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.

Regulation of Microtubule Assembly: The View From The End

2000 ◽  
Vol 6 (S2) ◽  
pp. 80-81
Author(s):  
L. Cassimeris ◽  
C. Spittle ◽  
M. Kratzer
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Dynamic Instability ◽  
Intrinsic Property ◽  
Microtubule Dynamics ◽  
Microtubule Assembly ◽  
Chromosome Movement ◽  
Spindle Formation ◽  
Associated Proteins

The mitotic spindle is responsible for chromosome movement during mitosis. It is composed of a dynamic array of microtubules and associated proteins whose assembly and constant turnover are required for both spindle formation and chromosome movement. Because microtubule assembly and turnover are necessary for chromosome segregation, we are studying how cells regulate microtubule dynamics. Microtubules are polarized polymers composed of tubulin subunits; they assemble by a process of dynamic instability where individual microtubules exist in persistent phases of elongation or rapid shortening with abrupt transitions between these two states. The switch from elongation to shortening is termed catastrophe, and the switch from shortening to elongation, rescue. Although dynamic instability is an intrinsic property of the tubulin subunits, cells use associated proteins to both speed elongation (∼ 10 fold) and regulate transitions.The only protein isolated to date capable of promoting fast polymerization consistent with rates in vivo is XMAP215, a 215 kD protein from Xenopus eggs.

scholarly journals csi2p modulates microtubule dynamics and organizes the bipolar spindle for chromosome segregation

2014 ◽  
Vol 25 (24) ◽  
pp. 3900-3908 ◽  
Author(s):  
Judite Costa ◽  
Chuanhai Fu ◽  
V. Mohini Khare ◽  
Phong T. Tran
Keyword(s):  
Fission Yeast ◽  
Chromosome Segregation ◽  
Spindle Assembly Checkpoint ◽  
Microtubule Dynamics ◽  
High Rate ◽  
Spindle Formation ◽  
Bipolar Spindle ◽  
Eukaryotic Chromosome ◽  
Relative Contribution ◽  
Multiple Phenotypes

Proper chromosome segregation is of paramount importance for proper genetic inheritance. Defects in chromosome segregation can lead to aneuploidy, which is a hallmark of cancer cells. Eukaryotic chromosome segregation is accomplished by the bipolar spindle. Additional mechanisms, such as the spindle assembly checkpoint and centromere positioning, further help to ensure complete segregation fidelity. Here we present the fission yeast csi2+. csi2p localizes to the spindle poles, where it regulates mitotic microtubule dynamics, bipolar spindle formation, and subsequent chromosome segregation. csi2 deletion (csi2Δ) results in abnormally long mitotic microtubules, high rate of transient monopolar spindles, and subsequent high rate of chromosome segregation defects. Because csi2Δ has multiple phenotypes, it enables estimates of the relative contribution of the different mechanisms to the overall chromosome segregation process. Centromere positioning, microtubule dynamics, and bipolar spindle formation can all contribute to chromosome segregation. However, the major determinant of chromosome segregation defects in fission yeast may be microtubule dynamic defects.

scholarly journals The Nek6 and Nek7 Protein Kinases Are Required for Robust Mitotic Spindle Formation and Cytokinesis

2009 ◽  
Vol 29 (14) ◽  
pp. 3975-3990 ◽  
Author(s):  
Laura O'Regan ◽  
Andrew M. Fry
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Mitotic Spindles ◽  
Mitotic Progression ◽  
Serine Threonine Kinase ◽  
Spindle Formation ◽  
Spindle Poles ◽  
And Function ◽  
Interfering Rna ◽  
Reduced Activity

ABSTRACT Nek6 and Nek7 are members of the NIMA-related serine/threonine kinase family. Previous work showed that they contribute to mitotic progression downstream of another NIMA-related kinase, Nek9, although the roles of these different kinases remain to be defined. Here, we carried out a comprehensive analysis of the regulation and function of Nek6 and Nek7 in human cells. By generating specific antibodies, we show that both Nek6 and Nek7 are activated in mitosis and that interfering with their activity by either depletion or expression of reduced-activity mutants leads to mitotic arrest and apoptosis. Interestingly, while completely inactive mutants and small interfering RNA-mediated depletion delay cells at metaphase with fragile mitotic spindles, hypomorphic mutants or RNA interference treatment combined with a spindle assembly checkpoint inhibitor delays cells at cytokinesis. Importantly, depletion of either Nek6 or Nek7 leads to defective mitotic progression, indicating that although highly similar, they are not redundant. Indeed, while both kinases localize to spindle poles, only Nek6 obviously localizes to spindle microtubules in metaphase and anaphase and to the midbody during cytokinesis. Together, these data lead us to propose that Nek6 and Nek7 play independent roles not only in robust mitotic spindle formation but also potentially in cytokinesis.

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 Altering microtubule dynamics is synergistically toxic with inhibition of the spindle checkpoint

2019 ◽  
Author(s):  
Klaske M. Schukken ◽  
Yi-Chih Lin ◽  
Michael Schubert ◽  
Stephanie F. Preuss ◽  
Judith E. Simon ◽  
...  
Keyword(s):  
Spindle Assembly Checkpoint ◽  
Kinase Inhibitor ◽  
Chromosome Instability ◽  
Src Kinase ◽  
Microtubule Dynamics ◽  
Target Cells ◽  
Microtubule Polymerization ◽  
Powerful Means ◽  
Small Molecule Compound ◽  
Microtubule Poisons

AbstractChromosome instability (CIN) and aneuploidy are hallmarks of cancer. As the majority of cancers are aneuploid, targeting aneuploidy or CIN may be an effective way to target a broad spectrum of cancers. Here, we perform two small molecule compound screens to identify drugs that selectively target cells that are aneuploid or exhibit a CIN phenotype. We find that aneuploid cells are much more sensitive to the energy metabolism regulating drug ZLN005 than their euploid counterparts. Furthermore, cells with an ongoing CIN phenotype, induced by spindle assembly checkpoint (SAC) alleviation, are significantly more sensitive to the Src kinase inhibitor SKI606. We show that inhibiting Src kinase increases microtubule polymerization rates and, more generally, that deregulating microtubule polymerization rates is particularly toxic to cells with a defective SAC. Our findings therefore suggest that tumors with a dysfunctional SAC are particularly sensitive to microtubule poisons and, vice versa, that compounds alleviating the SAC provide a powerful means to treat tumors with deregulated microtubule dynamics.

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 Dynamic microtubules are essential for efficient chromosome capture and biorientation in S. cerevisiae

2006 ◽  
Vol 175 (1) ◽  
pp. 17-23 ◽  
Author(s):  
Baoying Huang ◽  
Tim C. Huffaker
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Direct Evidence ◽  
Dimensional Space ◽  
Three Dimensional ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
Microtubule Dynamics ◽  
Three Dimensional Space

Attachment of chromosomes to the mitotic spindle has been proposed to require dynamic microtubules that randomly search three-dimensional space and become stabilized upon capture by kinetochores. In this study, we test this model by examining chromosome capture in Saccharomyces cerevisiae mutants with attenuated microtubule dynamics. Although viable, these cells are slow to progress through mitosis. Preanaphase cells contain a high proportion of chromosomes that are attached to only one spindle pole and missegregate in the absence of the spindle assembly checkpoint. Measurement of the rates of chromosome capture and biorientation demonstrate that both are severely decreased in the mutants. These results provide direct evidence that dynamic microtubules are critical for efficient chromosome capture and biorientation and support the hypothesis that microtubule search and capture plays a central role in assembly of the mitotic spindle.

scholarly journals Regulation of tau’s proline rich region by its N-terminal domain

2019 ◽  
Author(s):  
Kristen McKibben ◽  
Elizabeth Rhoades
Keyword(s):  
Binding Domain ◽  
Rich Region ◽  
Tau Function ◽  
Microtubule Binding ◽  
Intrinsically Disordered ◽  
Terminal Domain ◽  
Microtubule Polymerization ◽  
Tubulin Binding ◽  
Proline Rich Region

AbstractTau is an intrinsically disordered, microtubule-associated protein with a role in regulating microtubule dynamics. Despite intensive research, the molecular mechanisms of taumediated microtubule polymerization are poorly understood. Here we use single molecule fluorescence to investigate the role of tau’s N-terminal domain (NTD) and proline rich region (PRR) in regulating interactions of tau with soluble tubulin. Both full-length tau isoforms and truncated variants are assayed for their ability to bind soluble tubulin and stimulate microtubule polymerization. We describe a novel role for tau’s PRR as an independent tubulin-binding domain with polymerization capacity. In contrast to the relatively weak tubulin interactions distributed throughout the microtubule binding repeats (MTBR), resulting in heterogeneous tau:tubulin complexes, the PRR binds tubulin tightly and stoichiometrically. Moreover, we demonstrate that interactions between the PRR and MTBR are reduced by the NTD through a conserved conformational ensemble. Based on our data, we propose that tau’s PRR can serve as a core tubulin-binding domain, while the MTBR enhances polymerization capacity by increasing the local tubulin concentration. The NTD negatively regulates tubulin-binding interactions of both of these domains. This study draws attention to the central role of the PRR in tau function, as well as providing mechanistic insight into tau-mediated polymerization of tubulin.Significance StatementTau is an intrinsically disordered, microtubule associated protein linked to a number of neurodegenerative disorders. Here we identify tau’s proline rich region as having autonomous tubulin binding and polymerization capacity, which is enhanced by the flanking microtubule binding repeats. Moreover, we demonstrate that tau’s N-terminal domain negatively regulates both binding and polymerization. We propose a novel model for tau-mediated polymerization whereby the proline rich region serves as a core tubulin-binding domain, while the microtubule binding repeats increase the local concentration. Our work draws attention to the importance of the proline rich region and N-terminal domain in tau function, and highlights the proline rich region as a putative target for the development of therapeutics.

scholarly journals Ipl1/Aurora-dependent phosphorylation of Sli15/INCENP regulates CPC–spindle interaction to ensure proper microtubule dynamics

2011 ◽  
Vol 194 (1) ◽  
pp. 137-153 ◽  
Author(s):  
Yuko Nakajima ◽  
Anthony Cormier ◽  
Randall G. Tyers ◽  
Adrianne Pigula ◽  
Yutian Peng ◽  
...  
Keyword(s):  
Microtubule Dynamics ◽  
Central Spindle ◽  
Microtubule Polymerization ◽  
Microtubule Stability ◽  
Late Anaphase ◽  
Chromosomal Passenger ◽  
Chromosome Separation ◽  
Spindle Elongation ◽  
Spindle Function ◽  
Spatiotemporal Control

Dynamic microtubules facilitate chromosome arrangement before anaphase, whereas during anaphase microtubule stability assists chromosome separation. Changes in microtubule dynamics at the metaphase–anaphase transition are regulated by Cdk1. Cdk1-mediated phosphorylation of Sli15/INCENP promotes preanaphase microtubule dynamics by preventing chromosomal passenger complex (CPC; Sli15/INCENP, Bir1/Survivin, Nbl1/Borealin, Ipl1/Aurora) association with spindles. However, whether Cdk1 has sole control over microtubule dynamics, and how CPC–microtubule association influences microtubule behavior, are unclear. Here, we show that Ipl1/Aurora-dependent phosphorylation of Sli15/INCENP modulates microtubule dynamics by preventing CPC binding to the preanaphase spindle and to the central spindle until late anaphase, facilitating spatiotemporal control of microtubule dynamics required for proper metaphase centromere positioning and anaphase spindle elongation. Decreased Ipl1-dependent Sli15 phosphorylation drives direct CPC binding to microtubules, revealing how the CPC influences microtubule dynamics. We propose that Cdk1 and Ipl1/Aurora cooperatively modulate microtubule dynamics and that Ipl1/Aurora-dependent phosphorylation of Sli15 controls spindle function by excluding the CPC from spindle regions engaged in microtubule polymerization.

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