scholarly journals The kinesin-8 Kip3 scales anaphase spindle length by suppression of midzone microtubule polymerization

2014 ◽  
Vol 204 (6) ◽  
pp. 965-975 ◽  
Author(s):  
Rania S. Rizk ◽  
Katherine A. DiScipio ◽  
Kathleen G. Proudfoot ◽  
Mohan L. Gupta
Keyword(s):  
Mitotic Spindle ◽  
Molecular Mechanisms ◽  
Spindle Microtubule ◽  
Microtubule Polymerization ◽  
Sister Chromatids ◽  
Final Length ◽  
Spindle Microtubules ◽  
Spindle Elongation ◽  
Spindle Function ◽  
Yeast Mutants

Mitotic spindle function is critical for cell division and genomic stability. During anaphase, the elongating spindle physically segregates the sister chromatids. However, the molecular mechanisms that determine the extent of anaphase spindle elongation remain largely unclear. In a screen of yeast mutants with altered spindle length, we identified the kinesin-8 Kip3 as essential to scale spindle length with cell size. Kip3 is a multifunctional motor protein with microtubule depolymerase, plus-end motility, and antiparallel sliding activities. Here we demonstrate that the depolymerase activity is indispensable to control spindle length, whereas the motility and sliding activities are not sufficient. Furthermore, the microtubule-destabilizing activity is required to counteract Stu2/XMAP215-mediated microtubule polymerization so that spindle elongation terminates once spindles reach the appropriate final length. Our data support a model where Kip3 directly suppresses spindle microtubule polymerization, limiting midzone length. As a result, sliding forces within the midzone cannot buckle spindle microtubules, which allows the cell boundary to define the extent of spindle elongation.

scholarly journals Complexities of Microtubule Population Dynamics within the Mitotic Spindle

2019 ◽  
Author(s):  
Aaron R. Tipton ◽  
Gary J. Gorbsky
Keyword(s):  
Mitotic Spindle ◽  
Cell Cortex ◽  
Spindle Microtubule ◽  
Sister Chromatids ◽  
Two Component ◽  
Chromosome Attachment ◽  
Complex Landscape ◽  
Spindle Microtubules ◽  
Insight Into ◽  
Slow Turnover

AbstractThe mitotic spindle functions to move chromosomes to alignment at metaphase, then segregate sister chromatids during anaphase. Analysis of spindle microtubule kinetics utilizing fluorescence dissipation after photoactivation described two main populations, a slow and a fast turnover population, historically taken to reflect kinetochore versus non-kinetochore microtubules respectively. This two component demarcation seems likely oversimplified. Microtubule turnover may vary among different spindle microtubules, regulated by spatial distribution and interactions with other microtubules and with organelles such as kinetochores, chromosome arms, and the cell cortex. How turnover among various spindle microtubules is differentially regulated and its significance remains unclear. We tested the concept of kinetochore versus non-kinetochore microtubules by disrupting kinetochores through depletion of the Ndc80 complex. In the absence of functional kinetochores, microtubule dynamics still exhibited slow and fast turnover populations, though proportions and timings of turnover were altered. Importantly, the data obtained following Hec1/Ndc80 depletion suggests other sub-populations, in addition to kinetochore microtubules, contribute to the slow turnover population. Further manipulation of spindle microtubules revealed a complex landscape. Dissection of the dynamics of microtubule populations will provide a greater understanding of mitotic spindle kinetics and insight into roles in facilitating chromosome attachment, movement, and segregation during mitosis.

scholarly journals Molecular mechanisms facilitating the initial kinetochore encounter with spindle microtubules

2017 ◽  
Vol 216 (6) ◽  
pp. 1609-1622 ◽  
Author(s):  
Vanya Vasileva ◽  
Marek Gierlinski ◽  
Zuojun Yue ◽  
Nicola O’Reilly ◽  
Etsushi Kitamura ◽  
...  
Keyword(s):  
Half Life ◽  
Molecular Mechanisms ◽  
Significant Contribution ◽  
Budding Yeast ◽  
Computational Simulation ◽  
Spindle Microtubule ◽  
Multiple Factors ◽  
Spindle Microtubules ◽  
Rate Limiting

The initial kinetochore (KT) encounter with a spindle microtubule (MT; KT capture) is one of the rate-limiting steps in establishing proper KT–MT interaction during mitosis. KT capture is facilitated by multiple factors, such as MT extension in various directions, KT diffusion, and MT pivoting. In addition, KTs generate short MTs, which subsequently interact with a spindle MT. KT-derived MTs may facilitate KT capture, but their contribution is elusive. In this study, we find that Stu1 recruits Stu2 to budding yeast KTs, which promotes MT generation there. By removing Stu2 specifically from KTs, we show that KT-derived MTs shorten the half-life of noncaptured KTs from 48–49 s to 28–34 s. Using computational simulation, we found that multiple factors facilitate KT capture redundantly or synergistically. In particular, KT-derived MTs play important roles both by making a significant contribution on their own and by synergistically enhancing the effects of KT diffusion and MT pivoting. Our study reveals fundamental mechanisms facilitating the initial KT encounter with spindle MTs.

scholarly journals ULTRASTRUCTURAL ANALYSIS OF MITOTIC SPINDLE ELONGATION IN MAMMALIAN CELLS IN VITRO

1971 ◽  
Vol 50 (2) ◽  
pp. 416-431 ◽  
Author(s):  
B. R. Brinkley ◽  
Joiner Cartwright
Keyword(s):  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Spindle Axis ◽  
Hamster Strain ◽  
Early Anaphase ◽  
Spindle Microtubules ◽  
Spindle Elongation ◽  
Elongation Process

The mitotic spindle of many mammalian cells undergoes an abrupt elongation at anaphase. In both cultured rat kangaroo (strain PtK1) and Chinese hamster (strain Don-C) fibroblasts, the distance from pole to pole at metaphase doubles during anaphase and telophase. In order to determine the organization and distribution of spindle microtubules during the elongation process, cells were fixed and flat embedded in Epon 812. Selected cells were photographed with the phase-contrast microscope and then serially sectioned perpendicular to the major spindle axis. Microtubule profiles were counted in selected sections, and the number was plotted with respect to position along the spindle axis. Interpretation of the distribution profiles indicated that not all interpolar microtubules extended from pole to pole. It is estimated that 55–70% of the interpolar microtubules are overlapped at the cell equator while 30–45% extend across the equator into both half spindles. This arrangement appeared to persist from early anaphase (before elongation) until telophase after the elongation process. Although sliding or shearing of microtubules may occur in the spindle, such appears not to be the mechanism by which the spindle elongates in anaphase. Instead, our data support the hypothesis that spindle elongation occurs by growth of prepositioned microtubules which "push" the poles apart.

scholarly journals TPX2 phosphorylation maintains metaphase spindle length by regulating microtubule flux

2015 ◽  
Vol 210 (3) ◽  
pp. 373-383 ◽  
Author(s):  
Jingyan Fu ◽  
Minglei Bian ◽  
Guangwei Xin ◽  
Zhaoxuan Deng ◽  
Jia Luo ◽  
...  
Keyword(s):  
Steady State ◽  
Mitotic Spindle ◽  
Microtubule Dynamics ◽  
Flux Rate ◽  
Aurora A ◽  
Null Mutant ◽  
Constant Length ◽  
Microtubule Polymerization ◽  
Spindle Microtubules

A steady-state metaphase spindle maintains constant length, although the microtubules undergo intensive dynamics. Tubulin dimers are incorporated at plus ends of spindle microtubules while they are removed from the minus ends, resulting in poleward movement. Such microtubule flux is regulated by the microtubule rescue factors CLASPs at kinetochores and depolymerizing protein Kif2a at the poles, along with other regulators of microtubule dynamics. How microtubule polymerization and depolymerization are coordinated remains unclear. Here we show that TPX2, a microtubule-bundling protein and activator of Aurora A, plays an important role. TPX2 was phosphorylated by Aurora A during mitosis. Its phospho-null mutant caused short metaphase spindles coupled with low microtubule flux rate. Interestingly, phosphorylation of TPX2 regulated its interaction with CLASP1 but not Kif2a. The effect of its mutant in shortening the spindle could be rescued by codepletion of CLASP1 and Kif2a that abolished microtubule flux. Together we propose that Aurora A–dependent TPX2 phosphorylation controls mitotic spindle length through regulating microtubule flux.

scholarly journals Ensconsin/Map7 promotes microtubule growth and centrosome separation in Drosophila neural stem cells

2014 ◽  
Vol 204 (7) ◽  
pp. 1111-1121 ◽  
Author(s):  
Emmanuel Gallaud ◽  
Renaud Caous ◽  
Aude Pascal ◽  
Franck Bazile ◽  
Jean-Philippe Gagné ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Microtubule Associated Proteins ◽  
Microtubule Polymerization ◽  
Sister Chromatids ◽  
Centrosome Separation ◽  
Associated Proteins ◽  
Microtubule Growth ◽  
Mitotic Spindle Assembly

The mitotic spindle is crucial to achieve segregation of sister chromatids. To identify new mitotic spindle assembly regulators, we isolated 855 microtubule-associated proteins (MAPs) from Drosophila melanogaster mitotic or interphasic embryos. Using RNAi, we screened 96 poorly characterized genes in the Drosophila central nervous system to establish their possible role during spindle assembly. We found that Ensconsin/MAP7 mutant neuroblasts display shorter metaphase spindles, a defect caused by a reduced microtubule polymerization rate and enhanced by centrosome ablation. In agreement with a direct effect in regulating spindle length, Ensconsin overexpression triggered an increase in spindle length in S2 cells, whereas purified Ensconsin stimulated microtubule polymerization in vitro. Interestingly, ensc-null mutant flies also display defective centrosome separation and positioning during interphase, a phenotype also detected in kinesin-1 mutants. Collectively, our results suggest that Ensconsin cooperates with its binding partner Kinesin-1 during interphase to trigger centrosome separation. In addition, Ensconsin promotes microtubule polymerization during mitosis to control spindle length independent of Kinesin-1.

scholarly journals Spindle microtubule differentiation and deployment during micronuclear mitosis in Paramecium.

1985 ◽  
Vol 101 (5) ◽  
pp. 1966-1976 ◽  
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.

scholarly journals Kinesin-6 Klp9 orchestrates spindle elongation by regulating microtubule sliding and growth

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Lara Katharina Krüger ◽  
Matthieu Gélin ◽  
Liang Ji ◽  
Carlos Kikuti ◽  
Anne Houdusse ◽  
...  
Keyword(s):  
Dual Function ◽  
Tubulin Subunit ◽  
Microtubule Growth ◽  
Spindle Microtubules ◽  
Spindle Elongation ◽  
Spindle Stability ◽  
Spindle Function ◽  
Anaphase B

Mitotic spindle function depends on the precise regulation of microtubule dynamics and microtubule sliding. Throughout mitosis, both processes have to be orchestrated to establish and maintain spindle stability. We show that during anaphase B spindle elongation in S. pombe, the sliding motor Klp9 (kinesin-6) also promotes microtubule growth in vivo. In vitro, Klp9 can enhance and dampen microtubule growth, depending on the tubulin concentration. This indicates that the motor is able to promote and block tubulin subunit incorporation into the microtubule lattice in order to set a well-defined microtubule growth velocity. Moreover, Klp9 recruitment to spindle microtubules is dependent on its dephosphorylation mediated by XMAP215/Dis1, a microtubule polymerase, creating a link between the regulation of spindle length and spindle elongation velocity. Collectively, we unravel the mechanism of anaphase B, from Klp9 recruitment to the motors dual-function in regulating microtubule sliding and microtubule growth, allowing an inherent coordination of both processes.

scholarly journals Kinesin-5 promotes microtubule nucleation and assembly by stabilizing a lattice-competent conformation of tubulin

2019 ◽  
Author(s):  
Geng-Yuan Chen ◽  
Ana B. Asenjo ◽  
Yalei Chen ◽  
Jake Mascaro ◽  
David F. J. Arginteanu ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Polymerase Activity ◽  
Microtubule Nucleation ◽  
Domain Swap ◽  
Microtubule Polymerization ◽  
The Family ◽  
Microtubule Growth ◽  
Spindle Elongation ◽  
Necessary And Sufficient ◽  
Induced Changes

SummaryBesides sliding apart antiparallel microtubules during spindle elongation, the mitotic kinesin-5, Eg5 promotes microtubule polymerization, emphasizing its importance in mitotic spindle length control. Here, we characterize the Eg5 microtubule polymerase mechanism by assessing motor-induced changes in the longitudinal and lateral tubulin-tubulin bonds that form the microtubule lattice. Isolated Eg5 motor domains promote microtubule nucleation, growth and stability. Eg5 binds preferentially to microtubules over free tubulin, and colchicine-like inhibitors that stabilize the bent conformation of tubulin allosterically inhibit Eg5 binding, consistent with a model in which Eg5 induces a curved-to-straight transition in tubulin. Domain swap experiments establish that the family-specific Loop11, which resides near the nucleotide-sensing Switch-II domain, is necessary and sufficient for the polymerase activity of Eg5. Thus, we propose a microtubule polymerase mechanism in which Eg5 at the plus-end promotes a curved-to-straight transition in tubulin that enhances lateral bond formation and thereby promotes microtubule growth and stability.

scholarly journals Augmin: a protein complex required for centrosome-independent microtubule generation within the spindle

2008 ◽  
Vol 181 (3) ◽  
pp. 421-429 ◽  
Author(s):  
Gohta Goshima ◽  
Mirjam Mayer ◽  
Nan Zhang ◽  
Nico Stuurman ◽  
Ronald D. Vale
Keyword(s):  
Drosophila Melanogaster ◽  
Mitotic Spindle ◽  
Fiber Formation ◽  
Stable Complex ◽  
Human Homologue ◽  
Spindle Microtubule ◽  
Critical Function ◽  
Spindle Formation ◽  
Spindle Microtubules ◽  
And Function

Since the discovery of γ-tubulin, attention has focused on its involvement as a microtubule nucleator at the centrosome. However, mislocalization of γ-tubulin away from the centrosome does not inhibit mitotic spindle formation in Drosophila melanogaster, suggesting that a critical function for γ-tubulin might reside elsewhere. A previous RNA interference (RNAi) screen identified five genes (Dgt2–6) required for localizing γ-tubulin to spindle microtubules. We show that the Dgt proteins interact, forming a stable complex. We find that spindle microtubule generation is substantially reduced after knockdown of each Dgt protein by RNAi. Thus, the Dgt complex that we name “augmin” functions to increase microtubule number. Reduced spindle microtubule generation after augmin RNAi, particularly in the absence of functional centrosomes, has dramatic consequences on mitotic spindle formation and function, leading to reduced kinetochore fiber formation, chromosome misalignment, and spindle bipolarity defects. We also identify a functional human homologue of Dgt6. Our results suggest that an important mitotic function for γ-tubulin may lie within the spindle, where augmin and γ-tubulin function cooperatively to amplify the number of microtubules.

scholarly journals MCAK and Paclitaxel Have Differential Effects on Spindle Microtubule Organization and Dynamics

2009 ◽  
Vol 20 (6) ◽  
pp. 1639-1651 ◽  
Author(s):  
Rania S. Rizk ◽  
Kevin P. Bohannon ◽  
Laura A. Wetzel ◽  
James Powers ◽  
Sidney L. Shaw ◽  
...  
Keyword(s):  
Fluorescence Intensity ◽  
Mitotic Spindle ◽  
Fluorescence Recovery After Photobleaching ◽  
Microtubule Dynamics ◽  
Microtubule Organization ◽  
Spindle Microtubule ◽  
Quantitative Immunofluorescence ◽  
Spindle Microtubules ◽  
Low Levels ◽  
Paclitaxel Treatment

Within the mitotic spindle, there are multiple populations of microtubules with different turnover dynamics, but how these different dynamics are maintained is not fully understood. MCAK is a member of the kinesin-13 family of microtubule-destabilizing enzymes that is required for proper establishment and maintenance of the spindle. Using quantitative immunofluorescence and fluorescence recovery after photobleaching, we compared the differences in spindle organization caused by global suppression of microtubule dynamics, by treating cells with low levels of paclitaxel, versus specific perturbation of spindle microtubule subsets by MCAK inhibition. Paclitaxel treatment caused a disruption in spindle microtubule organization marked by a significant increase in microtubules near the poles and a reduction in K-fiber fluorescence intensity. This was correlated with a faster t1/2 of both spindle and K-fiber microtubules. In contrast, MCAK inhibition caused a dramatic reorganization of spindle microtubules with a significant increase in astral microtubules and reduction in K-fiber fluorescence intensity, which correlated with a slower t1/2 of K-fibers but no change in the t1/2 of spindle microtubules. Our data support the model that MCAK perturbs spindle organization by acting preferentially on a subset of microtubules, and they support the overall hypothesis that microtubule dynamics is differentially regulated in the spindle.

Sign in / Sign up

Export Citation Format

Share Document