Mechanisms of chromosome biorientation and bipolar spindle assembly analyzed by computational modeling

The essential functions required for mitotic spindle assembly and chromosome biorientation and segregation are not fully understood, despite extensive study. To illuminate the combinations of ingredients most important to align and segregate chromosomes and simultaneously assemble a bipolar spindle, we developed a computational model of fission-yeast mitosis. Robust chromosome biorientation requires progressive restriction of attachment geometry, destabilization of misaligned attachments, and attachment force dependence. Large spindle length fluctuations can occur when the kinetochore-microtubule attachment lifetime is long. The primary spindle force generators are kinesin-5 motors and crosslinkers in early mitosis, while interkinetochore stretch becomes important after biorientation. The same mechanisms that contribute to persistent biorientation lead to segregation of chromosomes to the poles after anaphase onset. This model therefore provides a framework to interrogate key requirements for robust chromosome biorientation, spindle length regulation, and force generation in the spindle.

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Mechanisms of chromosome biorientation and bipolar spindle assembly analyzed by computational modeling

10.1101/649913 ◽

2019 ◽

Cited By ~ 2

Author(s):

Christopher J. Edelmaier ◽

Adam R. Lamson ◽

Zachary R. Gergely ◽

Saad Ansari ◽

Robert Blackwell ◽

...

Keyword(s):

Mitotic Spindle ◽

Spindle Assembly ◽

Extensive Study ◽

Bipolar Spindle ◽

Microtubule Attachment ◽

Anaphase Onset ◽

Length Regulation ◽

Mitotic Spindle Assembly ◽

Early Mitosis ◽

Segregation Of Chromosomes

AbstractThe essential functions required for mitotic spindle assembly and chromosome biorientation and segregation are not fully understood, despite extensive study. To illuminate the combinations of ingredients most important to align and segregate chromosomes and simultaneously assemble a bipolar spindle, we developed a computational model of fission-yeast mitosis. Robust chromosome biorientation requires progressive restriction of attachment geometry, destabilization of misaligned attachments, and attachment force dependence. Large spindle length fluctuations can occur when the kinetochore-microtubule attachment lifetime is long. The primary spindle force generators are kinesin-5 motors and crosslinkers in early mitosis, while interkinetochore stretch becomes important after biorientation. The same mechanisms that contribute to persistent biorientation lead to segregation of chromosomes to the poles after anaphase onset. This model therefore provides a framework to interrogate key requirements for robust chromosome biorientation, spindle length regulation, and force generation in the spindle.

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

10.1101/438903 ◽

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.

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Thirty years of search and capture: The complex simplicity of mitotic spindle assembly

The Journal of Cell Biology ◽

10.1083/jcb.201510015 ◽

2015 ◽

Vol 211 (6) ◽

pp. 1103-1111 ◽

Cited By ~ 90

Author(s):

Rebecca Heald ◽

Alexey Khodjakov

Keyword(s):

Cell Division ◽

Mitotic Spindle ◽

Spindle Assembly ◽

Microtubule Dynamics ◽

Adaptive Changes ◽

Bipolar Spindle ◽

Chromosome Architecture ◽

Self Assembling ◽

Motor Activities ◽

Mitotic Spindle Assembly

Cell division is enacted by a microtubule-based, self-assembling macromolecular machine known as the mitotic spindle. In 1986, Kirschner and Mitchison proposed that by undergoing dynamic cycles of growth and disassembly, microtubules search for chromosomes. Capture of microtubules by the kinetochores progressively connects chromosomes to the bipolar spindle. 30 years later, “search and capture” remains the cornerstone of spindle assembly. However, a variety of facilitating mechanisms such as regulation of microtubule dynamics by diffusible gradients, spatially selective motor activities, and adaptive changes in chromosome architecture have been discovered. We discuss how these mechanisms ensure that the spindle assembles rapidly and with a minimal number of errors.

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Relative contributions of chromatin and kinetochores to mitotic spindle assembly

The Journal of Cell Biology ◽

10.1083/jcb.200903076 ◽

2009 ◽

Vol 187 (1) ◽

pp. 43-51 ◽

Cited By ~ 55

Author(s):

Christopher B. O'Connell ◽

Jadranka Lončarek ◽

Petr Kaláb ◽

Alexey Khodjakov

Keyword(s):

Mitotic Spindle ◽

Dominant Role ◽

Spindle Assembly ◽

High Concentration ◽

Animal Cells ◽

Microtubule Attachment ◽

Mitotic Spindle Assembly ◽

Assembly Pathways ◽

Mitosis And Meiosis ◽

Normal Mitosis

During mitosis and meiosis in animal cells, chromosomes actively participate in spindle assembly by generating a gradient of Ran guanosine triphosphate (RanGTP). A high concentration of RanGTP promotes microtubule nucleation and stabilization in the vicinity of chromatin. However, the relative contributions of chromosome arms and centromeres/kinetochores in this process are not known. In this study, we address this issue using cells undergoing mitosis with unreplicated genomes (MUG). During MUG, chromatin is rapidly separated from the forming spindle, and both centrosomal and noncentrosomal spindle assembly pathways are active. MUG chromatin is coated with RCC1 and establishes a RanGTP gradient. However, a robust spindle forms around kinetochores/centromeres outside of the gradient peak. When stable kinetochore microtubule attachment is prevented by Nuf2 depletion in both MUG and normal mitosis, chromatin attracts astral microtubules but cannot induce spindle assembly. These results support a model in which kinetochores play the dominant role in the chromosome-mediated pathway of mitotic spindle assembly.

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Nup98 regulates bipolar spindle assembly through association with microtubules and opposition of MCAK

Molecular Biology of the Cell ◽

10.1091/mbc.e10-06-0478 ◽

2011 ◽

Vol 22 (5) ◽

pp. 661-672 ◽

Cited By ~ 44

Author(s):

Marie K. Cross ◽

Maureen A. Powers

Keyword(s):

Nuclear Pore Complex ◽

Mitotic Spindle ◽

Spindle Assembly ◽

Microtubule Dynamics ◽

Nuclear Pore ◽

Bipolar Spindle ◽

Terminal Domain ◽

C Terminus ◽

Mitotic Spindle Assembly

During mitosis, the nuclear pore complex is disassembled and, increasingly, nucleoporins are proving to have mitotic functions when released from the pore. We find a contribution of the nucleoporin Nup98 to mitotic spindle assembly through regulation of microtubule dynamics. When added to Xenopus extract spindle assembly assays, the C-terminal domain of Nup98 stimulates uncontrolled growth of microtubules. Conversely, inhibition or depletion of Nup98 leads to formation of stable monopolar spindles. Spindle bipolarity is restored by addition of purified, recombinant Nup98 C-terminus. The minimal required region of Nup98 corresponds to a portion of the C-terminal domain lacking a previously characterized function. We show association between this region of the C-terminus of Nup98 and both Taxol-stabilized microtubules and the microtubule-depolymerizing mitotic centromere–associated kinesin (MCAK). Importantly, we demonstrate that this domain of Nup98 inhibits MCAK depolymerization activity in vitro. These data support a model in which Nup98 interacts with microtubules and antagonizes MCAK activity, thus promoting bipolar spindle assembly.

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Microtubule pivoting enables mitotic spindle assembly in S. cerevisiae

The Journal of Cell Biology ◽

10.1083/jcb.202007193 ◽

2021 ◽

Vol 220 (3) ◽

Author(s):

Kimberly K. Fong ◽

Trisha N. Davis ◽

Charles L. Asbury

Keyword(s):

Mitotic Spindle ◽

Spindle Assembly ◽

Force Generation ◽

Initial Contact ◽

Bipolar Spindle ◽

Spindle Poles ◽

Mitotic Spindle Assembly ◽

Spindle Microtubules ◽

Pole Component

To assemble a bipolar spindle, microtubules emanating from two poles must bundle into an antiparallel midzone, where plus end–directed motors generate outward pushing forces to drive pole separation. Midzone cross-linkers and motors display only modest preferences for antiparallel filaments, and duplicated poles are initially tethered together, an arrangement that instead favors parallel interactions. Pivoting of microtubules around spindle poles might help overcome this geometric bias, but the intrinsic pivoting flexibility of the microtubule–pole interface has not been directly measured, nor has its importance during early spindle assembly been tested. By measuring the pivoting of microtubules around isolated yeast spindle poles, we show that pivoting flexibility can be modified by mutating a microtubule-anchoring pole component, Spc110. By engineering mutants with different flexibilities, we establish the importance of pivoting in vivo for timely pole separation. Our results suggest that passive thermal pivoting can bring microtubules from side-by-side poles into initial contact, but active minus end–directed force generation will be needed to achieve antiparallel alignment.

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Deubiquitylase UCHL3 drives error correction at kinetochores and chromosome segregation independent of spindle assembly checkpoint

10.1101/2020.03.31.018077 ◽

2020 ◽

Author(s):

Katerina Jerabkova ◽

Yongrong Liao ◽

Charlotte Kleiss ◽

Sadek Fournane ◽

Matej Durik ◽

...

Keyword(s):

Error Correction ◽

Spindle Assembly Checkpoint ◽

Spindle Assembly ◽

Cellular Transformation ◽

Aurora B ◽

Mitotic Kinase ◽

Daughter Cells ◽

Microtubule Attachment ◽

Anaphase Onset ◽

Segregation Of Chromosomes

AbstractEqual segregation of chromosomes during mitosis ensures euploidy of daughter cells. Defects in this process may result in imbalance in chromosomal composition and cellular transformation. Two surveillance pathways, the spindle assembly checkpoint (SAC) and the error-correction (EC), exist at kinetochores that monitor microtubule attachment and faithful segregation of chromosomes at the metaphase to anaphase transition. However, the molecular understanding of the interplay between EC and SAC signaling remains limited. Here we describe a role of deubiquitylase UCHL3 in the regulation of EC pathway during mitosis. Downregulation or inhibition of UCHL3 leads to improper attachments of chromosomes to spindle microtubules and to chromosome alignment defects during metaphase. Frequent segregation errors during anaphase and consequently aneuploidy is also observed upon inactivation of UCHL3. Surprisingly, UCHL3 is not involved in SAC signaling as both recruitment of SAC proteins to kinetochores and timely anaphase onset are not perturbed in UCHL3-deficient cells. Mechanistically, UCHL3 interacts with and deubiquitylates the mitotic kinase Aurora B known to drive both SAC and EC signaling. UCHL3 promotes interaction of Aurora B with MCAK, important EC factor but does not regulate Aurora B binding to other interacting partners or subcellular localization of Aurora B. Our results thus suggest that UCHL3-mediated deubiquitylation functionally separates EC from SAC signaling during mitosis and is critical for maintenance of euploidy in human cells.

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Theory of cytoskeletal reorganization during crosslinker-mediated mitotic spindle assembly

10.1101/419135 ◽

2018 ◽

Cited By ~ 1

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.

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