An Inner Centromere Protein that Stimulates the Microtubule Depolymerizing Activity of a KinI Kinesin

AbstractMitosis requires precise control of microtubule dynamics. The KinI kinesin MCAK, a microtubule depolymerase, is critical for this regulation. In a screen to discover previously uncharacterized microtubule-associated proteins, we identified ICIS, a protein that stimulates MCAK activity in vitro. Consistent with this biochemical property, blocking ICIS function in Xenopus extracts with antibodies caused excessive microtubule growth and inhibited spindle formation. Prior to anaphase, ICIS localized in an MCAK-dependent manner to inner centromeres, the chromosomal region located in between sister kinetochores. From Xenopus extracts, ICIS coimmunoprecipitated MCAK and the inner centromere proteins INCENP and Aurora B, which are thought to promote chromosome biorientation. By immunoelectron microscopy, we found that ICIS is present on the surface of inner centromeres, placing it in an ideal location to depolymerize microtubules associated laterally with inner centromeres. At inner centromeres, MCAK-ICIS may destabilize these microtubules and provide a mechanism that prevents kinetochore-microtubule attachment errors

XRHAMM Functions in Ran-Dependent Microtubule Nucleation and Pole Formation during Anastral Spindle Assembly

Background: The regulated assembly of microtubules is essential for bipolar spindle formation. Depending on cell type, microtubules nucleate through two different pathways: centrosome-driven or chromatin-driven. The chromatin-driven pathway dominates in cells lacking centrosomes.Results: Human RHAMM (receptor for hyaluronic-acid-mediated motility) was originally implicated in hyaluronic-acid-induced motility but has since been shown to associate with centrosomes and play a role in astral spindle pole integrity in mitotic systems. We have identified the Xenopus ortholog of human RHAMM as a microtubule-associated protein that plays a role in focusing spindle poles and is essential for efficient microtubule nucleation during spindle assembly without centrosomes. XRHAMM associates both with γ-TuRC, a complex required for microtubule nucleation and with TPX2, a protein required for microtubule nucleation and spindle pole organization.Conclusions: XRHAMM facilitates Ran-dependent, chromatin-driven nucleation in a process that may require coordinate activation of TPX2 and γ-TuRC

The Chromosomal Passenger Complex Is Required for Chromatin-Induced Microtubule Stabilization and Spindle Assembly

AbstractIn cells lacking centrosomes, such as those found in female meiosis, chromosomes must nucleate and stabilize microtubules in order to form a bipolar spindle. Here we report the identification of Dasra A and Dasra B, two new components of the vertebrate chromosomal passenger complex containing Incenp, Survivin, and the kinase Aurora B, and demonstrate that this complex is required for chromatin-induced microtubule stabilization and spindle formation. The failure of microtubule stabilization caused by depletion of the chromosomal passenger complex was rescued by codepletion of the microtubule-depolymerizing kinesin MCAK, whose activity is negatively regulated by Aurora B. By contrast, we present evidence that the Ran-GTP pathway of chromatin-induced microtubule nucleation does not require the chromosomal passenger complex, indicating that the mechanisms of microtubule assembly by these two pathways are distinct. We propose that the chromosomal passenger complex regulates local MCAK activity to permit spindle formation via stabilization of chromatin-associated microtubules

Spindle assembly in the absence of a RanGTP gradient requires localized CPC activity

Author Posting. © The Author(s), 2009. This is the author's version of the work. It is posted here by permission of Elsevier B.V. for personal use, not for redistribution. The definitive version was published in Current Biology 19 (2009): 1210-1215, doi:10.1016/j.cub.2009.05.061.During animal cell division, a gradient of GTP-bound Ran is generated around mitotic chromatin. It is generally accepted that this RanGTP gradient is essential for organizing the spindle since it locally activates critical spindle assembly factors. Here, we show in Xenopus egg extract, where the gradient is best characterized, that spindles can assemble in the absence of a RanGTP gradient. Gradient-free spindle assembly occurred around sperm nuclei but not around chromatin-coated beads and required the chromosomal passenger complex (CPC). Artificial enrichment of CPC activity within hybrid bead arrays containing both immobilized chromatin and the CPC supported local microtubule assembly even in the absence of a RanGTP gradient. We conclude that RanGTP and the CPC constitute the two major molecular signals that spatially promote microtubule polymerization around chromatin. Furthermore, we hypothesize that the two signals mainly originate from discreet physical sites on the chromosomes to localize microtubule assembly around chromatin: a RanGTP signal from any chromatin, and a CPC-dependent signal predominantly generated from centromeric chromatin.This work was supported by the American Cancer Society (grant PF0711401 to T.J. Maresca), the National Cancer Institute (grant CA078048-09 to T.J. Mitchison) and the National Institutes of Health (grant F32GM080049 to J.C. Gatlin and grant GM24364 to E.D. Salmon)

Cdk1 phosphorylation of the kinetochore protein Nsk1 prevents error-prone chromosome segregation

Phosphorylation of the kinetochore component Nsk1 by Cdk1 antagonizes its localization to and function at the kinetochore and spindle during early mitosis

Ahead of the Curve: New Insights into Microtubule Dynamics [version 1; referees: 2 approved]

Microtubule dynamics are fundamental for many aspects of cell physiology, but their mechanistic underpinnings remain unclear despite 40 years of intense research. In recent years, the continued union of reconstitution biochemistry, structural biology, and modeling has yielded important discoveries that deepen our understanding of microtubule dynamics. These studies, which we review here, underscore the importance of GTP hydrolysis-induced changes in tubulin structure as microtubules assemble, and highlight the fact that each aspect of microtubule behavior is the output of complex, multi-step processes. Although this body of work moves us closer to appreciating the key features of microtubule biochemistry that drive dynamic instability, the divide between our understanding of microtubules in isolation versus within the cellular milieu remains vast. Bridging this gap will serve as fertile grounds of cytoskeleton-focused research for many years to come

CDK-1 Inhibition in G2 Stabilizes Kinetochore-Microtubules in the following Mitosis

<div><p>Cell proliferation is driven by cyclical activation of cyclin-dependent kinases (CDKs), which produce distinct biochemical cell cycle phases. Mitosis (M phase) is orchestrated by CDK-1, complexed with mitotic cyclins. During M phase, chromosomes are segregated by a bipolar array of microtubules called the mitotic spindle. The essential bipolarity of the mitotic spindle is established by the kinesin-5 Eg5, but factors influencing the maintenance of spindle bipolarity are not fully understood. Here, we describe an unexpected link between inhibiting CDK-1 before mitosis and bipolar spindle maintenance. Spindles in human RPE-1 cells normally collapse to monopolar structures when Eg5 is inhibited at metaphase. However, we found that inhibition of CDK-1 in the G2 phase of the cell cycle improved the ability of RPE-1 cells to maintain spindle bipolarity without Eg5 activity in the mitosis immediately after release from CDK-1 inhibition. This improved bipolarity maintenance correlated with an increase in the stability of kinetochore-microtubules, the subset of microtubules that link chromosomes to the spindle. The improvement in bipolarity maintenance after CDK-1 inhibition in G2 required both the kinesin-12 Kif15 and increased stability of kinetochore-microtubules. Consistent with increased kinetochore-microtubule stability, we find that inhibition of CDK-1 in G2 impairs mitotic fidelity by increasing the incidence of lagging chromosomes in anaphase. These results suggest that inhibition of CDK-1 in G2 causes unpredicted effects in mitosis, even after CDK-1 inhibition is relieved.</p></div

High K-MT stability is required for the bipolarity-protective effect of RO-3306.

<p>A) Representative images of spindles following transfection with control or HURP-targeting siRNA, followed by the drug treatments described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0157491#pone.0157491.g001" target="_blank">Fig 1A</a>. In overlay images, HURP is shown in magenta, tubulin is shown in green, and DNA is shown in blue; scale bar, 5 μm. B) Quantification of the percentage of mitotic cells with monopolar spindles following MG-STLC treatment as in (A). Bars represent the mean and error bars represent the s.e.m. at least 61 cells from at least 3 experiments. Among cells treated with DMSO, a statistically significant difference exists between cells subjected to control and HURP RNAi (p < 0.001); among cells treated with RO-3306, a statistically significant difference exists between cells subjected to control and HURP RNAi (p < 0.001); among cells subjected to control RNAi, a statistically significant difference exists between DMSO- and RO-3306-treated cells (p < 0.001); but among cells subjected to HURP RNAi, a statistically significant difference was not found between DMSO- and RO-3306-treated cells (p > 0.05). C) Quantification of the percentage of mitotic cells with monopolar spindles following MG-STLC treatment as in (A), but with the inclusion of 0.5 nM Taxol during MG-STLC treatment. A statistically significant difference exists between cells subjected to control RNAi and HURP RNAi (p < 0.001), and a statistically significant difference exists between DMSO- and RO-3306-treated cells (p = 0.005). D) Normalized immunofluorescence intensity of HURP relative to MTs on the spindle for untransfected cells treated with a double thymidine block, washout, 5 h recovery, 3 h RO-3306 or DMSO, washout, and 90 min MG-132 before fixation. Bars represent at least 39 cells from 4 experiments. Box-and-whisker plots indicate the 10^th, 25^th, 50^th, 75^th, and 90^th percentile as well as outliers. A statistically significant difference exists between DMSO- and RO-3306-treated cells (p < 0.05).</p