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.

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 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 Chromosomally unstable tumor cells specifically require KIF18A for proliferation

2020 ◽  
Author(s):  
Carolyn Marquis ◽  
Cindy L. Fonseca ◽  
Katelyn A. Queen ◽  
Lisa Wood ◽  
Sarah E. Vandal ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Mitotic Spindle ◽  
Microtubule Dynamics ◽  
Strongly Correlated ◽  
Spindle Microtubule ◽  
Multipolar Spindles ◽  
Dynamics And Control ◽  
Diploid Cells ◽  
And Control ◽  
Dynamics Characteristic

SummaryChromosomal instability (CIN), characterized by frequent missegregation of chromosomes during mitosis, is a hallmark of tumor cells caused by changes in the dynamics and control of microtubules that comprise the mitotic spindle1–3. Thus, CIN tumor cells may respond differently than normal diploid cells to treatments that target mitotic spindle regulation. We tested this idea by inhibiting a subset of kinesin motor proteins that control spindle microtubule dynamics and mechanics but are not required for the proliferation of near-diploid cells. Our results indicated that KIF18A was required for proliferation of CIN cells derived from triple negative breast cancer or colorectal cancer tumors but was not required in near-diploid cells. CIN tumor cells exhibited mitotic delays, multipolar spindles due to centrosome fragmentation, and increased cell death following inhibition of KIF18A. Sensitivity to KIF18A knockdown was strongly correlated with centrosome fragmentation, which required dynamic microtubules but did not depend on bipolar spindle formation or mitotic arrest. Our results indicate the altered spindle microtubule dynamics characteristic of CIN tumor cells can be exploited to reduce the proliferative capacity of CIN cells.

scholarly journals Spastin Interacts with CRMP5 to Promote  Spindle Organization of Mouse Oocytes by  Severing Microtubules.

2020 ◽  
Author(s):  
Hua-Feng Shou ◽  
Lei-lei Gao ◽  
Zhen Jin ◽  
Jin-Wei Liu ◽  
Shan-Shan Jiang ◽  
...  
Keyword(s):  
Developmental Disorders ◽  
Spindle Assembly ◽  
Microtubule Dynamics ◽  
Spindle Microtubule ◽  
Microtubule Severing ◽  
Abnormal Phenotype ◽  
Mammalian Oocyte ◽  
Spindle Microtubules ◽  
Normal Meiosis

Abstract Background ： Microtubule-severing protein (MTSP) is highly critical for the survival of both mitotic and post-mitotic cells.However, the study of MTSP in the meiosis of mammalian oocyte has not been reported. Results ：We found that spastin, a member of the MTSP family, was highly expressed in oocyte and aggregated in spindle microtubules. After knocking down spastin by specific siRNA, the spindle microtubule density of meiotic oocyte decreased significantly. When the oocyte was cultured in vitro, the oocyte lacking spastin showed obvious maturation obstacles. Combining with the microtubule severing activity of spastin, we speculate that spastin on spindle may increase the microtubule broken ends by severing microtubules, thus playing a nucleating role, promoting spindle assembly and ensuring normal meiosis. In addition, we found that there was co-localization and interaction between CRMP5 and spastin in oocyte. The knockdown of CRMP5 may also lead to spindle abnormalities and developmental disorders in oocyte. Overexpression of spastin may save the abnormal phenotype caused by deletion of CRMP5. Conclusions ：To sum up, our data support a model in which the interaction between spastin and CRMP5 promotes the assembly of spindle microtubules in oocyte by controlling microtubule dynamics, thus ensuring normal meiosis.

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 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.

Katanin inhibition prevents the redistribution of γ-tubulin at mitosis

2002 ◽  
Vol 115 (5) ◽  
pp. 1083-1092 ◽  
Author(s):  
Dan Buster ◽  
Karen McNally ◽  
Francis J. McNally
Keyword(s):  
Mitotic Spindle ◽  
Fluorescence Recovery After Photobleaching ◽  
Immunofluorescence Microscopy ◽  
Dominant Negative ◽  
Spindle Microtubule ◽  
Microtubule Severing ◽  
Spindle Poles ◽  
Spindle Disassembly ◽  
A Subunit ◽  
Ring Complexes

Katanin is a microtubule-severing protein that is concentrated at mitotic spindle poles but katanin's function in the mitotic spindle has not been previously reported. Inhibition of katanin with either of two dominant-negative proteins or a subunit-specific antibody prevented the redistribution of γ-tubulin from the centrosome to the spindle in prometaphase CV-1 cells as assayed by immunofluorescence microscopy. Becauseγ-tubulin complexes can bind to pre-existing microtubule minus ends,these results could be explained by a model in which the broad distribution ofγ-tubulin in the mitotic spindle is in part due to cytosolicγ-tubulin ring complexes binding to microtubule minus ends generated by katanin-mediated microtubule severing. Because microtubules depolymerize at their ends, we hypothesized that a greater number of microtubule ends generated by severing in the spindle would result in an increased rate of spindle disassembly when polymerization is blocked with nocodazole. Indeed,katanin inhibition slowed the rate of spindle microtubule disassembly in the presence of nocodazole. However, katanin inhibition did not affect the rate of exchange between polymerized and unpolymerized tubulin as assayed by fluorescence recovery after photobleaching. These results support a model in which katanin activity regulates the number of microtubule ends in the spindle.

scholarly journals Spatial regulation of astral microtubule dynamics by Kif18B in PtK cells

2016 ◽  
Vol 27 (20) ◽  
pp. 3021-3030 ◽  
Author(s):  
Claire E. Walczak ◽  
Hailing Zong ◽  
Sachin Jain ◽  
Jane R. Stout
Keyword(s):  
Chromosome Segregation ◽  
Microtubule Dynamics ◽  
Cell Cortex ◽  
Microtubule Organization ◽  
Spatial Regulation ◽  
Astral Microtubule ◽  
Chromosome Alignment ◽  
Spindle Microtubules ◽  
Kinesin Superfamily ◽  
Insight Into

The spatial and temporal control of microtubule dynamics is fundamentally important for proper spindle assembly and chromosome segregation. This is achieved, in part, by the multitude of proteins that bind to and regulate spindle microtubules, including kinesin superfamily members, which act as microtubule-destabilizing enzymes. These fall into two general classes: the kinesin-13 proteins, which directly depolymerize microtubules, and the kinesin-8 proteins, which are plus end–directed motors that either destabilize microtubules or cap the microtubule plus ends. Here we analyze the contribution of a PtK kinesin-8 protein, Kif18B, in the control of mitotic microtubule dynamics. Knockdown of Kif18B causes defects in spindle microtubule organization and a dramatic increase in astral microtubules. Kif18B-knockdown cells had defects in chromosome alignment, but there were no defects in chromosome segregation. The long astral microtubules that occur in the absence of Kif18B are limited in length by the cell cortex. Using EB1 tracking, we show that Kif18B activity is spatially controlled, as loss of Kif18B has the most dramatic effect on the lifetimes of astral microtubules that extend toward the cell cortex. Together our studies provide new insight into how diverse kinesins contribute to spatial microtubule organization in the spindle.

AtNUF2 modulates spindle microtubule organization and chromosome segregation during mitosis

2021 ◽  
Author(s):  
Jin Li ◽  
Yutao Wang ◽  
Wenxuan Zou ◽  
Liufang Jian ◽  
Ying Fu ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Microtubule Organization ◽  
Spindle Microtubule
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