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

2002 ◽  
Vol 115 (5) ◽  
pp. 931-940 ◽  
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.

The kinesin-like protein CENP-E is kinetochore-associated throughout poleward chromosome segregation during anaphase-A

1996 ◽  
Vol 109 (5) ◽  
pp. 961-969 ◽  
Author(s):  
K.D. Brown ◽  
K.W. Wood ◽  
D.W. Cleveland
Keyword(s):  
Chromosome Segregation ◽  
Chromosome Movement ◽  
Initial Alignment ◽  
Late Prophase ◽  
Nuclear Envelope Breakdown ◽  
Anaphase Onset ◽  
Spindle Elongation ◽  
Anaphase B

The kinesin-like protein CENP-E transiently associates with kinetochores following nuclear envelope breakdown in late prophase, remains bound throughout metaphase, but sometime after anaphase onset it releases and by telophase becomes bound to interzonal microtubules of the mitotic spindle. Inhibition of poleward chromosome movement in vitro by CENP-E antibodies and association of CENP-E with minus-end directed microtubule motility in vitro have combined to suggest a key role for CENP-E as an anaphase chromosome motor. For this to be plausible in vivo depends on whether CENP-E remains kinetochore associated during anaphase. Using Indian muntjac cells whose seven chromosomes have large, easily tracked kinetochores, we now show that CENP-E is kinetochore-associated throughout the entirety of anaphase-A (poleward chromosome movement), relocating gradually during spindle elongation (anaphase-B) to the interzonal microtubules. These observations support roles for CENP-E not only in the initial alignment of chromosomes at metaphase and in spindle elongation in anaphase-B, but also in poleward chromosome movement in anaphase-A.

scholarly journals Microtubule dynamics and chromosome motion visualized in living anaphase cells.

1988 ◽  
Vol 106 (4) ◽  
pp. 1185-1192 ◽  
Author(s):  
G J Gorbsky ◽  
P J Sammak ◽  
G G Borisy
Keyword(s):  
Chromosome Segregation ◽  
Digital Imaging ◽  
Living Cells ◽  
Microtubule Dynamics ◽  
Chromosome Movement ◽  
Animal Cells ◽  
Digital Imaging Microscopy ◽  
Opposite Pole ◽  
Anaphase B ◽  
Chromosome Motion

Chromosome segregation in most animal cells is brought about through two events: the movement of the chromosomes to the poles (anaphase A) and the movement of the poles away from each other (anaphase B). Essential to an understanding of the mechanism of mitosis is information on the relative movements of components of the spindle and identification of sites of subunit loss from shortening microtubules. Through use of tubulin derivatized with X-rhodamine, photobleaching, and digital imaging microscopy of living cells, we directly determined the relative movements of poles, chromosomes, and a marked domain on kinetochore fibers during anaphase. During chromosome movement and pole-pole separation, the marked domain did not move significantly with respect to the near pole. Therefore, the kinetochore microtubules were shortened by the loss of subunits at the kinetochore, although a small amount of subunit loss elsewhere was not excluded. In anaphase A, chromosomes moved on kinetochore microtubules that remained stationary with respect to the near pole. In anaphase B, the kinetochore fiber microtubules accompanied the near pole in its movement away from the opposite pole. These results eliminate models of anaphase in which microtubules are thought to be traction elements that are drawn to and depolymerized at the pole. Our results are compatible with models of anaphase in which the kinetochore fiber microtubules remain anchored at the pole and in which microtubule dynamics are centered at the kinetochore.

Mitotic motors and chromosome segregation: the mechanism of anaphase B

2011 ◽  
Vol 39 (5) ◽  
pp. 1149-1153 ◽  
Author(s):  
Ingrid Brust-Mascher ◽  
Jonathan M. Scholey
Keyword(s):  
Chromosome Segregation ◽  
Cyclin B ◽  
Spindle Poles ◽  
Mitotic Motors ◽  
Balance Of Forces ◽  
Spindle Elongation ◽  
Anaphase B ◽  
Relationship Of ◽  
The Relationship ◽  
Acting In Concert

Anaphase B spindle elongation plays an important role in chromosome segregation. In the present paper, we discuss our model for anaphase B in Drosophila syncytial embryos, in which spindle elongation depends on an ip (interpolar) MT (microtubule) sliding filament mechanism generated by homotetrameric kinesin-5 motors acting in concert with poleward ipMT flux, which acts as an ‘on/off’ switch. Specifically, the pre-anaphase B spindle is maintained at a steady-state length by the balance between ipMT sliding and ipMT depolymerization at spindle poles, producing poleward flux. Cyclin B degradation at anaphase B onset triggers: (i) an MT catastrophe gradient causing ipMT plus ends to invade the overlap zone where ipMT sliding forces are generated; and (ii) the inhibition of ipMT minus-end depolymerization so flux is turned ‘off’, tipping the balance of forces to allow outward ipMT sliding to push apart the spindle poles. We briefly comment on the relationship of this model to anaphase B in other systems.

scholarly journals Anaphase A: Melting Microtubules Move Chromosomes toward Spindle Poles

2017 ◽  
Author(s):  
Charles L. Asbury
Keyword(s):  
Fluorescence Microscopy ◽  
Mechanistic Models ◽  
Chromosome Movement ◽  
Load Bearing ◽  
Spindle Poles ◽  
Sister Chromatids ◽  
Spindle Elongation ◽  
Anaphase B ◽  
Chromosome Movements ◽  
Cellular Movement

The separation of sister chromatids during anaphase is the culmination of mitosis and one of the most strikingly beautiful examples of cellular movement. It consists of two distinct processes: Anaphase A, the movement of chromosomes toward spindle poles via shortening of the connecting fibers, and anaphase B, separation of the two poles from one another via spindle elongation. I focus here on anaphase A chromosome-to-pole movement. The chapter begins by summarizing classical observations of chromosome movements, which support the current understanding of anaphase mechanisms. Live cell fluorescence microscopy studies showed that poleward chromosome movement is associated with disassembly, or ‘melting’ of the kinetochore-attached microtubule fibers that link chromosomes to poles. Microtubule-marking techniques established that kinetochore-fiber disassembly often occurs through a ‘pac-man’ mechanism, where tubulin subunits are lost from kinetochore-attached plus ends and the kinetochore appears to consume its microtubule track as it moves poleward. In addition, kinetochore-fiber disassembly in many cells occurs partly through ‘flux’, where the microtubules flow continuously toward the poles and tubulin subunits are lost from minus ends. Molecular mechanistic models for how load-bearing attachments are maintained to disassembling microtubule ends, and how the forces are generated to drive pac-man and flux-based movements, are discussed.

scholarly journals The microtubule cross-linker Feo controls the midzone stability, motor composition, and elongation of the anaphase B spindle in Drosophila embryos

2015 ◽  
Vol 26 (8) ◽  
pp. 1452-1462 ◽  
Author(s):  
Haifeng Wang ◽  
Ingrid Brust-Mascher ◽  
Jonathan M. Scholey
Keyword(s):  
Chromosome Segregation ◽  
Cross Linking ◽  
Cross Link ◽  
Spindle Poles ◽  
Cross Linker ◽  
Spindle Elongation ◽  
Anaphase B ◽  
Drosophila Embryos ◽  
Poleward Flux

Chromosome segregation during anaphase depends on chromosome-to-pole motility and pole-to-pole separation. We propose that in Drosophila embryos, the latter process (anaphase B) depends on a persistent kinesin-5–generated interpolar (ip) microtubule (MT) sliding filament mechanism that “engages” to push apart the spindle poles when poleward flux is turned off. Here we investigated the contribution of the midzonal, antiparallel MT-cross-linking nonmotor MAP, Feo, to this “slide-and-flux-or-elongate” mechanism. Whereas Feo homologues in other systems enhance the midzone localization of the MT-MT cross-linking motors kinesin-4, -5 and -6, the midzone localization of these motors is respectively enhanced, reduced, and unaffected by Feo. Strikingly, kinesin-5 localizes all along ipMTs of the anaphase B spindle in the presence of Feo, including at the midzone, but the antibody-induced dissociation of Feo increases kinesin-5 association with the midzone, which becomes abnormally narrow, leading to impaired anaphase B and incomplete chromosome segregation. Thus, although Feo and kinesin-5 both preferentially cross-link MTs into antiparallel polarity patterns, kinesin-5 cannot substitute for loss of Feo function. We propose that Feo controls the organization, stability, and motor composition of antiparallel ipMTs at the midzone, thereby facilitating the kinesin-5–driven sliding filament mechanism underlying proper anaphase B spindle elongation and chromosome segregation.

scholarly journals Yeast Sphingolipid Phospholipase Gene ISC1 Regulates the Spindle Checkpoint by a CDC55-Dependent Mechanism

2020 ◽  
Vol 40 (12) ◽  
Author(s):  
Nabil Matmati ◽  
Bachar H. Hassan ◽  
Jihui Ren ◽  
Ashraf A. Shamssedine ◽  
Eunmi Jeong ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Regulation ◽  
Spindle Assembly Checkpoint ◽  
Synthetic Lethality ◽  
Spindle Checkpoint ◽  
Cellular Functions ◽  
Spindle Elongation ◽  
Synthetically Lethal ◽  
Cycle Regulation ◽  
Checkpoint Genes

ABSTRACT Defects in the spindle assembly checkpoint (SAC) can lead to aneuploidy and cancer. Sphingolipids have important roles in many cellular functions, including cell cycle regulation and apoptosis. However, the specific mechanisms and functions of sphingolipids in cell cycle regulation have not been elucidated. Using analysis of concordance for synthetic lethality for the yeast sphingolipid phospholipase ISC1, we identified two groups of genes. The first comprises genes involved in chromosome segregation and stability (CSM3, CTF4, YKE2, DCC1, and GIM4) as synthetically lethal with ISC1. The second group, to which ISC1 belongs, comprises genes involved in the spindle checkpoint (BUB1, MAD1, BIM1, and KAR3), and they all share the same synthetic lethality with the first group. We demonstrate that spindle checkpoint genes act upstream of Isc1, and their deletion phenocopies that of ISC1. Reciprocally, ISC1 deletion mutants were sensitive to benomyl, indicating a SAC defect. Similar to BUB1 deletion, ISC1 deletion prevents spindle elongation in hydroxyurea-treated cells. Mechanistically, PP2A-Cdc55 ceramide-activated phosphatase was found to act downstream of Isc1, thus coupling the spindle checkpoint genes and Isc1 to CDC55-mediated nuclear functions.

scholarly journals High-resolution imaging reveals how the spindle midzone impacts chromosome movement

2019 ◽  
Vol 218 (8) ◽  
pp. 2529-2544 ◽  
Author(s):  
Melissa C. Pamula ◽  
Lina Carlini ◽  
Scott Forth ◽  
Priyanka Verma ◽  
Subbulakshmi Suresh ◽  
...  
Keyword(s):  
High Resolution ◽  
Chromosome Segregation ◽  
Live Cell Imaging ◽  
Chromosome Movement ◽  
Daughter Cells ◽  
Filament Bundles ◽  
Spindle Midzone ◽  
Resolution Imaging ◽  
Spindle Elongation ◽  
Length Reduction

In the spindle midzone, microtubules from opposite half-spindles form bundles between segregating chromosomes. Microtubule bundles can either push or restrict chromosome movement during anaphase in different cellular contexts, but how these activities are achieved remains poorly understood. Here, we use high-resolution live-cell imaging to analyze individual microtubule bundles, growing filaments, and chromosome movement in dividing human cells. Within bundles, filament overlap length marked by the cross-linking protein PRC1 decreases during anaphase as chromosome segregation slows. Filament ends within microtubule bundles appear capped despite dynamic PRC1 turnover and submicrometer proximity to growing microtubules. Chromosome segregation distance and rate are increased in two human cell lines when microtubule bundle assembly is prevented via PRC1 knockdown. Upon expressing a mutant PRC1 with reduced microtubule affinity, bundles assemble but chromosome hypersegregation is still observed. We propose that microtubule overlap length reduction, typically linked to pushing forces generated within filament bundles, is needed to properly restrict spindle elongation and position chromosomes within daughter cells.

scholarly journals Evidence for anaphase pulling forces during C. elegans meiosis

2020 ◽  
Vol 219 (12) ◽  
Author(s):  
Brennan M. Danlasky ◽  
Michelle T. Panzica ◽  
Karen P. McNally ◽  
Elizabeth Vargas ◽  
Cynthia Bailey ◽  
...  
Keyword(s):  
Spindle Pole ◽  
Outer Face ◽  
Chromosome Movement ◽  
Female Meiosis ◽  
Homologous Chromosomes ◽  
C Elegans ◽  
Spindle Poles ◽  
Pulling Forces ◽  
Spindle Elongation ◽  
Anaphase B

Anaphase chromosome movement is thought to be mediated by pulling forces generated by end-on attachment of microtubules to the outer face of kinetochores. However, it has been suggested that during C. elegans female meiosis, anaphase is mediated by a kinetochore-independent pushing mechanism with microtubules only attached to the inner face of segregating chromosomes. We found that the kinetochore proteins KNL-1 and KNL-3 are required for preanaphase chromosome stretching, suggesting a role in pulling forces. In the absence of KNL-1,3, pairs of homologous chromosomes did not separate and did not move toward a spindle pole. Instead, each homolog pair moved together with the same spindle pole during anaphase B spindle elongation. Two masses of chromatin thus ended up at opposite spindle poles, giving the appearance of successful anaphase.

scholarly journals The Essentiality of the Fungus-Specific Dam1 Complex Is Correlated with a One-Kinetochore-One-Microtubule Interaction Present throughout the Cell Cycle, Independent of the Nature of a Centromere

2011 ◽  
Vol 10 (10) ◽  
pp. 1295-1305 ◽  
Author(s):  
Jitendra Thakur ◽  
Kaustuv Sanyal
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Candida Albicans ◽  
Fission Yeast ◽  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Mitotic Chromosome ◽  
Content Type ◽  
Spindle Elongation ◽  
Dam1 Complex

ABSTRACTA fungus-specific outer kinetochore complex, the Dam1 complex, is essential inSaccharomyces cerevisiae, nonessential in fission yeast, and absent from metazoans. The reason for the reductive evolution of the functionality of this complex remains unknown. BothCandida albicansandSchizosaccharomyces pombehave regional centromeres as opposed to the short-point centromeres ofS. cerevisiae. The interaction of one microtubule per kinetochore is established both inS. cerevisiaeandC. albicansearly during the cell cycle, which is in contrast to the multiple microtubules that bind to a kinetochore only during mitosis inS. pombe. Moreover, the Dam1 complex is associated with the kinetochore throughout the cell cycle inS. cerevisiaeandC. albicansbut only during mitosis inS. pombe. Here, we show that the Dam1 complex is essential for viability and indispensable for proper mitotic chromosome segregation inC. albicans. The kinetochore localization of the Dam1 complex is independent of the kinetochore-microtubule interaction, but the function of this complex is monitored by a spindle assembly checkpoint. Strikingly, the Dam1 complex is required to prevent precocious spindle elongation in premitotic phases. Thus, constitutive kinetochore localization associated with a one-microtubule-one kinetochore type of interaction, but not the length of a centromere, is correlated with the essentiality of the Dam1 complex.

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

2018 ◽  
Author(s):  
Masashi Yukawa ◽  
Masaki Okazaki ◽  
Yasuhiro Teratani ◽  
Ken’ya Furuta ◽  
Takashi Toda
Keyword(s):  
Fission Yeast ◽  
Structural Integrity ◽  
Coiled Coil ◽  
Cell Cortex ◽  
Mitotic Spindles ◽  
Motor Region ◽  
Spindle Microtubules ◽  
Spindle Elongation ◽  
Synthetically Lethal ◽  
Anaphase B

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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