scholarly journals Kinetochore-microtubule stability governs the metaphase requirement for Eg5

2014 ◽  
Vol 25 (13) ◽  
pp. 2051-2060 ◽  
Author(s):  
A. Sophia Gayek ◽  
Ryoma Ohi
Keyword(s):  
Physical Properties ◽  
Human Cell ◽  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Human Cell Lines ◽  
Bipolar Spindle ◽  
Daughter Cells ◽  
Microtubule Stability ◽  
The Stability ◽  
Bipolar Spindles

The mitotic spindle is a bipolar, microtubule (MT)-based cellular machine that segregates the duplicated genome into two daughter cells. The kinesin-5 Eg5 establishes the bipolar geometry of the mitotic spindle, but previous work in mammalian cells suggested that this motor is unimportant for the maintenance of spindle bipolarity. Although it is known that Kif15, a second mitotic kinesin, enforces spindle bipolarity in the absence of Eg5, how Kif15 functions in this capacity and/or whether other biochemical or physical properties of the spindle promote its bipolarity have been poorly studied. Here we report that not all human cell lines can efficiently maintain bipolarity without Eg5, despite their expressing Kif15. We show that the stability of chromosome-attached kinetochore-MTs (K-MTs) is important for bipolar spindle maintenance without Eg5. Cells that efficiently maintain bipolar spindles without Eg5 have more stable K-MTs than those that collapse without Eg5. Consistent with this observation, artificial destabilization of K-MTs promotes spindle collapse without Eg5, whereas stabilizing K-MTs improves bipolar spindle maintenance without Eg5. Our findings suggest that either rapid K-MT turnover pulls poles inward or slow K-MT turnover allows for greater resistance to inward-directed forces.

scholarly journals Kinesin-5 Is Dispensable for Bipolar Spindle Formation and Elongation in Candida albicans, but Simultaneous Loss of Kinesin-14 Activity Is Lethal

mSphere ◽  
2019 ◽  
Vol 4 (6) ◽  
Author(s):  
Irsa Shoukat ◽  
Corey Frazer ◽  
John S. Allingham
Keyword(s):  
Candida Albicans ◽  
Mitotic Spindle ◽  
Fungal Pathogens ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
Content Type ◽  
Bipolar Spindle ◽  
Spindle Elongation ◽  
Simultaneous Loss ◽  
Bipolar Spindles

ABSTRACT Mitotic spindles assume a bipolar architecture through the concerted actions of microtubules, motors, and cross-linking proteins. In most eukaryotes, kinesin-5 motors are essential to this process, and cells will fail to form a bipolar spindle without kinesin-5 activity. Remarkably, inactivation of kinesin-14 motors can rescue this kinesin-5 deficiency by reestablishing the balance of antagonistic forces needed to drive spindle pole separation and spindle assembly. We show that the yeast form of the opportunistic fungus Candida albicans assembles bipolar spindles in the absence of its sole kinesin-5, CaKip1, even though this motor exhibits stereotypical cell-cycle-dependent localization patterns within the mitotic spindle. However, cells lacking CaKip1 function have shorter metaphase spindles and longer and more numerous astral microtubules. They also show defective hyphal development. Interestingly, a small population of CaKip1-deficient spindles break apart and reform two bipolar spindles in a single nucleus. These spindles then separate, dividing the nucleus, and then elongate simultaneously in the mother and bud or across the bud neck, resulting in multinucleate cells. These data suggest that kinesin-5-independent mechanisms drive assembly and elongation of the mitotic spindle in C. albicans and that CaKip1 is important for bipolar spindle integrity. We also found that simultaneous loss of kinesin-5 and kinesin-14 (CaKar3Cik1) activity is lethal. This implies a divergence from the antagonistic force paradigm that has been ascribed to these motors, which could be linked to the high mitotic error rate that C. albicans experiences and often exploits as a generator of diversity. IMPORTANCE Candida albicans is one of the most prevalent fungal pathogens of humans and can infect a broad range of niches within its host. This organism frequently acquires resistance to antifungal agents through rapid generation of genetic diversity, with aneuploidy serving as a particularly important adaptive mechanism. This paper describes an investigation of the sole kinesin-5 in C. albicans, which is a major regulator of chromosome segregation. Contrary to other eukaryotes studied thus far, C. albicans does not require kinesin-5 function for bipolar spindle assembly or spindle elongation. Rather, this motor protein associates with the spindle throughout mitosis to maintain spindle integrity. Furthermore, kinesin-5 loss is synthetically lethal with loss of kinesin-14—canonically an opposing force producer to kinesin-5 in spindle assembly and anaphase. These results suggest a significant evolutionary rewiring of microtubule motor functions in the C. albicans mitotic spindle, which may have implications in the genetic instability of this pathogen.

The Role of the Centrosome in Development and Progression of Breast Cancer

2001 ◽  
Vol 7 (S2) ◽  
pp. 582-583
Author(s):  
W. Lingle ◽  
J. Salisbury ◽  
S. Barrett ◽  
V. Negron ◽  
C. Whitehead
Keyword(s):  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Microtubule Organizing Center ◽  
Daughter Cells ◽  
Spindle Poles ◽  
Sister Chromatids ◽  
Close Proximity ◽  
Interphase Cells ◽  
Organizing Center

The centrosome is the major microtubule organizing center in most mammalian cells, and as such it determines the number, polarity, and spatial distribution of microtubules (MTs). Interphase MTs, together with actin and intermediate filaments, constitute the cell's cytoskeleton, which dynamically maintains cell polarity and tissue architecture. Interphase cells begin Gl of the cell cycle with one centrosome. During S phase, the centrosome duplicates concomitantly with DNA replication. Duplicated centrosomes usually remain in close proximity to one another until late G2, at which time they separate and then move during prophase to become the poles that organize the bipolar mitotic spindle. During the G2/M transition, interphase MTs depolymerize and a new population of highly dynamic mitotic MTs are nucleated at the spindle poles. The bipolar mitotic spindle apparatus constitutes the machinery that partitions and separates sister chromatids equally between two daughter cells.

scholarly journals The RZZ complex facilitates Mad1 binding to Bub1 ensuring efficient checkpoint signaling

2018 ◽  
Author(s):  
Gang Zhang ◽  
Thomas Kruse ◽  
Jakob Nilsson
Keyword(s):  
Binding Site ◽  
Human Cell ◽  
Mammalian Cells ◽  
Primary Role ◽  
Human Cell Lines ◽  
Checkpoint Signaling ◽  
Checkpoint Protein ◽  
The Core ◽  
Exact Function

Introductory paragraphThe recruitment of Mad1 to unattached kinetochores is essential for generating a “wait anaphase” signal during mitosis yet Mad1 localization is poorly understood in mammalian cells. In yeast the Bub1 checkpoint protein is the sole Mad1 receptor but in mammalian cells the Rod-ZW10-Zwilch (RZZ) complex is also required for Mad1 kinetochore localization. The exact function of the two mammalian Mad1 receptors and whether there is any interplay between them is unclear. Here we use CRISPR genome editing to generate RNAi sensitized human cell lines revealing a strong requirement for both Rod and Bub1 in checkpoint signaling. We show that the RZZ complex facilitates Mad1 binding to Bub1 and that a region of Bub1 overlapping the Mad1 binding site stimulates RZZ kinetochore recruitment. The requirement for RZZ in the checkpoint, but not Bub1, can be bypassed by tethering Mad1 to kinetochores or by increasing the strength of the Bub1-Mad1 interaction. Our data support a model in which the primary role of RZZ is to localize Mad1 at kinetochores allowing for the efficient checkpoint generating Mad1-Bub1 interaction. As such, the core checkpoint principle is conserved from yeast to man.

scholarly journals Centrosomes control kinetochore-fiber plus-end dynamics via HURP to ensure symmetric divisions

2019 ◽  
Author(s):  
Damian Dudka ◽  
Nicolas Liaudet ◽  
Hélène Vassal ◽  
Patrick Meraldi
Keyword(s):  
Cell Division ◽  
Mitotic Spindle ◽  
Fiber Length ◽  
Molecular Mechanisms ◽  
Molecular Level ◽  
Asymmetric Cell Divisions ◽  
Fiber Dynamics ◽  
The Stability ◽  
Bipolar Spindles ◽  
Cancerous Cells

SUMMARYDuring mitosis centrosomes can affect the length of kinetochore-fibers (k-fibers) and the stability of kinetochore-microtubule attachments, implying that they regulate k-fiber dynamics. The exact cellular and molecular mechanisms by which centrosomes regulate k-fibers remain, however, unknown. Here, we created human non-cancerous cells with only one centrosome to investigate these mechanisms. Such cells formed highly asymmetric bipolar spindles that resulted in asymmetric cell divisions. K-fibers in acentrosomal spindles were shorter, more stable, had a reduced poleward microtubule flux at minus-ends, and more frequent pausing events at their plus-ends. This indicates that centrosomes regulate k-fiber dynamics both locally at minus-ends and far away at plus-ends. At the molecular level we find that the microtubule-stabilizing protein HURP is enriched on the k-fiber plus-ends in the acentrosomal spindles of cells with only one centrosome. HURP depletion rebalance k-fiber stability and dynamics in such cells, and improved spindle and cell division symmetry. Our data further indicate that HURP accumulates on k-fibers inversely proportionally to half-spindle length. We propose that centrosomes regulate k-fiber plus-ends indirectly via length-dependent accumulation of HURP. Thus by ensuring equal k-fiber length, centrosomes promote HURP symmetry, reinforcing the symmetry of the mitotic spindle and of cell division.

scholarly journals Cytoplasmic dynein plays a role in mammalian mitotic spindle formation.

1993 ◽  
Vol 123 (4) ◽  
pp. 849-858 ◽  
Author(s):  
E A Vaisberg ◽  
M P Koonce ◽  
J R McIntosh
Keyword(s):  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Cytoplasmic Dynein ◽  
Detectable Effect ◽  
Spindle Formation ◽  
Bipolar Spindle ◽  
Dynein Motor ◽  
Motor Enzymes ◽  
Chromosome Attachment

The formation and functioning of a mitotic spindle depends not only on the assembly/disassembly of microtubules but also on the action of motor enzymes. Cytoplasmic dynein has been localized to spindles, but whether or how it functions in mitotic processes is not yet known. We have cloned and expressed DNA fragments that encode the putative ATP-hydrolytic sites of the cytoplasmic dynein heavy chain from HeLa cells and from Dictyostelium. Monospecific antibodies have been raised to the resulting polypeptides, and these inhibit dynein motor activity in vitro. Their injection into mitotic mammalian cells blocks the formation of spindles in prophase or during recovery from nocodazole treatment at later stages of mitosis. Cells become arrested with unseparated centrosomes and form monopolar spindles. The injected antibodies have no detectable effect on chromosome attachment to a bipolar spindle or on motions during anaphase. These data suggest that cytoplasmic dynein plays a unique and important role in the initial events of bipolar spindle formation, while any later roles that it may play are redundant. Possible mechanisms of dynein's involvement in mitosis are discussed.

scholarly journals Kinesin-5 forms a stable bipolar spindle in a fast, irreversible snap

2018 ◽  
Author(s):  
Allen Leary ◽  
Elena Nazarova ◽  
Shannon Sim ◽  
Kristy Shulist ◽  
Paul Francois ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Electron Tomography ◽  
Maximal Extension ◽  
Spindle Formation ◽  
Bipolar Spindle ◽  
Spindle Poles ◽  
Equilibrium Length ◽  
Bipolar Spindles

SUMMARYGRAPHICAL ABSTRACTSeparation of duplicated spindle poles is the first step in forming the mitotic spindle. Kinesin-5 crosslinks and slides anti-parallel microtubules, but it is unclear how these two activities contribute to the first steps in spindle formation. In this study we report that in monopolar spindles, the duplicated spindle poles snap apart in a fast and irreversible step that produces a nascent bipolar spindle. Using mutations in Kinesin-5 that inhibit microtubule sliding, we show crosslinking alone drives the fast, irreversible pole separation. Electron tomography revealed microtubule pairs in monopolar spindles have short overlaps that intersect at high angles and are unsuited for ensemble Kinesin-5 sliding. However, maximal extension of a subset of microtubule pairs approaches the length of nascent bipolar spindles and is consistent with a Kinesin-5 crosslinking driven transition. Finally, stochastic microtubule sliding by Kinesin-5 stabilizes the nascent spindle and sets a stereotyped equilibrium length.

scholarly journals Centrosome age regulates kinetochore–microtubule stability and biases chromosome mis-segregation

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Ivana Gasic ◽  
Purnima Nerurkar ◽  
Patrick Meraldi
Keyword(s):  
Stem Cell ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Human Cells ◽  
Functional Asymmetry ◽  
Mitotic Spindles ◽  
Daughter Cells ◽  
Cell Divisions ◽  
Microtubule Stability ◽  
Stem Cell Divisions

The poles of the mitotic spindle contain one old and one young centrosome. In asymmetric stem cell divisions, the age of centrosomes affects their behaviour and their probability to remain in the stem cell. In contrast, in symmetric divisions, old and young centrosomes are thought to behave equally. This hypothesis is, however, untested. In this study, we show in symmetrically dividing human cells that kinetochore–microtubules associated to old centrosomes are more stable than those associated to young centrosomes, and that this difference favours the accumulation of premature end-on attachments that delay the alignment of polar chromosomes at old centrosomes. This differential microtubule stability depends on cenexin, a protein enriched on old centrosomes. It persists throughout mitosis, biasing chromosome segregation in anaphase by causing daughter cells with old centrosomes to retain non-disjoint chromosomes 85% of the time. We conclude that centrosome age imposes via cenexin a functional asymmetry on all mitotic spindles.

scholarly journals A computational model of the early stages of acentriolar meiotic spindle assembly

2019 ◽  
Vol 30 (7) ◽  
pp. 863-875 ◽  
Author(s):  
Gaelle Letort ◽  
Isma Bennabi ◽  
Serge Dmitrieff ◽  
François Nedelec ◽  
Marie-Hélène Verlhac ◽  
...  
Keyword(s):  
Spindle Assembly ◽  
Meiotic Spindle ◽  
Key Factors ◽  
Spatial Regulation ◽  
Daughter Cells ◽  
Early Stages ◽  
Microtubule Stability ◽  
Gradual Process ◽  
Associated Proteins ◽  
Bipolar Spindles

The mitotic spindle is an ensemble of microtubules responsible for the repartition of the chromosomal content between the two daughter cells during division. In metazoans, spindle assembly is a gradual process involving dynamic microtubules and recruitment of numerous associated proteins and motors. During mitosis, centrosomes organize and nucleate the majority of spindle microtubules. In contrast, oocytes lack canonical centrosomes but are still able to form bipolar spindles, starting from an initial ball that self-organizes in several hours. Interfering with early steps of meiotic spindle assembly can lead to erroneous chromosome segregation. Although not fully elucidated, this process is known to rely on antagonistic activities of plus end– and minus end–directed motors. We developed a model of early meiotic spindle assembly in mouse oocytes, including key factors such as microtubule dynamics and chromosome movement. We explored how the balance between plus end– and minus end–directed motors, as well as the influence of microtubule nucleation, impacts spindle morphology. In a refined model, we added spatial regulation of microtubule stability and minus-end clustering. We could reproduce the features of early stages of spindle assembly from 12 different experimental perturbations and predict eight additional perturbations. With its ability to characterize and predict chromosome individualization, this model can help deepen our understanding of spindle assembly.

scholarly journals Regulation of the cell cycle and centrosome biology by deubiquitylases

2017 ◽  
Vol 45 (5) ◽  
pp. 1125-1136 ◽  
Author(s):  
Sarah Darling ◽  
Andrew B. Fielding ◽  
Dorota Sabat-Pośpiech ◽  
Ian A. Prior ◽  
Judy M. Coulson
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Genetic Material ◽  
Post Translational Modification ◽  
Cellular Processes ◽  
Daughter Cells ◽  
Damage Response ◽  
A Cell

Post-translational modification of proteins by ubiquitylation is increasingly recognised as a highly complex code that contributes to the regulation of diverse cellular processes. In humans, a family of almost 100 deubiquitylase enzymes (DUBs) are assigned to six subfamilies and many of these DUBs can remove ubiquitin from proteins to reverse signals. Roles for individual DUBs have been delineated within specific cellular processes, including many that are dysregulated in diseases, particularly cancer. As potentially druggable enzymes, disease-associated DUBs are of increasing interest as pharmaceutical targets. The biology, structure and regulation of DUBs have been extensively reviewed elsewhere, so here we focus specifically on roles of DUBs in regulating cell cycle processes in mammalian cells. Over a quarter of all DUBs, representing four different families, have been shown to play roles either in the unidirectional progression of the cell cycle through specific checkpoints, or in the DNA damage response and repair pathways. We catalogue these roles and discuss specific examples. Centrosomes are the major microtubule nucleating centres within a cell and play a key role in forming the bipolar mitotic spindle required to accurately divide genetic material between daughter cells during cell division. To enable this mitotic role, centrosomes undergo a complex replication cycle that is intimately linked to the cell division cycle. Here, we also catalogue and discuss DUBs that have been linked to centrosome replication or function, including centrosome clustering, a mitotic survival strategy unique to cancer cells with supernumerary centrosomes.

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