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.

Driving chromosome segregation: lessons from the human and Drosophila centromere–kinetochore machinery

2010 ◽  
Vol 38 (6) ◽  
pp. 1667-1675 ◽  
Author(s):  
Bernardo Orr ◽  
Olga Afonso ◽  
Tália Feijão ◽  
Claudio E. Sunkel
Keyword(s):  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Genomic Stability ◽  
Mitotic Progression ◽  
Chromosomal Locus ◽  
Microtubule Attachment ◽  
Sister Chromatids ◽  
And Function ◽  
Drosophila Cells

The kinetochore is a complex molecular machine that serves as the interface between sister chromatids and the mitotic spindle. The kinetochore assembles at a particular chromosomal locus, the centromere, which is essential to maintain genomic stability during cell division. The kinetochore is a macromolecular puzzle of subcomplexes assembled in a hierarchical manner and fulfils three main functions: microtubule attachment, chromosome and sister chromatid movement, and regulation of mitotic progression though the spindle assembly checkpoint. In the present paper we compare recent results on the assembly, organization and function of the kinetochore in human and Drosophila cells and conclude that, although essential functions are highly conserved, there are important differences that might help define what is a minimal chromosome segregation machinery.

scholarly journals The Bipolar Kinesin, KLP61F, Cross-links Microtubules within Interpolar Microtubule Bundles of Drosophila Embryonic Mitotic Spindles

1999 ◽  
Vol 144 (1) ◽  
pp. 125-138 ◽  
Author(s):  
David J. Sharp ◽  
Kent L. McDonald ◽  
Heather M. Brown ◽  
Heinrich J. Matthies ◽  
Claire Walczak ◽  
...  
Keyword(s):  
Immunoelectron Microscopy ◽  
Mitotic Spindle ◽  
Cyclin B ◽  
Cytoplasmic Localization ◽  
Mitotic Spindles ◽  
Spindle Poles ◽  
Biochemical Studies ◽  
Spindle Midzone ◽  
Cross Links ◽  
And Function

Previous genetic and biochemical studies have led to the hypothesis that the essential mitotic bipolar kinesin, KLP61F, cross-links and slides microtubules (MTs) during spindle assembly and function. Here, we have tested this hypothesis by immunofluorescence and immunoelectron microscopy (immunoEM). We show that Drosophila embryonic spindles at metaphase and anaphase contain abundant bundles of MTs running between the spindle poles. These interpolar MT bundles are parallel near the poles and antiparallel in the midzone. We have observed that KLP61F motors, phosphorylated at a cdk1/cyclin B consensus domain within the BimC box (BCB), localize along the length of these interpolar MT bundles, being concentrated in the midzone region. Nonphosphorylated KLP61F motors, in contrast, are excluded from the spindle and display a cytoplasmic localization. Immunoelectron microscopy further suggested that phospho-KLP61F motors form cross-links between MTs within interpolar MT bundles. These bipolar KLP61F MT-MT cross-links should be capable of organizing parallel MTs into bundles within half spindles and sliding antiparallel MTs apart in the spindle midzone. Thus we propose that bipolar kinesin motors and MTs interact by a “sliding filament mechanism” during the formation and function of the mitotic spindle.

scholarly journals Systems cell biology of the mitotic spindle

2010 ◽  
Vol 188 (1) ◽  
pp. 7-9
Author(s):  
Ramsey A. Saleem ◽  
John D. Aitchison
Keyword(s):  
Cell Division ◽  
Mitotic Spindle ◽  
Cell Biology ◽  
Systems Genetics ◽  
Mitotic Spindles ◽  
High Content Imaging ◽  
Controlled Assembly ◽  
Daughter Cells ◽  
And Function ◽  
Insight Into

Cell division depends critically on the temporally controlled assembly of mitotic spindles, which are responsible for the distribution of duplicated chromosomes to each of the two daughter cells. To gain insight into the process, Vizeacoumar et al., in this issue (Vizeacoumar et al. 2010. J. Cell Biol. doi:10.1083/jcb.200909013), have combined systems genetics with high-throughput and high-content imaging to comprehensively identify and classify novel components that contribute to the morphology and function of the mitotic spindle.

scholarly journals Differing requirements for Augmin in male meiotic and mitotic spindle formation in Drosophila

Open Biology ◽  
2014 ◽  
Vol 4 (5) ◽  
pp. 140047 ◽  
Author(s):  
Matthew S. Savoian ◽  
David M. Glover
Keyword(s):  
Mitotic Spindle ◽  
Cell Types ◽  
Male Meiosis ◽  
Negative Regulation ◽  
Mitotic Spindles ◽  
Animal Cells ◽  
Spindle Formation ◽  
Formation Pathway ◽  
Fixed Cell ◽  
Mitotic Cells

Animal cells divide using a microtubule-based, bipolar spindle. Both somatic, mitotic cells and sperm-producing male meiotic spermatocytes use centrosome-dependent and acentrosomal spindle-forming mechanisms. Here, we characterize the largely undefined, centrosome-independent spindle formation pathway used during male meiosis. Our live and fixed cell analyses of Drosophila spermatocytes reveal that acentrosomal microtubules are nucleated at kinetochores and in the vicinity of chromatin and that together these assemble into functional spindles. Mutational studies indicate that γ-tubulin and its extra-centrosomal targeting complex, Augmin, are vital for this process. In addition, Augmin facilitates efficient spindle assembly in the presence of centrosomes. In contrast to the pronounced recruitment of Augmin on spindles in other cell types, the complex is absent from those of spermatocytes but does accumulate on kinetochores. Polo kinase facilitates this kinetochore recruitment while inhibiting Augmin's spindle association, and this in turn dictates γ-tubulin distribution and spindle density. Polo's negative regulation of Augmin in male meiosis contrasts with its requirement in loading Augmin along mitotic spindles in somatic Drosophila cells. Together our data identify a novel mechanism of acentrosomal spindle formation in spermatocytes and reveal its divergence from that used in mitotic cells.

scholarly journals Cell shape impacts on the positioning of the mitotic spindle with respect to the substratum

2015 ◽  
Vol 26 (7) ◽  
pp. 1286-1295 ◽  
Author(s):  
Francisco Lázaro-Diéguez ◽  
Iaroslav Ispolatov ◽  
Anne Müsch
Keyword(s):  
Mitotic Spindle ◽  
Cell Shape ◽  
Spindle Assembly Checkpoint ◽  
Mdck Cells ◽  
Spindle Pole ◽  
Spindle Orientation ◽  
Cell Cortex ◽  
Spindle Positioning ◽  
Spindle Poles ◽  
Spindle Position

All known mechanisms of mitotic spindle orientation rely on astral microtubules. We report that even in the absence of astral microtubules, metaphase spindles in MDCK and HeLa cells are not randomly positioned along their x-z dimension, but preferentially adopt shallow β angles between spindle pole axis and substratum. The nonrandom spindle positioning is due to constraints imposed by the cell cortex in flat cells that drive spindles that are longer and/or wider than the cell's height into a tilted, quasidiagonal x-z position. In rounder cells, which are taller, fewer cortical constraints make the x-z spindle position more random. Reestablishment of astral microtubule–mediated forces align the spindle poles with cortical cues parallel to the substratum in all cells. However, in flat cells, they frequently cause spindle deformations. Similar deformations are apparent when confined spindles rotate from tilted to parallel positions while MDCK cells progress from prometaphase to metaphase. The spindle disruptions cause the engagement of the spindle assembly checkpoint. We propose that cell rounding serves to maintain spindle integrity during its positioning.

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 Checkpoint Protein BubR1 Acts Synergistically with Mad2 to Inhibit Anaphase-promoting Complex

2002 ◽  
Vol 13 (3) ◽  
pp. 755-766 ◽  
Author(s):  
Guowei Fang
Keyword(s):  
Ubiquitin Ligase ◽  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Kinase Activity ◽  
Metaphase Plate ◽  
Anaphase Promoting Complex ◽  
Checkpoint Control ◽  
Sister Chromatids ◽  
Direct Binding ◽  
And Function

The spindle assembly checkpoint monitors the attachment of kinetochores to the mitotic spindle and the tension exerted on kinetochores by microtubules and delays the onset of anaphase until all the chromosomes are aligned at the metaphase plate. The target of the checkpoint control is the anaphase-promoting complex (APC)/cyclosome, a ubiquitin ligase whose activation by Cdc20 is required for separation of sister chromatids. In response to activation of the checkpoint, Mad2 binds to and inhibits Cdc20-APC. I show herein that in checkpoint-arrested cells, human Cdc20 forms two separate, inactive complexes, a lower affinity complex with Mad2 and a higher affinity complex with BubR1. Purified BubR1 binds to recombinant Cdc20 and this interaction is direct. Binding of BubR1 to Cdc20 inhibits activation of APC and this inhibition is independent of its kinase activity. Quantitative analysis indicates that BubR1 is 12-fold more potent than Mad2 as an inhibitor of Cdc20. Although at high protein concentrations BubR1 and Mad2 each is sufficient to inhibit Cdc20, BubR1 and Mad2 mutually promote each other's binding to Cdc20 and function synergistically at physiological concentrations to quantitatively inhibit Cdc20-APC. Thus, BubR1 and Mad2 act cooperatively to prevent premature separation of sister chromatids by directly inhibiting APC.

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 Modeling reveals cortical dynein-dependent oscillations in bipolar spindle length

2020 ◽  
Author(s):  
Dayna Mercadante ◽  
Amity Manning ◽  
Sarah Olson
Keyword(s):  
Experimental Data ◽  
Mitotic Spindle ◽  
Force Generation ◽  
Mitotic Progression ◽  
Computational Framework ◽  
Spindle Formation ◽  
Bipolar Spindle ◽  
Spindle Dynamics ◽  
Force Components ◽  
Over Time

AbstractProper formation and maintenance of the mitotic spindle is required for faithful cell division. While much work has been done to understand the roles of the key force components of the mitotic spindle, identifying the consequences of force perturbations in the spindle remains a challenge. We develop a computational framework accounting for the minimal force requirements of mitotic progression. To reflect early spindle formation, we account for microtubule dynamics and interactions with major force-generating motors, excluding chromosome interactions that dominate later in mitosis. We directly integrate our experimental data to define and validate the model, and then use simulations to analyze individual force components over time and their relationship to spindle dynamics, making it distinct from previously published models. Rather than achieving and maintaining a constant bipolar spindle length, oscillations in pole to pole distance occur that coincide with microtubule binding and force generation by cortical dynein. In the context of high kinesin-14 (HSET) activity, we identify the requirement of high cortical dynein activity for bipolar spindle formation.

Cytocentrin is a Ral-binding protein involved in the assembly and function of the mitotic apparatus

1999 ◽  
Vol 112 (5) ◽  
pp. 707-718
Author(s):  
A. Quaroni ◽  
E.C. Paul
Keyword(s):  
Mitotic Spindle ◽  
Binding Protein ◽  
Early Prophase ◽  
Mitotic Apparatus ◽  
Cytosolic Protein ◽  
Spindle Poles ◽  
Ral Gtpases ◽  
And Function ◽  
High Degree

Cytocentrin is a cytosolic protein that transiently associates with the mitotic spindle poles in early prophase, and dissociates from them after completion of mitosis. Cloning of its cDNA demonstrated a high degree of homology with three proteins known to specifically interact with an activated form of Ral. Herein we demonstrate that overexpression of cytocentrin inhibits assembly of the mitotic spindle without affecting polymerization or distribution of interphase microtubules. Conversely, loss of cytocentrin expression leads to formation of monopolar spindles. These results indicate that association of cytocentrin with the centrosome may be essential for a timely separation of the diplosomes. They also implicate Ral GTPases and their related pathways in the assembly and function of the mitotic apparatus.

Sign in / Sign up

Export Citation Format

Share Document