scholarly journals Poleward Transport of TPX2 in the Mammalian Mitotic Spindle Requires Dynein, Eg5, and Microtubule Flux

2010 ◽  
Vol 21 (6) ◽  
pp. 979-988 ◽  
Author(s):  
Nan Ma ◽  
U. S. Tulu ◽  
Nick P. Ferenz ◽  
Carey Fagerstrom ◽  
Andrew Wilde ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Fiber Formation ◽  
Aurora A ◽  
Mitotic Progression ◽  
Mitotic Kinase ◽  
Spindle Poles ◽  
C Terminus ◽  
Assembly Factor ◽  
Microtubule Formation

TPX2 is a Ran-regulated spindle assembly factor that is required for kinetochore fiber formation and activation of the mitotic kinase Aurora A. TPX2 is enriched near spindle poles and is required near kinetochores, suggesting that it undergoes dynamic relocalization throughout mitosis. Using photoactivation, we measured the movement of PA-GFP-TPX2 in the mitotic spindle. TPX2 moves poleward in the half-spindle and is static in the interzone and near spindle poles. Poleward transport of TPX2 is sensitive to inhibition of dynein or Eg5 and to suppression of microtubule flux with nocodazole or antibodies to Kif2a. Poleward transport requires the C terminus of TPX2, a domain that interacts with Eg5. Overexpression of TPX2 lacking this domain induced excessive microtubule formation near kinetochores, defects in spindle assembly and blocked mitotic progression. Our data support a model in which poleward transport of TPX2 down-regulates its microtubule nucleating activity near kinetochores and links microtubules generated at kinetochores to dynein for incorporation into the spindle.

scholarly journals TPX2 regulates the localization and activity of Eg5 in the mammalian mitotic spindle

2011 ◽  
Vol 195 (1) ◽  
pp. 87-98 ◽  
Author(s):  
Nan Ma ◽  
Janel Titus ◽  
Alyssa Gable ◽  
Jennifer L. Ross ◽  
Patricia Wadsworth
Keyword(s):  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Spindle Assembly ◽  
Artificial Chromosome ◽  
Fiber Formation ◽  
Interaction Domain ◽  
Spindle Poles ◽  
Spindle Elongation ◽  
Multiple Spindle ◽  
And Function

Mitotic spindle assembly requires the regulated activity of numerous spindle-associated proteins. In mammalian cells, the Kinesin-5 motor Eg5 interacts with the spindle assembly factor TPX2, but how this interaction contributes to spindle formation and function is not established. Using bacterial artificial chromosome technology, we generated cells expressing TPX2 lacking the Eg5 interaction domain. Spindles in these cells were highly disorganized with multiple spindle poles. The TPX2–Eg5 interaction was required for kinetochore fiber formation and contributed to Eg5 localization to spindle microtubules but not spindle poles. Microinjection of the Eg5-binding domain of TPX2 resulted in spindle elongation, indicating that the interaction of Eg5 with TPX2 reduces motor activity. Consistent with this possibility, we found that TPX2 reduced the velocity of Eg5-dependent microtubule gliding, inhibited microtubule sliding, and resulted in the accumulation of motor on microtubules. These results establish a novel function of TPX2 in regulating the location and activity of the mitotic motor Eg5.

scholarly journals The APC/C targets the Cep152-Cep63 complex at the centrosome to regulate mitotic spindle assembly

2021 ◽  
Author(s):  
Thomas Tischer ◽  
Jing Yang ◽  
David Barford
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Protein Abundance ◽  
Anaphase Promoting Complex ◽  
Mitotic Progression ◽  
Centrosomal Protein ◽  
Temporal Regulation ◽  
Spindle Poles ◽  
Main Protein ◽  
Mitotic Spindle Assembly

The control of protein abundance is a fundamental regulatory mechanism during mitosis. The anaphase promoting complex/cyclosome (APC/C) is the main protein ubiquitin ligase responsible for the temporal regulation of mitotic progression. It has been proposed that the APC/C might fulfil other functions including assembly of the mitotic spindle. Here, we show that the APC/C localizes to centrosomes, the organizers of the eukaryotic microtubule cytoskeleton, specifically during mitosis. Recruitment of the APC/C to spindle poles requires the centrosomal protein Cep152, and we identified Cep152 as both an APC/C interaction partner and as an APC/C substrate. Previous studies showed that Cep152 forms a complex with Cep57 and Cep63. The APC/C-mediated ubiquitination of Cep152 at the centrosome releases Cep57 from this inhibitory complex and enables its interaction with pericentrin, a critical step in promoting microtubule nucleation. Thus, our study extends the function of the APC/C from being a regulator of mitosis to also acting as a positive governor of spindle assembly. The APC/C thereby integrates control of these two important processes in a temporal manner.

scholarly journals Mitotic spindle assembly is controlled by the APC/C localized at the centrosome

2020 ◽  
Author(s):  
Thomas Tischer ◽  
Jing Yang ◽  
David Barford
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Anaphase Promoting Complex ◽  
Mitotic Progression ◽  
Centrosomal Protein ◽  
Temporal Regulation ◽  
Spindle Poles ◽  
Defective Mutant ◽  
Main Protein ◽  
Mitotic Spindle Assembly

AbstractThe control of protein abundance is a fundamental regulatory mechanism during mitosis. The anaphase promoting complex/cyclosome (APC/C) is the main protein ubiquitin ligase responsible for the temporal regulation of mitotic progression. It has long been speculated that the APC/C might fulfil other functions including assembly of the mitotic spindle. Here, we show that the APC/C localizes to centrosomes, the organizers of the eukaryotic microtubule cytoskeleton, specifically during mitosis. The APC/C is recruited to spindle poles by the centrosomal protein Cep152, and we identified Cep152 as both a novel APC/C interaction partner, and as an APC/C substrate. Importantly, this revealed a mitotic function of Cep152 that is reciprocally regulated by the APC/C. A destruction-defective mutant of Cep152 showed that the timely regulation of Cep152 levels at the centrosome controls spindle assembly and chromosome segregation. The APC/C-mediated degradation of Cep152 at the centrosome releases Cep57 from an inhibitory complex to enable its interaction with pericentrin, a critical step in promoting microtubule nucleation. Thus, our study extends the function of the APC/C from being a regulator of mitosis to also acting as a positive governor of spindle assembly. The APC/C thereby integrates control of these two important processes in a temporal manner.

scholarly journals Distinct roles for antiparallel microtubule pairing and overlap during early spindle assembly

2013 ◽  
Vol 24 (20) ◽  
pp. 3238-3250 ◽  
Author(s):  
Elena Nazarova ◽  
Eileen O'Toole ◽  
Susi Kaitna ◽  
Paul Francois ◽  
Mark Winey ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Dynamical Behavior ◽  
Three Dimensional ◽  
Spindle Assembly ◽  
Intermediate Step ◽  
High Angle ◽  
Normal Number ◽  
Spindle Poles ◽  
Evolutionarily Conserved ◽  
Microtubule Formation

During spindle assembly, microtubules may attach to kinetochores or pair to form antiparallel pairs or interpolar microtubules, which span the two spindle poles and contribute to mitotic pole separation and chromosome segregation. Events in the specification of the interpolar microtubules are poorly understood. Using three-dimensional electron tomography and analysis of spindle dynamical behavior in living cells, we investigated the process of spindle assembly. Unexpectedly, we found that the phosphorylation state of an evolutionarily conserved Cdk1 site (S360) in γ-tubulin is correlated with the number and organization of interpolar microtubules. Mimicking S360 phosphorylation (S360D) results in bipolar spindles with a normal number of microtubules but lacking interpolar microtubules. Inhibiting S360 phosphorylation (S360A) results in spindles with interpolar microtubules and high-angle, antiparallel microtubule pairs. The latter are also detected in wild-type spindles <1 μm in length, suggesting that high-angle microtubule pairing represents an intermediate step in interpolar microtubule formation. Correlation of spindle architecture with dynamical behavior suggests that microtubule pairing is sufficient to separate the spindle poles, whereas interpolar microtubules maintain the velocity of pole displacement during early spindle assembly. Our findings suggest that the number of interpolar microtubules formed during spindle assembly is controlled in part through activities at the spindle poles.

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 Adducin-1 is essential for mitotic spindle assembly through its interaction with myosin-X

2013 ◽  
Vol 204 (1) ◽  
pp. 19-28 ◽  
Author(s):  
Po-Chao Chan ◽  
Rosaline Y.C. Hsu ◽  
Chih-Wei Liu ◽  
Chien-Chen Lai ◽  
Hong-Chen Chen
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Actin Binding ◽  
Cyclin Dependent Kinase ◽  
Mitotic Spindles ◽  
Mitotic Progression ◽  
Filamentous Actin ◽  
Multipolar Spindles ◽  
Mitotic Spindle Assembly ◽  
Myosin X

Mitotic spindles are microtubule-based structures, but increasing evidence indicates that filamentous actin (F-actin) and F-actin–based motors are components of these structures. ADD1 (adducin-1) is an actin-binding protein that has been shown to play important roles in the stabilization of the membrane cortical cytoskeleton and cell–cell adhesions. In this study, we show that ADD1 associates with mitotic spindles and is crucial for proper spindle assembly and mitotic progression. Phosphorylation of ADD1 at Ser12 and Ser355 by cyclin-dependent kinase 1 enables ADD1 to bind to myosin-X (Myo10) and therefore to associate with mitotic spindles. ADD1 depletion resulted in distorted, elongated, and multipolar spindles, accompanied by aberrant chromosomal alignment. Remarkably, the mitotic defects caused by ADD1 depletion were rescued by reexpression of ADD1 but not of an ADD1 mutant defective in Myo10 binding. Together, our findings unveil a novel function for ADD1 in mitotic spindle assembly through its interaction with Myo10.

scholarly journals Spatial regulation of Aurora A activity during mitotic spindle assembly requires RHAMM to correctly localize TPX2

Cell Cycle ◽  
2014 ◽  
Vol 13 (14) ◽  
pp. 2248-2261 ◽  
Author(s):  
Helen Chen ◽  
Pooja Mohan ◽  
Jihong Jiang ◽  
Oksana Nemirovsky ◽  
Daniel He ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Aurora A ◽  
Spatial Regulation ◽  
Mitotic Spindle Assembly

scholarly journals Interactions between N-Terminal Modules in MPS1 Enable Spindle Checkpoint Silencing

2018 ◽  
Author(s):  
Spyridon T. Pachis ◽  
Yoshitaka Hiruma ◽  
Anastassis Perrakis ◽  
Geert J.P.L. Kops
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Intramolecular Interactions ◽  
Mitotic Progression ◽  
Microtubule Attachment ◽  
Anaphase Onset ◽  
Regulatory Input ◽  
Tpr Domain

ABSTRACTFaithful chromosome segregation relies on the ability of the spindle assembly checkpoint (SAC) to delay anaphase onset until all chromosomes are attached to the mitotic spindle via their kinetochores. MPS1 kinase is recruited to unattached kinetochores to initiate SAC signaling, and is removed from kinetochores once stable microtubule attachments have been formed to allow normal mitotic progression. Here we show that a helical fragment within the kinetochore-targeting NTE module of MPS1 is required for interactions with kinetochores, and also forms intramolecular interactions with its adjacent TPR domain. Bypassing this NTE-TPR interaction results in high MPS1 levels at kinetochores due to loss of regulatory input into MPS1 localization, ineffecient MPS1 delocalization from kinetochores upon microtubule attachment, and SAC silencing defects. These results show that SAC responsiveness to attachments relies on regulated intramolecular interactions in MPS1 and highlight the sensitivity of mitosis to perturbations in the dynamics of the MSP1-NDC80-C interactions.

scholarly journals Cyclin B1 scaffolds MAD1 at the corona to activate the spindle assembly checkpoint

2019 ◽  
Author(s):  
Lindsey A Allan ◽  
Magda Reis ◽  
Yahui Liu ◽  
Pim Huis in ’t Veld ◽  
Geert JPL Kops ◽  
...  
Keyword(s):  
Genome Stability ◽  
Spindle Assembly Checkpoint ◽  
Point Mutations ◽  
Spindle Assembly ◽  
Cyclin B1 ◽  
Cyclin B ◽  
Mitotic Progression ◽  
Mitotic Kinase ◽  
Binding Interface

ABSTRACTThe Cyclin B:CDK1 kinase complex is the master regulator of mitosis that phosphorylates hundreds of proteins to coordinate mitotic progression. We show here that, in addition to these kinase functions, Cyclin B also scaffolds a localised signalling pathway to help preserve genome stability. Cyclin B1 localises to an expanded region of the outer kinetochore, known as the corona, where it scaffolds the spindle assembly checkpoint (SAC) machinery by binding directly to MAD1. In vitro reconstitutions map the key binding interface to a few acidic residues in the N-terminus of MAD1, and point mutations in this region remove corona MAD1 and weaken the SAC. Therefore, Cyclin B1 is the long-sought-after scaffold that links MAD1 to the corona and this specific pool of MAD1 is needed to generate a robust SAC response. Robustness, in this context, arises because Cyclin B1-MAD1 localisation becomes MPS1-independent after the corona has been established. We demonstrate that this allows corona-MAD1 to persist at kinetochores when MPS1 activity falls, ensuring that it can still be phosphorylated on a key C-terminal catalytic site by MPS1. Therefore, this study explains how corona MAD1 generates a robust SAC signal and why stripping of this pool by dynein is essential for SAC silencing. It also reveals that the key mitotic kinase, Cyclin B1-Cdk1, scaffolds the pathway that inhibits its own degradation.

scholarly journals Two XMAP215/TOG microtubule polymerases, Alp14 and Dis1, play non-exchangeable, distinct roles in microtubule organisation in fission yeast

2019 ◽  
Author(s):  
Masashi Yukawa ◽  
Tomoki Kawakami ◽  
Corinne Pinder ◽  
Takashi Toda
Keyword(s):  
Fission Yeast ◽  
Chromosome Segregation ◽  
Genome Stability ◽  
Spindle Assembly ◽  
Mitotic Progression ◽  
Spindle Microtubule ◽  
Microtubule Associated Proteins ◽  
Spatial Regulation ◽  
Associated Proteins ◽  
Microtubule Formation

AbstractProper bipolar spindle assembly underlies accurate chromosome segregation. A cohort of microtubule-associated proteins orchestrates spindle microtubule formation in a spatiotemporally coordinated manner. Among them, the conserved XMAP215/TOG family of microtubule polymerase plays a central role in spindle assembly. In fission yeast, two XMAP215/TOG members, Alp14 and Dis1, share essential roles in cell viability; however how these two proteins functionally collaborate remains undetermined. Here we show the functional interplay and specification of Alp14 and Dis1. Creation of new mutant alleles of alp14, which display temperature sensitivity in the absence of Dis1, enabled us to conduct detailed analyses of a double mutant. We have found that simultaneous inactivation of Alp14 and Dis1 results in early mitotic arrest with very short, fragile spindles. Intriguingly, these cells often undergo spindle collapse, leading to a lethal “cut” phenotype. By implementing an artificial targetting system, we have shown that Alp14 and Dis1 are not functionally exchangeable and as such are not merely redundant paralogues. Intriguingly, while Alp14 promotes microtubule nucleation, Dis1 does not. Our results uncover that the intrinsic specification, not the spatial regulation, between Alp14 and Dis1 underlies the collaborative actions of these two XMAP215/TOG members in mitotic progression, spindle integrity and genome stability.

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