◽

Spindle Elongation ◽

Anaphase B

We have used time-lapse digital- and video-enhanced differential interference contrast (DE-DIC, VE-DIC) microscopy to study the role of dynein in spindle and nuclear dynamics in the yeast Saccharomyces cerevisiae. The real-time analysis reveals six stages in the spindle cycle. Anaphase B onset appears marked by a rapid phase of spindle elongation, simultaneous with nuclear migration into the daughter cell. The onset and kinetics of rapid spindle elongation are identical in wild type and dynein mutants. In the absence of dynein the nucleus does not migrate as close to the neck as in wild-type cells and initial spindle elongation is confined primarily to the mother cell. Rapid oscillations of the elongating spindle between the mother and bud are observed in wild-type cells, followed by a slower growth phase until the spindle reaches its maximal length. This stage is protracted in the dynein mutants and devoid of oscillatory motion. Thus dynein is required for rapid penetration of the nucleus into the bud and anaphase B spindle dynamics. Genetic analysis reveals that in the absence of a functional central spindle (ndcl), dynein is essential for chromosome movement into the bud. Immunofluorescent localization of dynein-beta-galactosidase fusion proteins reveals that dynein is associated with spindle pole bodies and the cell cortex: with spindle pole body localization dependent on intact microtubules. A kinetic analysis of nuclear movement also revealed that cytokinesis is delayed until nuclear translocation is completed, indicative of a surveillance pathway monitoring nuclear transit into the bud.

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Central-spindle microtubules are strongly coupled to chromosomes during both anaphase A and anaphase B

Molecular Biology of the Cell ◽

10.1091/mbc.e19-01-0074 ◽

2019 ◽

Vol 30 (19) ◽

pp. 2503-2514 ◽

Cited By ~ 11

Author(s):

Che-Hang Yu ◽

Stefanie Redemann ◽

Hai-Yin Wu ◽

Robert Kiewisz ◽

Tae Yeon Yoo ◽

...

Keyword(s):

Chromosome Segregation ◽

Tomographic Reconstruction ◽

Mitotic Spindles ◽

Strongly Coupled ◽

Central Spindle ◽

Spindle Poles ◽

Meiotic Spindles ◽

Spindle Microtubules ◽

Anaphase B ◽

Chromosome Motion

Spindle microtubules, whose dynamics vary over time and at different locations, cooperatively drive chromosome segregation. Measurements of microtubule dynamics and spindle ultrastructure can provide insight into the behaviors of microtubules, helping elucidate the mechanism of chromosome segregation. Much work has focused on the dynamics and organization of kinetochore microtubules, that is, on the region between chromosomes and poles. In comparison, microtubules in the central-spindle region, between segregating chromosomes, have been less thoroughly characterized. Here, we report measurements of the movement of central-spindle microtubules during chromosome segregation in human mitotic spindles and Caenorhabditis elegans mitotic and female meiotic spindles. We found that these central-spindle microtubules slide apart at the same speed as chromosomes, even as chromosomes move toward spindle poles. In these systems, damaging central-spindle microtubules by laser ablation caused an immediate and complete cessation of chromosome motion, suggesting a strong coupling between central-spindle microtubules and chromosomes. Electron tomographic reconstruction revealed that the analyzed anaphase spindles all contain microtubules with both ends between segregating chromosomes. Our results provide new dynamical, functional, and ultrastructural characterizations of central-spindle microtubules during chromosome segregation in diverse spindles and suggest that central-spindle microtubules and chromosomes are strongly coupled in anaphase.

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Interdependency of Fission Yeast Alp14/TOG and Coiled Coil Protein Alp7 in Microtubule Localization and Bipolar Spindle Formation

Molecular Biology of the Cell ◽

10.1091/mbc.e03-11-0837 ◽

2004 ◽

Vol 15 (4) ◽

pp. 1609-1622 ◽

Cited By ~ 61

Author(s):

Masamitsu Sato ◽

Leah Vardy ◽

Miguel Angel Garcia ◽

Nirada Koonrugsa ◽

Takashi Toda

Keyword(s):

Fission Yeast ◽

Spindle Pole Body ◽

Coiled Coil ◽

Spindle Pole ◽

Mitotic Spindles ◽

Microtubule Organization ◽

Spindle Formation ◽

Bipolar Spindle ◽

C Terminus ◽

Step Model

The Dis1/TOG family plays a pivotal role in microtubule organization. In fission yeast, Alp14 and Dis1 share an essential function in bipolar spindle formation. Here, we characterize Alp7, a novel coiled-coil protein that is required for organization of bipolar spindles. Both Alp7 and Alp14 colocalize to the spindle pole body (SPB) and mitotic spindles. Alp14 localization to these sites is fully dependent upon Alp7. Conversely, in the absence of Alp14, Alp7 localizes to the SPBs, but not mitotic spindles. Alp7 forms a complex with Alp14, where the C-terminal region of Alp14 interacts with the coiled-coil domain of Alp7. Intriguingly, this Alp14 C terminus is necessary and sufficient for mitotic spindle localization. Overproduction of either full-length or coiled-coil region of Alp7 results in abnormal V-shaped spindles and stabilization of interphase microtubules, which is induced independent of Alp14. Alp7 may be a functional homologue of animal TACC. Our results shed light on an interdependent relationship between Alp14/TOG and Alp7. We propose a two-step model that accounts for the recruitment of Alp7 and Alp14 to the SPB and microtubules.

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Central spindle microtubules are strongly coupled to chromosomes during both anaphase A and anaphase B

10.1101/537290 ◽

2019 ◽

Cited By ~ 2

Author(s):

Che-Hang Yu ◽

Stefanie Redemann ◽

Hai-Yin Wu ◽

Robert Kiewisz ◽

Tae Yeon Yoo ◽

...

Keyword(s):

Chromosome Segregation ◽

Tomographic Reconstruction ◽

Mitotic Spindles ◽

Strongly Coupled ◽

Central Spindle ◽

Spindle Poles ◽

Meiotic Spindles ◽

Spindle Microtubules ◽

Anaphase B ◽

Chromosome Motion

AbstractSpindle microtubules, whose dynamics vary over time and at different locations, cooperatively drive chromosome segregation. Measurements of microtubule dynamics and spindle ultrastructure can provide insight into the behaviors of microtubules, helping elucidate the mechanism of chromosome segregation. Much work has focused on the dynamics and organization of kinetochore microtubules, i.e. on the region between chromosomes and poles. In comparison, microtubules in the central spindle region, between segregating chromosomes, have been less thoroughly characterized. Here, we report measurements of the movement of central spindle microtubules during chromosome segregation in human mitotic spindles, and Caenorhabditis elegans mitotic and female meiotic spindles. We found that these central spindle microtubules slide apart at the same speed as chromosomes, even as chromosomes move towards spindle poles. In these systems, damaging central spindle microtubules by laser ablation caused an immediate and complete cessation of chromosome motion, suggesting a strong coupling between central spindle microtubules and chromosomes. Electron tomographic reconstruction revealed that the analyzed anaphase spindles all contain microtubules with both ends between segregating chromosomes. Our results provide new dynamical, functional, and ultrastructural characterizations of central spindle microtubules during chromosome segregation in diverse spindles, and suggest that central spindle microtubules and chromosomes are strongly coupled in anaphase.

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Kinesinsklp5+ andklp6+ are required for normal chromosome movement in mitosis

Journal of Cell Science ◽

10.1242/jcs.115.5.931 ◽

2002 ◽

Vol 115 (5) ◽

pp. 931-940 ◽

Cited By ~ 13

Author(s):

Robert R. West ◽

Terra Malmstrom ◽

J. Richard McIntosh

Keyword(s):

Chromosome Segregation ◽

Spindle Assembly Checkpoint ◽

Extended Period ◽

Normal Chromosome ◽

Chromosome Movement ◽

Dynamic Interactions ◽

Spindle Elongation ◽

Synthetically Lethal ◽

Anaphase B ◽

Null Mutants

Proper mitotic chromosome segregation requires dynamic interactions between spindle microtubules and kinetochores. Here we demonstrate that two related fission yeast kinesins, klp5+ and klp6+, are required for normal chromosome segregation in mitosis. Null mutants frequently lack a normal metaphase chromosome alignment. Chromosome pairs move back and forth along the spindle for an extended period prior to sister chromatid separation, a phenotype reminiscent of the loss of CENP-E in metazoans. Ultimately, sister chromatids segregate, regardless of chromosome position along the spindle, and viable daughter cells are usually produced. The initiation of anaphase B is sometimes delayed, but the rate of spindle elongation is similar to wildtype. Despite a delay, anaphase B often begins before anaphase A is completed. The klp5Δ and klp6Δ null mutants are synthetically lethal with a deletion of the spindle assembly checkpoint gene, bub1+, several mutants in components of the anaphase promoting complex, and a cold sensitive allele of the kinetochore and microtubule-binding protein, Dis1p. Klp5p-GFP and Klp6p-GFP localize to kinetochores from prophase to the onset of anaphase A, but relocalize to the spindle midzone during anaphase B. These data indicate that Klp5p and Klp6p are kinetochore kinesins required for normal chromosome movement in prometaphase.

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Spindle microtubule differentiation and deployment during micronuclear mitosis in Paramecium.

The Journal of Cell Biology ◽

10.1083/jcb.101.5.1966 ◽

1985 ◽

Vol 101 (5) ◽

pp. 1966-1976 ◽

Cited By ~ 18

Author(s):

J B Tucker ◽

S A Mathews ◽

K A Hendry ◽

J B Mackie ◽

D L Roche

Keyword(s):

Nuclear Envelope ◽

Early Stage ◽

Microtubule Assembly ◽

Membrane Association ◽

Paramecium Tetraurelia ◽

Spindle Microtubule ◽

Spatial Control ◽

Spindle Microtubules ◽

Spindle Elongation ◽

Anaphase B

Spindles underwent a 12-fold elongation before anaphase B was completed during the closed mitoses of micronuclei in Paramecium tetraurelia. Two main classes of spindle microtubules have been identified. A peripheral sheath of microtubules with diameters of 27-32 nm was found to be associated with the nuclear envelope and confined to the midportion of each spindle. Most of the other microtubules had diameters of approximately 24 nm and were present along the entire lengths of spindles. Nearly all of the 24-nm microtubules were eliminated from spindle midportions (largely because of microtubule disassembly) at a relatively early stage of spindle elongation. Disassembly of some of these microtubules also occurred at the ends of spindles. About 60% of the total microtubule content of spindles was lost at this stage. Most, perhaps all, peripheral sheath microtubules remained intact. Many of them detached from the nuclear envelope and regrouped to form a compact microtubule bundle in the spindle midportion. There was little, if any, further polymerization of 24-nm microtubules after the disassembly phase. Polymerization of microtubules with diameters of 27-32 nm continued as spindle elongation progressed. Most microtubules in the midportions of well-elongated spindles were constructed from 14-16 protofilaments. A few 24-nm microtubules with 13 protofilaments were also present. The implications of these findings for spatial control of microtubule assembly, disassembly, positioning, and membrane association, that apparently discriminate between microtubules with different protofilament numbers have been explored. The possibility that microtubule sliding occurs during spindle elongation has also been considered.

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Intramitotic controls in the fission yeast Schizosaccharomyces pombe: the effect of cell size on spindle length and the timing of mitotic events.

The Journal of Cell Biology ◽

10.1083/jcb.110.5.1617 ◽

1990 ◽

Vol 110 (5) ◽

pp. 1617-1621 ◽

Cited By ~ 25

Author(s):

I M Hagan ◽

P N Riddle ◽

J S Hyams

Keyword(s):

Schizosaccharomyces Pombe ◽

Fission Yeast ◽

Average Length ◽

Nuclear Migration ◽

Yeast Cells ◽

Reverse Direction ◽

Wild Type ◽

Spindle Elongation ◽

Anaphase B ◽

In Cells

We have used a new cinemicroscopy technique in combination with antitubulin immunofluorescence microscopy to investigate the timing of mitotic events in cells of the fission yeast Schizosaccharomyces pombe having lengths at division between 7 and 60 microns. Wild-type fission yeast cells divide at a length of 14 microns. Separation of daughter nuclei (anaphase B) proceeds at a rate of 1.6 +/- 0.2 microns min-1, until the spindle extends the length of the cell. Coincident with spindle depolymerization, the nuclei reverse direction and take up positions that will become the center of the two daughter cells. This post-mitotic nuclear migration occurs at a rate of 1.4 +/- 0.5 microns-1. In cells in which the weel+ gene is overexpressed fivefold and that have an average length at mitosis of 28 microns, the rate of nuclear separation was only slightly reduced but, as spindles in these cells measure 20-22 microns, the duration of anaphase B was extended by approximately 40%. By contrast, in the mutant weel.50, which divides at 7 microns, both the rate and duration of anaphase B were indistinguishable from wild type. Nuclei reach the ends of these cells earlier but remain there until a point corresponding to the time of postmitotic nuclear migration in wild type. Thus, the events of mitosis can be extended but not abbreviated. These results are discussed in terms of a mitotic termination control that monitors many different events, one of which is spindle elongation.

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Drosophila EB1 is important for proper assembly, dynamics, and positioning of the mitotic spindle

The Journal of Cell Biology ◽

10.1083/jcb.200202032 ◽

2002 ◽

Vol 158 (5) ◽

pp. 873-884 ◽

Cited By ~ 265

Author(s):

Stephen L. Rogers ◽

Gregory C. Rogers ◽

David J. Sharp ◽

Ronald D. Vale

Keyword(s):

Cell Attachment ◽

Cell Cortex ◽

Mitotic Spindles ◽

Microtubule Organization ◽

Chromosomal Segregation ◽

Spindle Poles ◽

Evolutionarily Conserved ◽

Interphase Cells ◽

Spindle Elongation ◽

Drosophila Embryos

EB1 is an evolutionarily conserved protein that localizes to the plus ends of growing microtubules. In yeast, the EB1 homologue (BIM1) has been shown to modulate microtubule dynamics and link microtubules to the cortex, but the functions of metazoan EB1 proteins remain unknown. Using a novel preparation of the Drosophila S2 cell line that promotes cell attachment and spreading, we visualized dynamics of single microtubules in real time and found that depletion of EB1 by RNA-mediated inhibition (RNAi) in interphase cells causes a dramatic increase in nondynamic microtubules (neither growing nor shrinking), but does not alter overall microtubule organization. In contrast, several defects in microtubule organization are observed in RNAi-treated mitotic cells, including a drastic reduction in astral microtubules, malformed mitotic spindles, defocused spindle poles, and mispositioning of spindles away from the cell center. Similar phenotypes were observed in mitotic spindles of Drosophila embryos that were microinjected with anti-EB1 antibodies. In addition, live cell imaging of mitosis in Drosophila embryos reveals defective spindle elongation and chromosomal segregation during anaphase after antibody injection. Our results reveal crucial roles for EB1 in mitosis, which we postulate involves its ability to promote the growth and interactions of microtubules within the central spindle and at the cell cortex.

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In vitro reactivation of spindle elongation in fission yeast nuc2 mutant cells.

The Journal of Cell Biology ◽

10.1083/jcb.110.2.417 ◽

1990 ◽

Vol 110 (2) ◽

pp. 417-425 ◽

Cited By ~ 35

Author(s):

H Masuda ◽

T Hirano ◽

M Yanagida ◽

W Z Cande

Keyword(s):

Fission Yeast ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Sensitive Mutant ◽

Vitro Assay ◽

Chromosome Separation ◽

Spindle Microtubules ◽

Spindle Elongation ◽

Mutant Cells

To investigate the mechanisms of spindle elongation and chromosome separation in the fission yeast Schizosaccharomyces pombe, we have developed an in vitro assay using a temperature-sensitive mutant strain, nuc2. At the restrictive temperature, nuc2 cells are arrested at a metaphase-like stage with short spindles and condensed chromosomes. After permeabilization of spheroplasts of the arrested cells, spindle elongation was reactivated by addition of ATP and neurotubulin both at the restrictive and the permissive temperatures, but chromosome separation was not. This suggests that the nuc2 cells are impaired in function at a stage before sister chromatid disjunction. Spindle elongation required both ATP and exogenous tubulin and was inhibited by adenylyl imidodiphosphate (AMPPNP) or vanadate. The ends of yeast half-spindle microtubules pulse-labeled with biotinylated tubulin moved past each other during spindle elongation and a gap formed between the original half-spindles. These results suggest that the primary mechanochemical event responsible for spindle elongation is the sliding apart of antiparallel microtubules of the two half-spindles.

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