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 EB1 Targets to Kinetochores with Attached, Polymerizing Microtubules

2002 ◽  
Vol 13 (12) ◽  
pp. 4308-4316 ◽  
Author(s):  
Jennifer S. Tirnauer ◽  
Julie C. Canman ◽  
E.D. Salmon ◽  
Timothy J. Mitchison
Keyword(s):  
Spindle Checkpoint ◽  
Time Lapse ◽  
Microtubule Dynamics ◽  
Microtubule Polymerization ◽  
Spindle Microtubules ◽  
Chromosome Movements

Microtubule polymerization dynamics at kinetochores is coupled to chromosome movements, but its regulation there is poorly understood. The plus end tracking protein EB1 is required both for regulating microtubule dynamics and for maintaining a euploid genome. To address the role of EB1 in aneuploidy, we visualized its targeting in mitotic PtK1 cells. Fluorescent EB1, which localized to polymerizing ends of astral and spindle microtubules, was used to track their polymerization. EB1 also associated with a subset of attached kinetochores in late prometaphase and metaphase, and rarely in anaphase. Localization occurred in a narrow crescent, concave toward the centromere, consistent with targeting to the microtubule plus end–kinetochore interface. EB1 did not localize to kinetochores lacking attached kinetochore microtubules in prophase or early prometaphase, or upon nocodazole treatment. By time lapse, EB1 specifically targeted to kinetochores moving antipoleward, coupled to microtubule plus end polymerization, and not during plus end depolymerization. It localized independently of spindle bipolarity, the spindle checkpoint, and dynein/dynactin function. EB1 is the first protein whose targeting reflects kinetochore directionality, unlike other plus end tracking proteins that show enhanced kinetochore binding in the absence of microtubules. Our results suggest EB1 may modulate kinetochore microtubule polymerization and/or attachment.

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 Roles of Polymerization Dynamics, Opposed Motors, and a Tensile Element in Governing the Length of Xenopus Extract Meiotic Spindles

2005 ◽  
Vol 16 (6) ◽  
pp. 3064-3076 ◽  
Author(s):  
T. J. Mitchison ◽  
P. Maddox ◽  
J. Gaetz ◽  
A. Groen ◽  
M. Shirasu ◽  
...  
Keyword(s):  
Steady State ◽  
Relative Stability ◽  
Motor Proteins ◽  
Microtubule Dynamics ◽  
Microtubule Polymerization ◽  
Hexylene Glycol ◽  
Meiotic Spindles

Metaphase spindles assemble to a steady state in length by mechanisms that involve microtubule dynamics and motor proteins, but they are incompletely understood. We found that Xenopus extract spindles recapitulate the length of egg meiosis II spindles, by using mechanisms intrinsic to the spindle. To probe these mechanisms, we perturbed microtubule polymerization dynamics and opposed motor proteins and measured effects on spindle morphology and dynamics. Microtubules were stabilized by hexylene glycol and inhibition of the catastrophe factor mitotic centromere-associated kinesin (MCAK) (a kinesin 13, previously called XKCM) and destabilized by depolymerizing drugs. The opposed motors Eg5 and dynein were inhibited separately and together. Our results are consistent with important roles for polymerization dynamics in regulating spindle length, and for opposed motors in regulating the relative stability of bipolar versus monopolar organization. The response to microtubule destabilization suggests that an unidentified tensile element acts in parallel with these conventional factors, generating spindle shortening force.

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 Theory of microtubule length regulation in antiparallel overlaps

2019 ◽  
Author(s):  
Hui-Shun Kuan ◽  
Meredith D. Betterton
Keyword(s):  
Steady State ◽  
Cell Division ◽  
Mitotic Spindle ◽  
Motor Proteins ◽  
Critical Concentration ◽  
Binding Kinetics ◽  
Kinesin Motor ◽  
Microtubule Polymerization ◽  
Length Regulation ◽  
Theory And Experiment

AbstractDuring cell division, microtubules in the mitotic spindle form antiparallel overlaps near the center of the spindle. Kinesin motor proteins alter microtubule polymerization dynamics to regulate the length of these overlaps to maintain spindle integrity. Length regulation of antiparallel overlaps has been reconstituted with purified microtubules, crosslinkers, and motors. Here we develop a theory of steady-state overlap length which depends on the filament plus-end motor concentration, determined by a balance between motor arrival (motor binding and stepping in the overlap) and motor departure (motor unbinding from filament tips during depolymerization) in the absence of motor-driven sliding. Assuming that motors processively depolymerize and exhibit altered binding kinetics near MT plus-ends improves the agreement between theory and experiment. Our theory explains the origin of the experimentally observed critical concentration, a minimum motor concentration to observe a steady-state overlap length.

scholarly journals Aurora at the pole and equator: overlapping functions of Aurora kinases in the mitotic spindle

Open Biology ◽  
2013 ◽  
Vol 3 (3) ◽  
pp. 120185 ◽  
Author(s):  
Helfrid Hochegger ◽  
Nadia Hégarat ◽  
Jose B. Pereira-Leal
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Aurora Kinase ◽  
Spindle Pole ◽  
Aurora A ◽  
Mitotic Progression ◽  
Aurora Kinases ◽  
Mitotic Kinases ◽  
Chromosome Alignment ◽  
Spindle Microtubules

The correct assembly and timely disassembly of the mitotic spindle is crucial for the propagation of the genome during cell division. Aurora kinases play a central role in orchestrating bipolar spindle establishment, chromosome alignment and segregation. In most eukaryotes, ranging from amoebas to humans, Aurora activity appears to be required both at the spindle pole and the kinetochore, and these activities are often split between two different Aurora paralogues, termed Aurora A and B. Polar and equatorial functions of Aurora kinases have generally been considered separately, with Aurora A being mostly involved in centrosome dynamics, whereas Aurora B coordinates kinetochore attachment and cytokinesis. However, double inactivation of both Aurora A and B results in a dramatic synergy that abolishes chromosome segregation. This suggests that these two activities jointly coordinate mitotic progression. Accordingly, recent evidence suggests that Aurora A and B work together in both spindle assembly in metaphase and disassembly in anaphase. Here, we provide an outlook on these shared functions of the Auroras, discuss the evolution of this family of mitotic kinases and speculate why Aurora kinase activity may be required at both ends of the spindle 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.

scholarly journals TOG–tubulin binding specificity promotes microtubule dynamics and mitotic spindle formation

2017 ◽  
Vol 216 (6) ◽  
pp. 1641-1657 ◽  
Author(s):  
Amy E. Byrnes ◽  
Kevin C. Slep
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Polymerase Activity ◽  
Microtubule Dynamics ◽  
Spindle Formation ◽  
Microtubule Binding ◽  
Microtubule Polymerization ◽  
Tubulin Binding ◽  
Specific Order ◽  
Spindle Function

XMAP215, CLASP, and Crescerin use arrayed tubulin-binding tumor overexpressed gene (TOG) domains to modulate microtubule dynamics. We hypothesized that TOGs have distinct architectures and tubulin-binding properties that underlie each family’s ability to promote microtubule polymerization or pause. As a model, we investigated the pentameric TOG array of a Drosophila melanogaster XMAP215 member, Msps. We found that Msps TOGs have distinct architectures that bind either free or polymerized tubulin, and that a polarized array drives microtubule polymerization. An engineered TOG1-2-5 array fully supported Msps-dependent microtubule polymerase activity. Requisite for this activity was a TOG5-specific N-terminal HEAT repeat that engaged microtubule lattice-incorporated tubulin. TOG5–microtubule binding maintained mitotic spindle formation as deleting or mutating TOG5 compromised spindle architecture and increased the mitotic index. Mad2 knockdown released the spindle assembly checkpoint triggered when TOG5–microtubule binding was compromised, indicating that TOG5 is essential for spindle function. Our results reveal a TOG5-specific role in mitotic fidelity and support our hypothesis that architecturally distinct TOGs arranged in a sequence-specific order underlie TOG array microtubule regulator activity.

scholarly journals Microtubule rescue at midzone edges promotes overlap stability and prevents spindle collapse during anaphase B

2021 ◽  
Author(s):  
Manuel Lera-Ramirez ◽  
Francois Nedelec ◽  
Phong T Tran
Keyword(s):  
Fission Yeast ◽  
Nuclear Membrane ◽  
Molecular Motors ◽  
Mitotic Spindle ◽  
Microtubule Dynamics ◽  
Growth Speed ◽  
Constant Length ◽  
Spindle Midzone ◽  
Microtubule Growth ◽  
Anaphase B

During anaphase B, molecular motors slide interpolar microtubules to elongate the mitotic spindle, contributing to the separation of chromosomes. However, sliding of antiparallel microtubules reduces their overlap, which may lead to spindle breakage, unless microtubules grow to compensate sliding. How sliding and growth are coordinated is still poorly understood. In this study, we have used the fission yeast S. pombe to measure microtubule dynamics during anaphase B. We report that the coordination of microtubule growth and sliding relies on promoting rescues at the midzone edges. This makes microtubules stable from pole to midzone, while their distal parts including the plus ends alternate between assembly and disassembly. Consequently, the midzone keeps a constant length throughout anaphase, enabling sustained sliding without the need for a precise regulation of microtubule growth speed. Additionally, we found that in S. pombe, which undergoes closed mitosis, microtubule growth speed decreases when the nuclear membrane wraps around the spindle midzone.

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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