scholarly journals Ran-GTP is non-essential to activate NuMA for spindle pole focusing, but dynamically polarizes HURP to control mitotic spindle length

2018 ◽  
Author(s):  
Kenta Tsuchiya ◽  
Hisato Hayashi ◽  
Momoko Nishina ◽  
Masako Okumura ◽  
Yoshikatsu Sato ◽  
...  
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Nucleocytoplasmic Transport ◽  
Spindle Pole ◽  
Human Cells ◽  
Fiber Formation ◽  
List Type ◽  
Mitotic Spindle Assembly ◽  
Ran Gtp ◽  
Dynamic Microtubule

AbstractDuring mitosis, a bipolar spindle is assembled around chromosomes to efficiently capture chromosomes. Previous work proposed that a chromosome-derived Ran-GTP gradient promotes spindle assembly around chromosomes by liberating spindle assembly factors (SAFs) from inhibitory importins. However, Ran’s dual functions in interphase nucleocytoplasmic transport and mitotic spindle assembly have made it difficult to assess its mitotic roles in somatic cells. Here, using auxin-inducible degron technology in human cells, we developed acute mitotic degradation assays to dissect Ran’s mitotic roles systematically and separately from its interphase function. In contrast to the prevailing model, we found that the Ran pathway is not essential for spindle assembly activities that occur at sites spatially separated from chromosomes, including activating NuMA for spindle pole focusing or for targeting TPX2. In contrast, Ran-GTP is required to localize HURP and HSET specifically at chromosome-proximal regions. We demonstrated that Ran-GTP and importin-β coordinately promote HURP’s dynamic microtubule binding-dissociation cycle near chromosomes, which results in stable kinetochore-fiber formation. Intriguingly, this pathway acts to establish proper spindle length preferentially during prometaphase, rather than metaphase. Together, we propose that the Ran pathway is required to activate SAFs specifically near chromosomes, but not generally during human mitotic spindle assembly. Ran-dependent spindle assembly is likely coupled with parallel pathways to activate SAFs, including NuMA, for spindle pole focusing away from chromosomes.HighlightsUsing auxin-inducible degron technology, we developed mitotic degradation assays for the Ran pathway in human cells.The Ran pathway is non-essential to activate NuMA for spindle pole focusing.The Ran pathway dynamically polarizes HURP and defines mitotic spindle length preferentially during prometaphase.Ran-GTP is required to activate SAFs specifically near chromosomes, but not generally, in human mitotic cells.

scholarly journals Targeting Alp7/TACC to the spindle pole body is essential for mitotic spindle assembly in fission yeast

FEBS Letters ◽  
2014 ◽  
Vol 588 (17) ◽  
pp. 2814-2821 ◽  
Author(s):  
Ngang Heok Tang ◽  
Naoyuki Okada ◽  
Chii Shyang Fong ◽  
Kunio Arai ◽  
Masamitsu Sato ◽  
...  
Keyword(s):  
Fission Yeast ◽  
Mitotic Spindle ◽  
Spindle Pole Body ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
Pole Body ◽  
Mitotic Spindle Assembly

scholarly journals Kinetochore-driven formation of kinetochore fibers contributes to spindle assembly during animal mitosis

2004 ◽  
Vol 167 (5) ◽  
pp. 831-840 ◽  
Author(s):  
Helder Maiato ◽  
Conly L. Rieder ◽  
Alexey Khodjakov
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
S2 Cells ◽  
Spindle Poles ◽  
Drosophila S2 Cells ◽  
Mitotic Spindle Assembly ◽  
Normal Mitosis ◽  
Proper Orientation ◽  
Dependent Mechanism

It is now clear that a centrosome-independent pathway for mitotic spindle assembly exists even in cells that normally possess centrosomes. The question remains, however, whether this pathway only activates when centrosome activity is compromised, or whether it contributes to spindle morphogenesis during a normal mitosis. Here, we show that many of the kinetochore fibers (K-fibers) in centrosomal Drosophila S2 cells are formed by the kinetochores. Initially, kinetochore-formed K-fibers are not oriented toward a spindle pole but, as they grow, their minus ends are captured by astral microtubules (MTs) and transported poleward through a dynein-dependent mechanism. This poleward transport results in chromosome bi-orientation and congression. Furthermore, when individual K-fibers are severed by laser microsurgery, they regrow from the kinetochore outward via MT plus-end polymerization at the kinetochore. Thus, even in the presence of centrosomes, the formation of some K-fibers is initiated by the kinetochores. However, centrosomes facilitate the proper orientation of K-fibers toward spindle poles by integrating them into a common spindle.

scholarly journals Cooperative Accumulation of Dynein-Dynactin at Microtubule Minus-Ends Drives Microtubule Network Reorganization

2017 ◽  
Author(s):  
Ruensern Tan ◽  
Peter J. Foster ◽  
Daniel J. Needleman ◽  
Richard J. McKenney
Keyword(s):  
Mitotic Spindle ◽  
Large Scale ◽  
Spindle Assembly ◽  
Cytoplasmic Dynein ◽  
List Type ◽  
Microtubule Network ◽  
Cellular Processes ◽  
Dynein Motor ◽  
Mitotic Spindle Assembly ◽  
Orientation Bias

SummaryCytoplasmic dynein-1 (dynein) is minus-end directed motor protein that transports cargo over long distances and organizes microtubules (MTs) during critical cellular processes such as mitotic spindle assembly. How dynein motor activity is harnessed for these diverse functions remains unknown. Here, we have uncovered a mechanism for how processive dynein-dynactin complexes drive MT-MT sliding, reorganization, and focusing, activities required for mitotic spindle assembly. We find that motors cooperatively accumulate, in limited numbers, at MT minus-ends. Minus-end accumulations drive MT-MT sliding, independent of MT orientation, and this activity always results in the clustering of MT minus-ends. At a mesoscale level, activated dynein-dynactin drives the formation and coalescence of MT asters. Macroscopically, dynein-dynactin activity leads to bulk contraction of millimeter-scale MT networks, demonstrating that minus-end accumulations produce network scale contractile stresses. Our data provides a model for how localized dynein activity is harnessed by cells to produce contractile stresses within the mitotic spindle.HighlightsProcessive dynein-dynactin complexes cooperatively form stable accumulations at MT minus-ends.Minus-end accumulations of motors slide MTs without orientation bias, leading to minus-end focusing.Minus-end accumulations of motors organize dynamic MTs into asters.Minus-end accumulations of motors drive bulk contractions of large-scale MT networks.

scholarly journals Theory of cytoskeletal reorganization during crosslinker-mediated mitotic spindle assembly

2018 ◽  
Author(s):  
A. R. Lamson ◽  
C. J. Edelmaier ◽  
M. A. Glaser ◽  
M. D. Betterton
Keyword(s):  
Mitotic Spindle ◽  
Large Scale ◽  
Dynamic Instability ◽  
Kinetic Monte Carlo ◽  
Spindle Assembly ◽  
Spindle Pole ◽  
Balance Model ◽  
Cytoskeletal Reorganization ◽  
Bipolar Spindle ◽  
Mitotic Spindle Assembly

AbstractCells grow, move, and respond to outside stimuli by large-scale cytoskeletal reorganization. A prototypical example of cytoskeletal remodeling is mitotic spindle assembly, during which micro-tubules nucleate, undergo dynamic instability, bundle, and organize into a bipolar spindle. Key mechanisms of this process include regulated filament polymerization, crosslinking, and motor-protein activity. Remarkably, using passive crosslinkers, fission yeast can assemble a bipolar spindle in the absence of motor proteins. We develop a torque-balance model that describes this reorganization due to dynamic microtubule bundles, spindle-pole bodies, the nuclear envelope, and passive crosslinkers to predict spindle-assembly dynamics. We compare these results to those obtained with kinetic Monte Carlo-Brownian dynamics simulations, which include crosslinker-binding kinetics and other stochastic effects. Our results show that rapid crosslinker reorganization to microtubule overlaps facilitates crosslinker-driven spindle assembly, a testable prediction for future experiments. Combining these two modeling techniques, we illustrate a general method for studying cytoskeletal network reorganization.

Ran–GTP coordinates regulation of microtubule nucleation and dynamics during mitotic-spindle assembly

2001 ◽  
Vol 3 (3) ◽  
pp. 228-234 ◽  
Author(s):  
Rafael E. Carazo-Salas ◽  
Oliver J. Gruss ◽  
Iain W. Mattaj ◽  
Eric Karsenti
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Microtubule Nucleation ◽  
Mitotic Spindle Assembly ◽  
Ran Gtp

scholarly journals Targeting of RCC1 to Chromosomes Is Required for Proper Mitotic Spindle Assembly in Human Cells

2002 ◽  
Vol 12 (16) ◽  
pp. 1442-1447 ◽  
Author(s):  
William J Moore ◽  
Chuanmao Zhang ◽  
Paul R Clarke
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Human Cells ◽  
Mitotic Spindle Assembly

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.

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