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Eg5 Inhibitors Have Contrasting Effects on Microtubule Stability and Metaphase Spindle Integrity.
Chen GY, Kang YJ, Gayek AS, Youyen W, Tüzel E, Ohi R, Hancock WO
(2017) ACS Chem Biol 12: 1038-1046
MeSH Terms: Animals, Cell Line, Humans, Kinesin, Metaphase, Microtubules, Spindle Apparatus
Show Abstract · Added April 18, 2017
To uncover their contrasting mechanisms, antimitotic drugs that inhibit Eg5 (kinesin-5) were analyzed in mixed-motor gliding assays of kinesin-1 and Eg5 motors in which Eg5 "braking" dominates motility. Loop-5 inhibitors (monastrol, STLC, ispinesib, and filanesib) increased gliding speeds, consistent with inducing a weak-binding state in Eg5, whereas BRD9876 slowed gliding, consistent with locking Eg5 in a rigor state. Biochemical and single-molecule assays demonstrated that BRD9876 acts as an ATP- and ADP-competitive inhibitor with 4 nM K. Consistent with its microtubule polymerase activity, Eg5 was shown to stabilize microtubules against depolymerization. This stabilization activity was eliminated in monastrol but was enhanced by BRD9876. Finally, in metaphase-arrested RPE-1 cells, STLC promoted spindle collapse, whereas BRD9876 did not. Thus, different Eg5 inhibitors impact spindle assembly and architecture through contrasting mechanisms, and rigor inhibitors may paradoxically have the capacity to stabilize microtubule arrays in cells.
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7 MeSH Terms
Resistance is not futile: Surviving Eg5 inhibition.
Dumas ME, Sturgill EG, Ohi R
(2016) Cell Cycle 15: 2845-2847
MeSH Terms: Kinesin, Microtubules, Mitosis, Spindle Apparatus
Added April 18, 2017
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1 Members
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4 MeSH Terms
CDK-1 Inhibition in G2 Stabilizes Kinetochore-Microtubules in the following Mitosis.
Gayek AS, Ohi R
(2016) PLoS One 11: e0157491
MeSH Terms: Anaphase, CDC2 Protein Kinase, Cell Line, Transformed, Chromosomes, Human, Cyclin-Dependent Kinases, G2 Phase, Humans, Kinesin, Kinetochores, Microtubules
Show Abstract · Added April 18, 2017
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.
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10 MeSH Terms
Kinesin-5 inhibitor resistance is driven by kinesin-12.
Sturgill EG, Norris SR, Guo Y, Ohi R
(2016) J Cell Biol 213: 213-27
MeSH Terms: Cysteine, HeLa Cells, Humans, Kinesin, Spindle Apparatus
Show Abstract · Added April 18, 2017
The microtubule (MT) cytoskeleton bipolarizes at the onset of mitosis to form the spindle. In animal cells, the kinesin-5 Eg5 primarily drives this reorganization by actively sliding MTs apart. Its primacy during spindle assembly renders Eg5 essential for mitotic progression, demonstrated by the lethal effects of kinesin-5/Eg5 inhibitors (K5Is) administered in cell culture. However, cultured cells can acquire resistance to K5Is, indicative of alternative spindle assembly mechanisms and/or pharmacological failure. Through characterization of novel K5I-resistant cell lines, we unveil an Eg5 motility-independent spindle assembly pathway that involves both an Eg5 rigor mutant and the kinesin-12 Kif15. This pathway centers on spindle MT bundling instead of Kif15 overexpression, distinguishing it from those previously described. We further show that large populations (∼10(7) cells) of HeLa cells require Kif15 to survive K5I treatment. Overall, this study provides insight into the functional plasticity of mitotic kinesins during spindle assembly and has important implications for the development of antimitotic regimens that target this process.
© 2016 Sturgill et al.
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5 MeSH Terms
Biased Brownian motion as a mechanism to facilitate nanometer-scale exploration of the microtubule plus end by a kinesin-8.
Shin Y, Du Y, Collier SE, Ohi MD, Lang MJ, Ohi R
(2015) Proc Natl Acad Sci U S A 112: E3826-35
MeSH Terms: Biophysical Phenomena, Cell Tracking, Diffusion, HeLa Cells, Humans, Kinesin, Kinetics, Microtubules, Motion, Mutant Proteins, Protein Binding, Protein Multimerization, Protein Structure, Tertiary, Video Recording
Show Abstract · Added April 18, 2017
Kinesin-8s are plus-end-directed motors that negatively regulate microtubule (MT) length. Well-characterized members of this subfamily (Kip3, Kif18A) exhibit two important properties: (i) They are "ultraprocessive," a feature enabled by a second MT-binding site that tethers the motors to a MT track, and (ii) they dissociate infrequently from the plus end. Together, these characteristics combined with their plus-end motility cause Kip3 and Kif18A to enrich preferentially at the plus ends of long MTs, promoting MT catastrophes or pausing. Kif18B, an understudied human kinesin-8, also limits MT growth during mitosis. In contrast to Kif18A and Kip3, localization of Kif18B to plus ends relies on binding to the plus-end tracking protein EB1, making the relationship between its potential plus-end-directed motility and plus-end accumulation unclear. Using single-molecule assays, we show that Kif18B is only modestly processive and that the motor switches frequently between directed and diffusive modes of motility. Diffusion is promoted by the tail domain, which also contains a second MT-binding site that decreases the off rate of the motor from the MT lattice. In cells, Kif18B concentrates at the extreme tip of a subset of MTs, superseding EB1. Our data demonstrate that kinesin-8 motors use diverse design principles to target MT plus ends, which likely target them to the plus ends of distinct MT subpopulations in the mitotic spindle.
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14 MeSH Terms
Kinesin-12 Kif15 targets kinetochore fibers through an intrinsic two-step mechanism.
Sturgill EG, Das DK, Takizawa Y, Shin Y, Collier SE, Ohi MD, Hwang W, Lang MJ, Ohi R
(2014) Curr Biol 24: 2307-13
MeSH Terms: Cell Cycle, HeLa Cells, Humans, Immunoblotting, Kinesin, Kinetochores, Microscopy, Fluorescence, Microtubules, Mitosis, Spindle Apparatus
Show Abstract · Added January 20, 2015
Proteins that recognize and act on specific subsets of microtubules (MTs) enable the varied functions of the MT cytoskeleton. We recently discovered that Kif15 localizes exclusively to kinetochore fibers (K-fibers) or bundles of kinetochore-MTs within the mitotic spindle. It is currently speculated that the MT-associated protein TPX2 loads Kif15 onto spindle MTs, but this model has not been rigorously tested. Here, we show that Kif15 accumulates on MT bundles as a consequence of two inherent biochemical properties. First, Kif15 is self-repressed by its C terminus. Second, Kif15 harbors a nonmotor MT-binding site, enabling dimeric Kif15 to crosslink and slide MTs. Two-MT binding activates Kif15, resulting in its accumulation on and motility within MT bundles but not on individual MTs. We propose that Kif15 targets K-fibers via an intrinsic two-step mechanism involving molecular unfolding and two-MT binding. This work challenges the current model of Kif15 regulation and provides the first account of a kinesin that specifically recognizes a higher-order MT array.
Copyright © 2014 Elsevier Ltd. All rights reserved.
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2 Members
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10 MeSH Terms
Kinetochore-microtubule stability governs the metaphase requirement for Eg5.
Gayek AS, Ohi R
(2014) Mol Biol Cell 25: 2051-60
MeSH Terms: Cell Line, Tumor, Humans, Kinesin, Kinetochores, Metaphase, Microtubules, Protein Stability, Spindle Apparatus
Show Abstract · Added May 19, 2014
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.
© 2014 Gayek and Ohi. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).
0 Communities
1 Members
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8 MeSH Terms
Microtubule-regulating kinesins.
Sturgill EG, Ohi R
(2013) Curr Biol 23: R946-8
MeSH Terms: Eukaryota, Humans, Kinesin, Microtubules, Tubulin, Yeasts
Added March 7, 2014
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1 Members
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6 MeSH Terms
Individual pericentromeres display coordinated motion and stretching in the yeast spindle.
Stephens AD, Snider CE, Haase J, Haggerty RA, Vasquez PA, Forest MG, Bloom K
(2013) J Cell Biol 203: 407-16
MeSH Terms: Adenosine Triphosphatases, Cell Cycle Proteins, Centromere, Chromatids, Chromatin, Chromosomal Proteins, Non-Histone, Chromosome Segregation, DNA-Binding Proteins, Kinesin, Kinetochores, Microtubules, Mitosis, Multiprotein Complexes, Nuclear Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Spindle Apparatus, Stress, Physiological
Show Abstract · Added January 25, 2016
The mitotic segregation apparatus composed of microtubules and chromatin functions to faithfully partition a duplicated genome into two daughter cells. Microtubules exert extensional pulling force on sister chromatids toward opposite poles, whereas pericentric chromatin resists with contractile springlike properties. Tension generated from these opposing forces silences the spindle checkpoint to ensure accurate chromosome segregation. It is unknown how the cell senses tension across multiple microtubule attachment sites, considering the stochastic dynamics of microtubule growth and shortening. In budding yeast, there is one microtubule attachment site per chromosome. By labeling several chromosomes, we find that pericentromeres display coordinated motion and stretching in metaphase. The pericentromeres of different chromosomes exhibit physical linkage dependent on centromere function and structural maintenance of chromosomes complexes. Coordinated motion is dependent on condensin and the kinesin motor Cin8, whereas coordinated stretching is dependent on pericentric cohesin and Cin8. Linking of pericentric chromatin through cohesin, condensin, and kinetochore microtubules functions to coordinate dynamics across multiple attachment sites.
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1 Members
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18 MeSH Terms
Microtubule-depolymerizing kinesins.
Walczak CE, Gayek S, Ohi R
(2013) Annu Rev Cell Dev Biol 29: 417-41
MeSH Terms: Animals, Humans, Kinesin, Microtubules, Molecular Motor Proteins, Phylogeny, Spindle Apparatus
Show Abstract · Added March 7, 2014
The microtubule (MT) cytoskeleton supports a broad range of cellular functions, from providing tracks for intracellular transport, to supporting movement of cilia and flagella, to segregating chromosomes in mitosis. These functions are facilitated by the organizational and dynamic plasticity of MT networks. An important class of enzymes that alters MT dynamics is the depolymerizing kinesin-like proteins, which use their catalytic activities to regulate MT end dynamics. In this review, we discuss four topics surrounding these MT-depolymerizing kinesins. We provide a historical overview of studies focused on these motors and discuss their phylogeny. In the second half, we discuss their enzymology and biophysics and give an overview of their known cellular functions. This discussion highlights the fact that MT-depolymerizing kinesins exhibit a diverse range of design principles, which in turn increases their functional versatility in cells.
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7 MeSH Terms