scholarly journals Microtubule poleward flux in human cells is driven by the coordinated action of four kinesins

2020 ◽  
Author(s):  
Yulia Steblyanko ◽  
Girish Rajendraprasad ◽  
Mariana Osswald ◽  
Susana Eibes ◽  
Stephan Geley ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Driving Force ◽  
Molecular Mechanisms ◽  
Human Cells ◽  
Flux Rate ◽  
Loss Of Function ◽  
Coordinated Action ◽  
Spindle Microtubules ◽  
And Function ◽  
Poleward Flux

AbstractMitotic spindle microtubules (MTs) undergo continuous poleward flux, whose driving force and function in humans remain unclear. Here, we combined loss-of-function screenings with analysis of MT dynamics in human cells to investigate the molecular mechanisms underlying MT-flux. We report that kinesin-7/CENP-E at kinetochores (KTs) is the predominant driver of MT-flux in early prometaphase, while kinesin-4/KIF4A on chromosome arms facilitates MT-flux during late prometaphase and metaphase. We show that both of these activities work in coordination with MT-crosslinking motors kinesin-5/EG5 and kinesin-12/KIF15. Our data further indicate that MT-flux driving force is transmitted from non-KT MTs to KT-MTs via MT-coupling by HSET and NuMA. Moreover, we found that MT-flux rate correlates with spindle size and this correlation depends on the establishment of stable end-on KT-MT attachments. Strikingly, we revealed that flux is required to counteract the kinesin 13/MCAK-dependent MT-depolymerization to regulate spindle length. Thus, our study demonstrates that MT-flux in human cells is driven by the coordinated action of four kinesins, and is required to regulate mitotic spindle size in response to MCAK-mediated MT-depolymerizing activity at KTs.

Katanin, the microtubule-severing ATPase, is concentrated at centrosomes

1996 ◽  
Vol 109 (3) ◽  
pp. 561-567 ◽  
Author(s):  
F.J. McNally ◽  
K. Okawa ◽  
A. Iwamatsu ◽  
R.D. Vale
Keyword(s):  
Mitotic Spindle ◽  
Dynamic Properties ◽  
Mitotic Spindles ◽  
Microtubule Severing ◽  
Spindle Microtubules ◽  
Gamma Tubulin ◽  
And Function ◽  
Poleward Flux

The assembly and function of the mitotic spindle involve specific changes in the dynamic properties of microtubules. One such change results in the poleward flux of tubulin in which spindle microtubules polymerize at their kinetochore-attached plus ends while they shorten at their centrosome-attached minus ends. Since free microtubule minus ends do not depolymerize in vivo, the poleward flux of tubulin suggests that spindle microtubules are actively disassembled at or near their centrosomal attachment points. The microtubule-severing ATPase, katanin, has the ability actively to sever and disassemble microtubules and is thus a candidate for the role of a protein mediating the poleward flux of tubulin. Here we determine the subcellular localization of katanin by immunofluorescence as a preliminary step in determining whether katanin mediates the poleward flux of tubulin. We find that katanin is highly concentrated at centrosomes throughout the cell cycle. Katanin's localization is different from that of gamma-tubulin in that microtubules are required to maintain the centrosomal localization of katanin. Direct comparison of the localization of katanin and gamma-tubulin reveals that katanin is localized in a region surrounding the gamma-tubulin-containing pericentriolar region in detergent-extracted mitotic spindles. The centrosomal localization of katanin is consistent with the hypothesis that katanin mediates the disassembly of microtubule minus ends during poleward flux.

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 Poleward Tubulin Flux in Spindles: Regulation and Function in Mitotic Cells

2007 ◽  
Vol 18 (8) ◽  
pp. 3094-3104 ◽  
Author(s):  
Daniel W. Buster ◽  
Dong Zhang ◽  
David J. Sharp
Keyword(s):  
Rna Interference ◽  
Microtubule Dynamics ◽  
S2 Cells ◽  
Flux Rate ◽  
Chromosome Congression ◽  
Functional Antagonism ◽  
Spindle Microtubules ◽  
And Function ◽  
Molecular Components ◽  
Mitotic Cells

The poleward flux of tubulin subunits through spindle microtubules is a striking and conserved phenomenon whose function and molecular components remain poorly understood. To screen for novel components of the flux machinery, we utilized RNA interference to deplete regulators of microtubule dynamics, individually and in various combinations, from S2 cells and examined the resulting impact on flux rate. This led to the identification of two previously unknown flux inhibitors, KLP59C and KLP67A, and a flux promoter, Mini-spindles. Furthermore, we find that flux rate is regulated by functional antagonism among microtubule stabilizers and destabilizers specifically at plus ends. Finally, by examining mitosis on spindles in which flux has been up- or down-regulated or restored after the codepletion of antagonistic flux regulators, we show that flux is an integral contributor to anaphase A but is not responsible for chromosome congression, interkinetochore tension, or the establishment of normal spindle length during prometaphase/metaphase.

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 Molecular Mechanisms and Therapeutics for SBMA/Kennedy’s Disease

2019 ◽  
Vol 16 (4) ◽  
pp. 928-947 ◽  
Author(s):  
Frederick J. Arnold ◽  
Diane E. Merry
Keyword(s):  
Motor Neurons ◽  
Molecular Mechanisms ◽  
Muscular Atrophy ◽  
Structure And Function ◽  
Loss Of Function ◽  
Mechanisms Of Toxicity ◽  
Current Review ◽  
And Function ◽  
Positive Results ◽  
Polyq Expansion

Abstract Spinal and bulbar muscular atrophy (SBMA) is a neuromuscular disease caused by a polyglutamine (polyQ) expansion in the androgen receptor (AR). Despite the fact that the monogenic cause of SBMA has been known for nearly 3 decades, there is no effective treatment for this disease, underscoring the complexity of the pathogenic mechanisms that lead to a loss of motor neurons and muscle in SBMA patients. In the current review, we provide an overview of the system-wide clinical features of SBMA, summarize the structure and function of the AR, discuss both gain-of-function and loss-of-function mechanisms of toxicity caused by polyQ-expanded AR, and describe the cell and animal models utilized in the study of SBMA. Additionally, we summarize previously conducted clinical trials which, despite being based on positive results from preclinical studies, proved to be largely ineffective in the treatment of SBMA; nonetheless, these studies provide important insights as researchers develop the next generation of therapies.

scholarly journals The Zebra fish cassiopeia Mutant Reveals that SIL Is Required for Mitotic Spindle Organization

2007 ◽  
Vol 27 (16) ◽  
pp. 5887-5897 ◽  
Author(s):  
Kathleen L. Pfaff ◽  
Christian T. Straub ◽  
Ken Chiang ◽  
Daniel M. Bear ◽  
Yi Zhou ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Human Cells ◽  
Zebra Fish ◽  
Loss Of Function ◽  
Mitotic Spindles ◽  
Hairpin Rna ◽  
Chromosome Separation ◽  
Mitotic Spindle Assembly ◽  
Short Hairpin ◽  
Rna Knockdown

ABSTRACT A critical step in cell division is formation of the mitotic spindle, which is a bipolar array of microtubules that mediates chromosome separation. Here, we report that the SCL-interrupting locus (SIL), a vertebrate-specific cytosolic protein, is necessary for proper mitotic spindle organization in zebrafish and human cells. A homozygous lethal zebrafish mutant, cassiopeia (csp), was identified by a genetic screen for mitotic mutant. csp mutant embryos have an increased mitotic index, have highly disorganized mitotic spindles, and often lack one or both centrosomes. These phenotypes are caused by a loss-of-function mutation in zebrafish sil. To determine if the requirement for SIL in mitotic spindle organization is conserved in mammals, we generated an antibody against human SIL, which revealed that SIL localizes to the poles of the mitotic spindle during metaphase. Furthermore, short hairpin RNA knockdown of SIL in human cells recapitulates the zebrafish csp mitotic spindle defects. These data, taken together, identify SIL as a novel, vertebrate-specific regulator of mitotic spindle assembly.

scholarly journals Cohesion promotes nucleolar structure and function

2014 ◽  
Vol 25 (3) ◽  
pp. 337-346 ◽  
Author(s):  
Bethany Harris ◽  
Tania Bose ◽  
Kenneth K. Lee ◽  
Fei Wang ◽  
Shuai Lu ◽  
...  
Keyword(s):  
Ribosome Biogenesis ◽  
Molecular Mechanisms ◽  
Human Cells ◽  
Rna Polymerase I ◽  
Nucleolar Structure ◽  
Roberts Syndrome ◽  
Evolutionarily Conserved ◽  
Rrna Cleavage ◽  
And Function ◽  
Polymerase I

The cohesin complex contributes to ribosome function, although the molecular mechanisms involved are unclear. Compromised cohesin function is associated with a class of diseases known as cohesinopathies. One cohesinopathy, Roberts syndrome (RBS), occurs when a mutation reduces acetylation of the cohesin Smc3 subunit. Mutation of the cohesin acetyltransferase is associated with impaired rRNA production, ribosome biogenesis, and protein synthesis in yeast and human cells. Cohesin binding to the ribosomal DNA (rDNA) is evolutionarily conserved from bacteria to human cells. We report that the RBS mutation in yeast (eco1-W216G) exhibits a disorganized nucleolus and reduced looping at the rDNA. RNA polymerase I occupancy of the genes remains normal, suggesting that recruitment is not impaired. Impaired rRNA production in the RBS mutant coincides with slower rRNA cleavage. In addition to the RBS mutation, mutations in any subunit of the cohesin ring are associated with defects in ribosome biogenesis. Depletion or artificial destruction of cohesion in a single cell cycle is associated with loss of nucleolar integrity, demonstrating that the defects at the rDNA can be directly attributed to loss of cohesion. Our results strongly suggest that organization of the rDNA provided by cohesion is critical for formation and function of the nucleolus.

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 Aurora A contributes to p150glued phosphorylation and function during mitosis

2010 ◽  
Vol 189 (4) ◽  
pp. 651-659 ◽  
Author(s):  
Pierre Romé ◽  
Emilie Montembault ◽  
Nathalie Franck ◽  
Aude Pascal ◽  
David M. Glover ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Microtubule Binding ◽  
Microtubule Binding Domain ◽  
Spindle Microtubules ◽  
And Function

Aurora A is a spindle pole–associated protein kinase required for mitotic spindle assembly and chromosome segregation. In this study, we show that Drosophila melanogaster aurora A phosphorylates the dynactin subunit p150glued on sites required for its association with the mitotic spindle. Dynactin strongly accumulates on microtubules during prophase but disappears as soon as the nuclear envelope breaks down, suggesting that its spindle localization is tightly regulated. If aurora A's function is compromised, dynactin and dynein become enriched on mitotic spindle microtubules. Phosphorylation sites are localized within the conserved microtubule-binding domain (MBD) of the p150glued. Although wild-type p150glued binds weakly to spindle microtubules, a variant that can no longer be phosphorylated by aurora A remains associated with spindle microtubules and fails to rescue depletion of endogenous p150glued. Our results suggest that aurora A kinase participates in vivo to the phosphoregulation of the p150glued MBD to limit the microtubule binding of the dynein–dynactin complex and thus regulates spindle assembly.

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