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1
Adib, Rozita, Jessica M. Montgomery, Joseph Atherton, Laura O’Regan, Mark W. Richards, Kees R. Straatman, Daniel Roth, et al. "Mitotic phosphorylation by NEK6 and NEK7 reduces the microtubule affinity of EML4 to promote chromosome congression." Science Signaling 12, no. 594 (August 13, 2019): eaaw2939. http://dx.doi.org/10.1126/scisignal.aaw2939.
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EML4 is a microtubule-associated protein that promotes microtubule stability. We investigated its regulation across the cell cycle and found that EML4 was distributed as punctate foci along the microtubule lattice in interphase but exhibited reduced association with spindle microtubules in mitosis. Microtubule sedimentation and cryo–electron microscopy with 3D reconstruction revealed that the basic N-terminal domain of EML4 mediated its binding to the acidic C-terminal tails of α- and β-tubulin on the microtubule surface. The mitotic kinases NEK6 and NEK7 phosphorylated the EML4 N-terminal domain at Ser144 and Ser146 in vitro, and depletion of these kinases in cells led to increased EML4 binding to microtubules in mitosis. An S144A-S146A double mutant not only bound inappropriately to mitotic microtubules but also increased their stability and interfered with chromosome congression. In addition, constitutive activation of NEK6 or NEK7 reduced the association of EML4 with interphase microtubules. Together, these data support a model in which NEK6- and NEK7-dependent phosphorylation promotes the dissociation of EML4 from microtubules in mitosis in a manner that is required for efficient chromosome congression.
2
Jordan, M. A., D. Thrower, and L. Wilson. "Effects of vinblastine, podophyllotoxin and nocodazole on mitotic spindles. Implications for the role of microtubule dynamics in mitosis." Journal of Cell Science 102, no. 3 (July 1, 1992): 401–16. http://dx.doi.org/10.1242/jcs.102.3.401.
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Inhibition of mitosis by many drugs that bind to tubulin has been attributed to depolymerization of microtubules. However, we found previously that low concentrations of vinblastine and vincristine blocked mitosis in HeLa cells with little or no depolymerization of spindle microtubules, and spindles appeared morphologically normal or nearly normal. In the present study, we characterized the effects of vinblastine, podophyllotoxin and nocodazole over broad concentration ranges on mitotic spindle organization in HeLa cells. These three drugs are known to affect the dynamics of microtubule polymerization in vitro and to depolymerize microtubules in cells. We wanted to probe further whether mitotic inhibition by these drugs is brought about by a more subtle effect on the microtubules than net microtubule depolymerization. We compared the effects of vinblastine, podophyllotoxin and nocodazole on the organization of spindle microtubules, chromosomes and centrosomes, and on the total mass of microtubules. Spindle organization was examined by immunofluorescence microscopy, and microtubule polymer mass was assayed on isolated cytoskeletons by a quantitative enzyme-linked immunoadsorbence assay for tubulin. As the drug concentration was increased, the organization of mitotic spindles changed in the same way with all three drugs. The changes were associated with mitotic arrest, but were not necessarily accompanied by net microtubule depolymerization. With podophyllotoxin, mitotic arrest was accompanied by microtubule depolymerization. In contrast, with vinblastine and nocodazole, mitotic arrest occurred in the presence of a full complement of spindle microtubules. All three drugs induced a nearly identical rearrangement of spindle microtubules, an increasingly aberrant organization of metaphase chromosomes, and fragmentation of centrosomes. The data suggest that these anti-mitotic drugs block mitosis primarily by inhibiting the dynamics of spindle microtubules rather than by simply depolymerizing the microtubules.
3
Vedrenne, Cécile, Dieter R. Klopfenstein, and Hans-Peter Hauri. "Phosphorylation Controls CLIMP-63–mediated Anchoring of the Endoplasmic Reticulum to Microtubules." Molecular Biology of the Cell 16, no. 4 (April 2005): 1928–37. http://dx.doi.org/10.1091/mbc.e04-07-0554.
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The microtubule-binding 63-kDa cytoskeleton-linking membrane protein (CLIMP-63) is an integral membrane protein that links the endoplasmic reticulum (ER) to microtubules. Here, we tested whether this interaction is regulated by phosphorylation. Metabolic labeling with 32P showed that CLIMP-63 is a phosphoprotein with increased phosphorylation during mitosis. CLIMP-63 of mitotic cells is unable to bind to microtubules in vitro. Mitotic phosphorylation can be prevented by mutation of serines 3, 17, and 19 in the cytoplasmic domain of CLIMP-63. When these residues are mutated to glutamic acid, and hence mimic mitotic phosphorylation, CLIMP-63 does no longer bind to microtubules in vitro. Overexpression of the phospho-mimicking mitotic form of CLIMP-63 in interphase cells leads to a collapse of the ER around the nucleus, leaving the microtubular network intact. The results suggest that CLIMP-63–mediated stable anchoring of the ER to microtubules is required to maintain the spatial distribution of the ER during interphase and that this interaction is abolished by phosphorylation of CLIMP-63 during mitosis.
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Roychoudhury, Sonali, and Martha J. Powell. "Ultrastructure of mitosis in the algal parasitic fungus Polyphagus euglenae." Canadian Journal of Botany 69, no. 10 (October 1, 1991): 2201–14. http://dx.doi.org/10.1139/b91-277.
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Ultrastructure of mitosis in the parasitic fungus, Polyphagus euglenae, was investigated with emphasis on centrosome structure and prophase events. The interphase centrosome included a diplosome, scattered electron-dense satellites, and multiple ring-shaped microtubule foci. As centrosomes separated during prophase, microtubular arrays extended between the replicated centrosomes and radiated out along the outer surface of the nuclear envelope. The asymmetric configuration of these microtubular arrays suggests that intersecting microtubules provide tension forces on elongating centrosome to centrosome microtubules during centrosome separation. After centrosome migration, multiple microtubule foci appeared to fuse into crescent-shaped microtubule organizing centers. Condensing chromatin was concentrated in the region of the future equatorial plane of the mitotic spindle prior to the appearance of discontinuities in the nuclear envelope and incursion of the spindle. The nucleolus fragmented during prometaphase, and fragments were discarded with the interzonal region during telophase. Nucleoli appeared in daughter nuclei before chromatin became diffuse. Similarities in the mitotic apparatus of P. euglenae with that previously reported for Monoblepharella sp. support a phylogenetic affinity between members of the orders Chytridiales and Monoblepharidales. Key words: mitosis, Polyphagus euglenae, Chytridiales, centrosomes, phylogeny, ultrastructure.
5
Andreassen, P. R., and R. L. Margolis. "Microtubule dependency of p34cdc2 inactivation and mitotic exit in mammalian cells." Journal of Cell Biology 127, no. 3 (November 1, 1994): 789–802. http://dx.doi.org/10.1083/jcb.127.3.789.
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The protein kinase inhibitor 2-aminopurine induces checkpoint override and mitotic exit in BHK cells which have been arrested in mitosis by inhibitors of microtubule function (Andreassen, P. R., and R. L. Margolis. 1991. J. Cell Sci. 100:299-310). Mitotic exit is monitored by loss of MPM-2 antigen, by the reformation of nuclei, and by the extinction of p34cdc2-dependent H1 kinase activity. 2-AP-induced inactivation of p34cdc2 and mitotic exit depend on the assembly state of microtubules. During mitotic arrest generated by the microtubule assembly inhibitor nocodazole, the rate of mitotic exit induced by 2-AP decreases proportionally with increasing nocodazole concentrations. At nocodazole concentrations of 0.12 microgram/ml or greater, 2-AP induces no apparent exit through 75 min of treatment. In contrast, 2-AP brings about a rapid exit (t1/2 = 20 min) from mitotic arrest by taxol, a drug which causes inappropriate overassembly of microtubules. In control mitotic cells, p34cdc2 localizes to kinetochores, centrosomes, and spindle microtubules. We find that efficient exit from mitosis occurs under conditions where p34cdc2 remains associated with centrosomal microtubules, suggesting it must be present on these microtubules in order to be inactivated. Mitotic slippage, the natural reentry of cells into G1 during prolonged mitotic block, is also microtubule dependent. At high nocodazole concentrations slippage is prevented and mitotic arrest approaches 100%. We conclude that essential components of the machinery for exit from mitosis are present on the mitotic spindle, and that normal mitotic exit thereby may be regulated by the microtubule assembly state.
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Masson, D., and T. E. Kreis. "Binding of E-MAP-115 to microtubules is regulated by cell cycle-dependent phosphorylation." Journal of Cell Biology 131, no. 4 (November 15, 1995): 1015–24. http://dx.doi.org/10.1083/jcb.131.4.1015.
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Expression levels of E-MAP-115, a microtubule-associated protein that stabilizes microtubules, increase with epithelial cell polarization and differentiation (Masson and Kreis, 1993). Although polarizing cells contain significant amounts of this protein, they can still divide and thus all stabilized microtubules must disassemble at the onset of mitosis to allow formation of the dynamic mitotic spindle. We show here that binding of E-MAP-115 to microtubules is regulated by phosphorylation during the cell cycle. Immunolabeling of HeLa cells for E-MAP-115 indicates that the protein is absent from microtubules during early prophase and progressively reassociates with microtubules after late prophase. A fraction of E-MAP-115 from HeLa cells released from a block at the G1/S boundary runs with higher apparent molecular weight on SDS-PAGE, with a peak correlating with the maximal number of cells in early stages of mitosis. E-MAP-115 from nocodazole-arrested mitotic cells, which can be obtained in larger amounts, displays identical modifications and was used for further biochemical characterization. The level of incorporation of 32P into mitotic E-MAP-115 is about 15-fold higher than into the interphase protein. Specific threonine phosphorylation occurs in mitosis, and the amount of phosphate associated with serine also increases. Hyperphosphorylated E-MAP-115 from mitotic cells cannot bind stably to microtubules in vitro. These results suggest that phosphorylation of E-MAP-115 is a prerequisite for increasing the dynamic properties of the interphase microtubules which leads to the assembly of the mitotic spindle at the onset of mitosis. Microtubule-associated proteins are thus most likely key targets for kinases which control changes in microtubule dynamic properties at the G2- to M-phase transition.
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Gable, Alyssa, Minhua Qiu, Janel Titus, Sai Balchand, Nick P. Ferenz, Nan Ma, Elizabeth S. Collins, et al. "Dynamic reorganization of Eg5 in the mammalian spindle throughout mitosis requires dynein and TPX2." Molecular Biology of the Cell 23, no. 7 (April 2012): 1254–66. http://dx.doi.org/10.1091/mbc.e11-09-0820.
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Kinesin-5 is an essential mitotic motor. However, how its spatial–temporal distribution is regulated in mitosis remains poorly understood. We expressed localization and affinity purification–tagged Eg5 from a mouse bacterial artificial chromosome (this construct was called mEg5) and found its distribution to be tightly regulated throughout mitosis. Fluorescence recovery after photobleaching analysis showed rapid Eg5 turnover throughout mitosis, which cannot be accounted for by microtubule turnover. Total internal reflection fluorescence microscopy and high-resolution, single-particle tracking revealed that mEg5 punctae on both astral and midzone microtubules rapidly bind and unbind. mEg5 punctae on midzone microtubules moved transiently both toward and away from spindle poles. In contrast, mEg5 punctae on astral microtubules moved transiently toward microtubule minus ends during early mitosis but switched to plus end–directed motion during anaphase. These observations explain the poleward accumulation of Eg5 in early mitosis and its redistribution in anaphase. Inhibition of dynein blocked mEg5 movement on astral microtubules, whereas depletion of the Eg5-binding protein TPX2 resulted in plus end–directed mEg5 movement. However, motion of Eg5 on midzone microtubules was not altered. Our results reveal differential and precise spatial and temporal regulation of Eg5 in the spindle mediated by dynein and TPX2.
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Rogers, Stephen L., Gregory C. Rogers, David J. Sharp, and Ronald D. Vale. "Drosophila EB1 is important for proper assembly, dynamics, and positioning of the mitotic spindle." Journal of Cell Biology 158, no. 5 (September 2, 2002): 873–84. http://dx.doi.org/10.1083/jcb.200202032.
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EB1 is an evolutionarily conserved protein that localizes to the plus ends of growing microtubules. In yeast, the EB1 homologue (BIM1) has been shown to modulate microtubule dynamics and link microtubules to the cortex, but the functions of metazoan EB1 proteins remain unknown. Using a novel preparation of the Drosophila S2 cell line that promotes cell attachment and spreading, we visualized dynamics of single microtubules in real time and found that depletion of EB1 by RNA-mediated inhibition (RNAi) in interphase cells causes a dramatic increase in nondynamic microtubules (neither growing nor shrinking), but does not alter overall microtubule organization. In contrast, several defects in microtubule organization are observed in RNAi-treated mitotic cells, including a drastic reduction in astral microtubules, malformed mitotic spindles, defocused spindle poles, and mispositioning of spindles away from the cell center. Similar phenotypes were observed in mitotic spindles of Drosophila embryos that were microinjected with anti-EB1 antibodies. In addition, live cell imaging of mitosis in Drosophila embryos reveals defective spindle elongation and chromosomal segregation during anaphase after antibody injection. Our results reveal crucial roles for EB1 in mitosis, which we postulate involves its ability to promote the growth and interactions of microtubules within the central spindle and at the cell cortex.
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Osmani, Aysha H., Jonathan Davies, C. Elizabeth Oakley, Berl R. Oakley, and Stephen A. Osmani. "TINA Interacts with the NIMA Kinase in Aspergillus nidulans and Negatively Regulates Astral Microtubules during Metaphase Arrest." Molecular Biology of the Cell 14, no. 8 (August 2003): 3169–79. http://dx.doi.org/10.1091/mbc.e02-11-0715.
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The tinA gene of Aspergillus nidulans encodes a protein that interacts with the NIMA mitotic protein kinase in a cell cycle-specific manner. Highly similar proteins are encoded in Neurospora crassa and Aspergillus fumigatus. TINA and NIMA preferentially interact in interphase and larger forms of TINA are generated during mitosis. Localization studies indicate that TINA is specifically localized to the spindle pole bodies only during mitosis in a microtubule-dependent manner. Deletion of tinA alone is not lethal but displays synthetic lethality in combination with the anaphase-promoting complex/cyclosome mutation bimE7. At the bimE7 metaphase arrest point, lack of TINA enhanced the nucleation of bundles of cytoplasmic microtubules from the spindle pole bodies. These microtubules interacted to form spindles joined in series via astral microtubules as revealed by live cell imaging. Because TINA is modified and localizes to the spindle pole bodies at mitosis, and lack of TINA causes enhanced production of cytoplasmic microtubules at metaphase arrest, we suggest TINA is involved in negative regulation of the astral microtubule organizing capacity of the spindle pole bodies during metaphase.
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Tipton, Aaron R., Jonathan D. Wren, John R. Daum, Joseph C. Siefert, and Gary J. Gorbsky. "GTSE1 regulates spindle microtubule dynamics to control Aurora B kinase and Kif4A chromokinesin on chromosome arms." Journal of Cell Biology 216, no. 10 (August 18, 2017): 3117–32. http://dx.doi.org/10.1083/jcb.201610012.
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In mitosis, the dynamic assembly and disassembly of microtubules are critical for normal chromosome movement and segregation. Microtubule turnover varies among different mitotic spindle microtubules, dictated by their spatial distribution within the spindle. How turnover among the various classes of spindle microtubules is differentially regulated and the resulting significance of differential turnover for chromosome movement remains a mystery. As a new tactic, we used global microarray meta-analysis (GAMMA), a bioinformatic method, to identify novel regulators of mitosis, and in this study, we describe G2- and S phase–expressed protein 1 (GTSE1). GTSE1 is expressed exclusively in late G2 and M phase. From nuclear envelope breakdown until anaphase onset, GTSE1 binds preferentially to the most stable mitotic spindle microtubules and promotes their turnover. Cells depleted of GTSE1 show defects in chromosome alignment at the metaphase plate and in spindle pole integrity. These defects are coupled with an increase in the proportion of stable mitotic spindle microtubules. A consequence of this reduced microtubule turnover is diminished recruitment and activity of Aurora B kinase on chromosome arms. This decrease in Aurora B results in diminished binding of the chromokinesin Kif4A to chromosome arms.
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Vantard, M., A. M. Lambert, J. De Mey, P. Picquot, and L. J. Van Eldik. "Characterization and immunocytochemical distribution of calmodulin in higher plant endosperm cells: localization in the mitotic apparatus." Journal of Cell Biology 101, no. 2 (August 1, 1985): 488–99. http://dx.doi.org/10.1083/jcb.101.2.488.
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In this study we have examined the immunocytochemical distribution of calmodulin during mitosis of higher plant endosperm cells. Spindle development in these cells occurs without centrioles. Instead, asterlike microtubule converging centers appear to be involved in establishing spindle polarity. By indirect immunofluorescence and immunogold staining methods with anti-calmodulin antibodies, we found endosperm calmodulin to be associated with the mitotic apparatus, particularly with asterlike and/or polar microtubule converging centers and kinetochore microtubules, in an immunocytochemical pattern distinct from that of tubulin. In addition, endosperm calmodulin and calcium showed analogous distribution profiles during mitosis. Previous reports have demonstrated that calmodulin is associated with the mitotic apparatus in animal cells. The present observation that calmodulin is also associated with the mitotic apparatus in acentriolar, higher plant endosperm cells suggests that some of the regulatory mechanisms involved in spindle formation, microtubule disassembly, and chromosome movement in plant cells may be similar to those in animal cells. However, unlike animal cell calmodulin, endosperm calmodulin appears to associate with kinetochore microtubules throughout mitosis, which suggests a specialized role for higher plant calmodulin in the regulation of kinetochore microtubule dynamics.
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Tucker, J. B., S. A. Mathews, K. A. Hendry, J. B. Mackie, and D. L. Roche. "Spindle microtubule differentiation and deployment during micronuclear mitosis in Paramecium." Journal of Cell Biology 101, no. 5 (November 1, 1985): 1966–76. http://dx.doi.org/10.1083/jcb.101.5.1966.
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Spindles underwent a 12-fold elongation before anaphase B was completed during the closed mitoses of micronuclei in Paramecium tetraurelia. Two main classes of spindle microtubules have been identified. A peripheral sheath of microtubules with diameters of 27-32 nm was found to be associated with the nuclear envelope and confined to the midportion of each spindle. Most of the other microtubules had diameters of approximately 24 nm and were present along the entire lengths of spindles. Nearly all of the 24-nm microtubules were eliminated from spindle midportions (largely because of microtubule disassembly) at a relatively early stage of spindle elongation. Disassembly of some of these microtubules also occurred at the ends of spindles. About 60% of the total microtubule content of spindles was lost at this stage. Most, perhaps all, peripheral sheath microtubules remained intact. Many of them detached from the nuclear envelope and regrouped to form a compact microtubule bundle in the spindle midportion. There was little, if any, further polymerization of 24-nm microtubules after the disassembly phase. Polymerization of microtubules with diameters of 27-32 nm continued as spindle elongation progressed. Most microtubules in the midportions of well-elongated spindles were constructed from 14-16 protofilaments. A few 24-nm microtubules with 13 protofilaments were also present. The implications of these findings for spatial control of microtubule assembly, disassembly, positioning, and membrane association, that apparently discriminate between microtubules with different protofilament numbers have been explored. The possibility that microtubule sliding occurs during spindle elongation has also been considered.
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Nahaboo, Wallis, Melissa Zouak, Peter Askjaer, and Marie Delattre. "Chromatids segregate without centrosomes during Caenorhabditis elegans mitosis in a Ran- and CLASP-dependent manner." Molecular Biology of the Cell 26, no. 11 (June 2015): 2020–29. http://dx.doi.org/10.1091/mbc.e14-12-1577.
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During mitosis, chromosomes are connected to a microtubule-based spindle. Current models propose that displacement of the spindle poles and/or the activity of kinetochore microtubules generate mechanical forces that segregate sister chromatids. Using laser destruction of the centrosomes during Caenorhabditis elegans mitosis, we show that neither of these mechanisms is necessary to achieve proper chromatid segregation. Our results strongly suggest that an outward force generated by the spindle midzone, independently of centrosomes, is sufficient to segregate chromosomes in mitotic cells. Using mutant and RNAi analysis, we show that the microtubule-bundling protein SPD-1/MAP-65 and BMK-1/kinesin-5 act as a brake opposing the force generated by the spindle midzone. Conversely, we identify a novel role for two microtubule-growth and nucleation agents, Ran and CLASP, in the establishment of the centrosome-independent force during anaphase. Their involvement raises the interesting possibility that microtubule polymerization of midzone microtubules is continuously required to sustain chromosome segregation during mitosis.
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Fourest-Lieuvin, Anne, Leticia Peris, Vincent Gache, Isabel Garcia-Saez, Céline Juillan-Binard, Violaine Lantez, and Didier Job. "Microtubule Regulation in Mitosis: Tubulin Phosphorylation by the Cyclin-dependent Kinase Cdk1." Molecular Biology of the Cell 17, no. 3 (March 2006): 1041–50. http://dx.doi.org/10.1091/mbc.e05-07-0621.
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The activation of the cyclin-depdndent kinase Cdk1 at the transition from interphase to mitosis induces important changes in microtubule dynamics. Cdk1 phosphorylates a number of microtubule- or tubulin-binding proteins but, hitherto, tubulin itself has not been detected as a Cdk1 substrate. Here we show that Cdk1 phosphorylates β-tubulin both in vitro and in vivo. Phosphorylation occurs on Ser172 of β-tubulin, a site that is well conserved in evolution. Using a phosphopeptide antibody, we find that a fraction of the cell tubulin is phosphorylated during mitosis, and this tubulin phosphorylation is inhibited by the Cdk1 inhibitor roscovitine. In mitotic cells, phosphorylated tubulin is excluded from microtubules, being present in the soluble tubulin fraction. Consistent with this distribution in cells, the incorporation of Cdk1-phosphorylated tubulin into growing microtubules is impaired in vitro. Additionally, EGFP-β3-tubulinS172D/E mutants that mimic phosphorylated tubulin are unable to incorporate into microtubules when expressed in cells. Modeling shows that the presence of a phosphoserine at position 172 may impair both GTP binding to β-tubulin and interactions between tubulin dimers. These data indicate that phosphorylation of tubulin by Cdk1 could be involved in the regulation of microtubule dynamics during mitosis.
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Rischitor, Patricia E., Sven Konzack, and Reinhard Fischer. "The Kip3-Like Kinesin KipB Moves along Microtubules and Determines Spindle Position during Synchronized Mitoses in Aspergillus nidulans Hyphae." Eukaryotic Cell 3, no. 3 (June 2004): 632–45. http://dx.doi.org/10.1128/ec.3.3.632-645.2004.
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ABSTRACT Kinesins are motor proteins which are classified into 11 different families. We identified 11 kinesin-like proteins in the genome of the filamentous fungus Aspergillus nidulans. Relatedness analyses based on the motor domains grouped them into nine families. In this paper, we characterize KipB as a member of the Kip3 family of microtubule depolymerases. The closest homologues of KipB are Saccharomyces cerevisiae Kip3 and Schizosaccharomyces pombe Klp5 and Klp6, but sequence similarities outside the motor domain are very low. A disruption of kipB demonstrated that it is not essential for vegetative growth. kipB mutant strains were resistant to high concentrations of the microtubule-destabilizing drug benomyl, suggesting that KipB destabilizes microtubules. kipB mutations caused a failure of spindle positioning in the cell, a delay in mitotic progression, an increased number of bent mitotic spindles, and a decrease in the depolymerization of cytoplasmic microtubules during interphase and mitosis. Meiosis and ascospore formation were not affected. Disruption of the kipB gene was synthetically lethal in combination with the temperature-sensitive mitotic kinesin motor mutation bimC4, suggesting an important but redundant role of KipB in mitosis. KipB localized to cytoplasmic, astral, and mitotic microtubules in a discontinuous pattern, and spots of green fluorescent protein moved along microtubules toward the plus ends.
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Loïodice, Isabelle, Jayme Staub, Thanuja Gangi Setty, Nam-Phuong T. Nguyen, Anne Paoletti, and P. T. Tran. "Ase1p Organizes Antiparallel Microtubule Arrays during Interphase and Mitosis in Fission Yeast." Molecular Biology of the Cell 16, no. 4 (April 2005): 1756–68. http://dx.doi.org/10.1091/mbc.e04-10-0899.
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Proper microtubule organization is essential for cellular processes such as organelle positioning during interphase and spindle formation during mitosis. The fission yeast Schizosaccharomyces pombe presents a good model for understanding microtubule organization. We identify fission yeast ase1p, a member of the conserved ASE1/PRC1/MAP65 family of microtubule bundling proteins, which functions in organizing the spindle midzone during mitosis. Using fluorescence live cell imaging, we show that ase1p localizes to sites of microtubule overlaps associated with microtubule organizing centers at both interphase and mitosis. ase1Δ mutants fail to form overlapping antiparallel microtubule bundles, leading to interphase nuclear positioning defects, and premature mitotic spindle collapse. FRAP analysis revealed that interphase ase1p at overlapping microtubule minus ends is highly dynamic. In contrast, mitotic ase1p at microtubule plus ends at the spindle midzone is more stable. We propose that ase1p functions to organize microtubules into overlapping antiparallel bundles both in interphase and mitosis and that ase1p may be differentially regulated through the cell cycle.
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Rusan, Nasser M., Carey J. Fagerstrom, Anne-Marie C. Yvon, and Patricia Wadsworth. "Cell Cycle-Dependent Changes in Microtubule Dynamics in Living Cells Expressing Green Fluorescent Protein-α Tubulin." Molecular Biology of the Cell 12, no. 4 (April 2001): 971–80. http://dx.doi.org/10.1091/mbc.12.4.971.
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LLCPK-1 cells were transfected with a green fluorescent protein (GFP)-α tubulin construct and a cell line permanently expressing GFP-α tubulin was established (LLCPK-1α). The mitotic index and doubling time for LLCPK-1α were not significantly different from parental cells. Quantitative immunoblotting showed that 17% of the tubulin in LLCPK-1α cells was GFP-tubulin; the level of unlabeled tubulin was reduced to 82% of that in parental cells. The parameters of microtubule dynamic instability were compared for interphase LLCPK-1α and parental cells injected with rhodamine-labeled tubulin. Dynamic instability was very similar in the two cases, demonstrating that LLCPK-1α cells are a useful tool for analysis of microtubule dynamics throughout the cell cycle. Comparison of astral microtubule behavior in mitosis with microtubule behavior in interphase demonstrated that the frequency of catastrophe increased twofold and that the frequency of rescue decreased nearly fourfold in mitotic compared with interphase cells. The percentage of time that microtubules spent in an attenuated state, or pause, was also dramatically reduced, from 73.5% in interphase to 11.4% in mitosis. The rates of microtubule elongation and rapid shortening were not changed; overall dynamicity increased 3.6-fold in mitosis. Microtubule release from the centrosome and a subset of differentially stable astral microtubules were also observed. The results provide the first quantitative measurements of mitotic microtubule dynamics in mammalian cells.
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Raemaekers, Tim, Katharina Ribbeck, Joël Beaudouin, Wim Annaert, Mark Van Camp, Ingrid Stockmans, Nico Smets, Roger Bouillon, Jan Ellenberg, and Geert Carmeliet. "NuSAP, a novel microtubule-associated protein involved in mitotic spindle organization." Journal of Cell Biology 162, no. 6 (September 8, 2003): 1017–29. http://dx.doi.org/10.1083/jcb.200302129.
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Here, we report on the identification of nucleolar spindle–associated protein (NuSAP), a novel 55-kD vertebrate protein with selective expression in proliferating cells. Its mRNA and protein levels peak at the transition of G2 to mitosis and abruptly decline after cell division. Microscopic analysis of both fixed and live mammalian cells showed that NuSAP is primarily nucleolar in interphase, and localizes prominently to central spindle microtubules during mitosis. Direct interaction of NuSAP with microtubules was demonstrated in vitro. Overexpression of NuSAP caused profound bundling of cytoplasmic microtubules in interphase cells, and this relied on a COOH-terminal microtubule-binding domain. In contrast, depletion of NuSAP by RNA interference resulted in aberrant mitotic spindles, defective chromosome segregation, and cytokinesis. In addition, many NuSAP-depleted interphase cells had deformed nuclei. Both overexpression and knockdown of NuSAP impaired cell proliferation. These results suggest a crucial role for NuSAP in spindle microtubule organization.
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Svoboda, Augustin, and Iva Slaninová. "Colocalization of microtubules and mitochondria in the yeast Schizosaccharomyces japonicus var. versatilis." Canadian Journal of Microbiology 43, no. 10 (October 1, 1997): 945–53. http://dx.doi.org/10.1139/m97-136.
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Both living and fixed cells of Schizosaccharomyces japonicus var. versatilis showed thread-like mitochondria when studied by phase-contrast and fluorescence microscopy. In the interphase cells, mitochondria extended from pole to pole and converged towards the growing tips. The mitochondrial threads did not disrupt but persisted during mitosis and, subsequently, their bundle was split between the two daughter cells by a concentrically growing septum. Mitochondria in the interphase cells were accompanied by cytoplasmic microtubules. These disappeared during mitosis and, instead, spindle microtubules were formed in the nucleus. The cytoplasmic microtubules reappeared after anaphase B, again in coaligment with mitochondria. Protoplasting as well as the action of microtubule inhibitors methyl-1-(butylcarbamoyl)-2-benzimidazolecarbamate (benomyl) and 2-methylbenzimidazole (MBC) resulted in rapid disintegration of microtubules and, suprisingly, also in disruption of mitochondria into small bodies. Removal of the inhibitors or a short regeneration of protoplasts allowed both the cytoplasmic microtubules and the thread-like mitochondria to reaggregate into the original pattern. Cytochalasin D treatment caused a complete disintegration of actin filaments, while the cytoplasmic microtubules and mitochondria remained intact. These findings of a transient close association of mitochondria and microtubules and their relative independence of the arrangement of actin filaments suggest that microtubules, but not actin cables, form supports for positioning or movement of mitochondria along the cylindrical cells. The persistence of mitochondria in the cell centre during mitosis may be accounted for by the fact that disrupted microtubules fail to provide support for mitochondrial movement towards the cell poles.Key words: microtubules, mitochondria, yeast, actin, microtubular drugs, cell cycle, Schizosaccharomyces japonicus var. versatilis.
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Lee, G. M., J. Diguiseppi, G. M. Gawdi, and B. Herman. "Chloral hydrate disrupts mitosis by increasing intracellular free calcium." Journal of Cell Science 88, no. 5 (December 1, 1987): 603–12. http://dx.doi.org/10.1242/jcs.88.5.603.
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In examining how chloral hydrate affects mitosis, we found that extracellular application of 0.1% chloral hydrate produced an abrupt rise in cytosolic free Ca2+. Digitized fluorescence microscopy of Fura-2-loaded, mitotic and interphase PtK cells revealed that Ca2+ rose 15 s after chloral hydrate application, peaked within 1 min at a concentration two- to sevenfold above the basal level and then slowly dropped. Bathing cells in 0.1% chloral hydrate caused metaphase spindles to shorten, starting in 1–2 min, and inhibited spindle elongation without affecting chromosome-to-pole movement during anaphase, as determined by phase-contrast observation of living cells. Spindle elongation and chromosome movement were unaffected by intracellular injection of 7.5% chloral hydrate. Extensive mitotic microtubule breakdown occurred after cells were bathed for 7 min in 0.1% chloral hydrate, while interphase microtubules were unaffected as determined by immunofluorescence. The chloral hydrate-induced microtubule breakdown and metaphase spindle shortening were prevented by 10 mM-CoCl2, which has previously been shown to block Ca2+ influx and to stabilize microtubules in vitro. These results imply that disruption of mitotic spindle function and structure by chloral hydrate is due to a rise in cytosolic free Ca2+, and also indicate that mitotic microtubules are more Ca2+-labile than interphase microtubules.
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Kallajoki, M., K. Weber, and M. Osborn. "Ability to organize microtubules in taxol-treated mitotic PtK2 cells goes with the SPN antigen and not with the centrosome." Journal of Cell Science 102, no. 1 (May 1, 1992): 91–102. http://dx.doi.org/10.1242/jcs.102.1.91.
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The SPN antigen plays an essential role in mitosis, since microinjection of antibodies causes mitotic arrest. Here we show, by examination of the relative locations of SPN antigen, the centrosomal 5051 antigen and tubulin in normal mitotic, and in taxol-treated mitotic cells, that the SPN antigen is involved in organizing the microtubules of the spindle. The 210 kDa protein defined as SPN antigen relocates from the nuclear matrix to the centrosome at prophase, remains associated with the poles at metaphase and anaphase, and dissociates from the centrosomes in telophase. In taxol-treated mitotic cells, SPN staining shows a striking redistribution while 5051 antigen remains associated with centrosomes. SPN antigen is seen at the plasma membrane end of the rearranged microtubules. SPN antigen is always at the center of the multiple microtubule asters (5 to 20 per cell) induced by taxol, whereas 5051 again remains associated with the centrosomal complex (1 to 2 foci per cell). Microtubule nucleation is associated with the SPN antigen rather than with the 5051 antigen. Microinjection of SPN-3 antibody into taxol-treated mitotic PtK2 cells causes disruption of the asters as judged by tubulin staining of the same cells. Finally, SPN antigen extracted in soluble form from synchronized mitotic HeLa cells binds to, and sediments with, pig brain microtubules stabilized by taxol. This association of SPN antigen with microtubules is partially dissociated by 0.5 M NaCl but not by 5 mM ATP. Thus SPN antigen binds to microtubules in vitro and seems to act as a microtubular minus-end organizer in mitotic cells in vivo.
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Schatten, Heide, Christopher N. Hueser, and Amitabha Chakrabarti. "Centrosome Structure And Function Is Altered By Experimental Manipulations With Formamide: Implications For Abnormal Cell Divisions During Cancer." Microscopy and Microanalysis 5, S2 (August 1999): 1286–87. http://dx.doi.org/10.1017/s1431927600019759.
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The formation of abnormal mitosis associated with cancer has been intriguing for many decades. While microtubules had been the focus of previous studies, recent research has focused on centrosomes, microtubule organizing centers which organize the mitotic apparatus during cell division. During normal mitosis centrosomes form two poles but in cancer, centrosomes can form three, four, or more poles, and organize tripolar, quadripolar, and multipolar mitoses, respectively. This has severe consequences for genomic stability because chromosomes are separated unequally to three, four, or more poles. This can result in aneuploidy and gene amplifications with multiple defects in cellular regulation. It can result in malignancy that is accompanied by cell cycle imbalances and abnormal cell proliferation. While radiation and chemical agents are known to damage DNA and can lead to cell cycle abnormalities, the damage of centrosome structure leading to abnormal mitosis deserves also consideration.
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Schmit, A. C., and A. M. Lambert. "Characterization and dynamics of cytoplasmic F-actin in higher plant endosperm cells during interphase, mitosis, and cytokinesis." Journal of Cell Biology 105, no. 5 (November 1, 1987): 2157–66. http://dx.doi.org/10.1083/jcb.105.5.2157.
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We have identified an F-actin cytoskeletal network that remains throughout interphase, mitosis, and cytokinesis of higher plant endosperm cells. Fluorescent labeling was obtained using actin monoclonal antibodies and/or rhodamine-phalloidin. Video-enhanced microscopy and ultrastructural observations of immunogold-labeled preparations illustrated microfilament-microtubule co-distribution and interactions. Actin was also identified in cell crude extract with Western blotting. During interphase, microfilament and microtubule arrays formed two distinct networks that intermingled. At the onset of mitosis, when microtubules rearranged into the mitotic spindle, microfilaments were redistributed to the cell cortex, while few microfilaments remained in the spindle. During mitosis, the cortical actin network remained as an elastic cage around the mitotic apparatus and was stretched parallel to the spindle axis during poleward movement of chromosomes. This suggested the presence of dynamic cross-links that rearrange when they are submitted to slow and regular mitotic forces. At the poles, the regular network is maintained. After midanaphase, new, short microfilaments invaded the equator when interzonal vesicles were transported along the phragmoplast microtubules. Colchicine did not affect actin distribution, and cytochalasin B or D did not inhibit chromosome transport. Our data on endosperm cells suggested that plant cytoplasmic actin has an important role in the cell cortex integrity and in the structural dynamics of the poorly understood cytoplasm-mitotic spindle interface. F-actin may contribute to the regulatory mechanisms of microtubule-dependent or guided transport of vesicles during mitosis and cytokinesis in higher plant cells.
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Berlin, V., C. A. Styles, and G. R. Fink. "BIK1, a protein required for microtubule function during mating and mitosis in Saccharomyces cerevisiae, colocalizes with tubulin." Journal of Cell Biology 111, no. 6 (December 1, 1990): 2573–86. http://dx.doi.org/10.1083/jcb.111.6.2573.
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BIK1 function is required for nuclear fusion, chromosome disjunction, and nuclear segregation during mitosis. The BIK1 protein colocalizes with tubulin to the spindle pole body and mitotic spindle. Synthetic lethality observed in double mutant strains containing a mutation in the BIK1 gene and in the gene for alpha- or beta-tubulin is consistent with a physical interaction between BIK1 and tubulin. Furthermore, over- or underexpression of BIK1 causes aberrant microtubule assembly and function, bik1 null mutants are viable but contain very short or undetectable cytoplasmic microtubules. Spindle formation often occurs strictly within the mother cell, probably accounting for the many multinucleate and anucleate bik1 cells. Elevated levels of chromosome loss in bik1 cells are indicative of defective spindle function. Nuclear fusion is blocked in bik1 x bik1 zygotes, which have truncated cytoplasmic microtubules. Cells overexpressing BIK1 initially have abnormally short or nonexistent spindle microtubules and long cytoplasmic microtubules. Subsequently, cells lose all microtubule structures, coincident with the arrest of division. Based on these results, we propose that BIK1 is required stoichiometrically for the formation or stabilization of microtubules during mitosis and for spindle pole body fusion during conjugation.
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Sakai, Hikoichi. "Microtubules in Mitosis." Cell Structure and Function 19, no. 2 (1994): 57–62. http://dx.doi.org/10.1247/csf.19.57.
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Inoue, Yoshihiro H., Maria do Carmo Avides, Michina Shiraki, Peter Deak, Masamitsu Yamaguchi, Yoshio Nishimoto, Akio Matsukage, and David M. Glover. "Orbit, a Novel Microtubule-Associated Protein Essential for Mitosis in Drosophila melanogaster." Journal of Cell Biology 149, no. 1 (April 3, 2000): 153–66. http://dx.doi.org/10.1083/jcb.149.1.153.
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We describe a Drosophila gene, orbit, that encodes a conserved 165-kD microtubule-associated protein (MAP) with GTP binding motifs. Hypomorphic mutations in orbit lead to a maternal effect resulting in branched and bent mitotic spindles in the syncytial embryo. In the larval central nervous system, such mutants have an elevated mitotic index with some mitotic cells showing an increase in ploidy. Amorphic alleles show late lethality and greater frequencies of hyperploid mitotic cells. The presence of cells in the hypomorphic mutant in which the chromosomes can be arranged, either in a circular metaphase or an anaphase-like configuration on monopolar spindles, suggests that polyploidy arises through spindle and chromosome segregation defects rather than defects in cytokinesis. A role for the Orbit protein in regulating microtubule behavior in mitosis is suggested by its association with microtubules throughout the spindle at all mitotic stages, by its copurification with microtubules from embryonic extracts, and by the finding that the Orbit protein directly binds to MAP-free microtubules in a GTP-dependent manner.
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DeLuca, Jennifer G., Ben Moree, Jennifer M. Hickey, John V. Kilmartin, and E. D. Salmon. "hNuf2 inhibition blocks stable kinetochore–microtubule attachment and induces mitotic cell death in HeLa cells." Journal of Cell Biology 159, no. 4 (November 18, 2002): 549–55. http://dx.doi.org/10.1083/jcb.200208159.
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Identification of proteins that couple kinetochores to spindle microtubules is critical for understanding how accurate chromosome segregation is achieved in mitosis. Here we show that the protein hNuf2 specifically functions at kinetochores for stable microtubule attachment in HeLa cells. When hNuf2 is depleted by RNA interference, spindle formation occurs normally as cells enter mitosis, but kinetochores fail to form their attachments to spindle microtubules and cells block in prometaphase with an active spindle checkpoint. Kinetochores depleted of hNuf2 retain the microtubule motors CENP-E and cytoplasmic dynein, proteins previously implicated in recruiting kinetochore microtubules. Kinetochores also retain detectable levels of the spindle checkpoint proteins Mad2 and BubR1, as expected for activation of the spindle checkpoint by unattached kinetochores. In addition, the cell cycle block produced by hNuf2 depletion induces mitotic cells to undergo cell death. These data highlight a specific role for hNuf2 in kinetochore–microtubule attachment and suggest that hNuf2 is part of a molecular linker between the kinetochore attachment site and tubulin subunits within the lattice of attached plus ends.
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Scholey, Jonathan M., Gregory C. Rogers, and David J. Sharp. "Mitosis, microtubules, and the matrix." Journal of Cell Biology 154, no. 2 (July 23, 2001): 261–66. http://dx.doi.org/10.1083/jcb.200101097.
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Haren, Laurence, and Andreas Merdes. "Direct binding of NuMA to tubulin is mediated by a novel sequence motif in the tail domain that bundles and stabilizes microtubules." Journal of Cell Science 115, no. 9 (May 1, 2002): 1815–24. http://dx.doi.org/10.1242/jcs.115.9.1815.
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In mitosis, NuMA localises to spindle poles where it contributes to the formation and maintenance of focussed microtubule arrays. Previous work has shown that NuMA is transported to the poles by dynein and dynactin. So far, it is unclear how NuMA accumulates at the spindle poles following transport and how it remains associated throughout mitosis. We show here that NuMA can bind to microtubules independently of dynein/dynactin. We characterise a 100-residue domain located within the C-terminal tail of NuMA that mediates a direct interaction with tubulin in vitro and that is necessary for NuMA association with tubulin in vivo. Moreover, this domain induces bundling and stabilisation of microtubules when expressed in cultured cells and leads to formation of abnormal mitotic spindles with increased microtubule asters or multiple poles. Our results suggest that NuMA organises the poles by stable crosslinking of the microtubule fibers.
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McNally, Francis J., and Susan Thomas. "Katanin Is Responsible for the M-Phase Microtubule-severing Activity in Xenopus Eggs." Molecular Biology of the Cell 9, no. 7 (July 1998): 1847–61. http://dx.doi.org/10.1091/mbc.9.7.1847.
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Microtubules are dynamic structures whose proper rearrangement during the cell cycle is essential for the positioning of membranes during interphase and for chromosome segregation during mitosis. The previous discovery of a cyclin B/cdc2-activated microtubule-severing activity in M-phase Xenopus egg extracts suggested that a microtubule-severing protein might play an important role in cell cycle-dependent changes in microtubule dynamics and organization. However, the isolation of three different microtubule-severing proteins, p56, EF1α, and katanin, has only confused the issue because none of these proteins is directly activated by cyclin B/cdc2. Here we use immunodepletion with antibodies specific for a vertebrate katanin homologue to demonstrate that katanin is responsible for the majority of M-phase severing activity inXenopus eggs. This result suggests that katanin is responsible for changes in microtubules occurring at mitosis. Immunofluorescence analysis demonstrated that katanin is concentrated at a microtubule-dependent structure at mitotic spindle poles inXenopus A6 cells and in human fibroblasts, suggesting a specific role in microtubule disassembly at spindle poles. Surprisingly, katanin was also found in adult mouse brain, indicating that katanin may have other functions distinct from its mitotic role.
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Silk, Alain D., Andrew J. Holland, and Don W. Cleveland. "Requirements for NuMA in maintenance and establishment of mammalian spindle poles." Journal of Cell Biology 184, no. 5 (March 2, 2009): 677–90. http://dx.doi.org/10.1083/jcb.200810091.
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Microtubules of the mitotic spindle in mammalian somatic cells are focused at spindle poles, a process thought to include direct capture by astral microtubules of kinetochores and/or noncentrosomally nucleated microtubule bundles. By construction and analysis of a conditional loss of mitotic function allele of the nuclear mitotic apparatus (NuMA) protein in mice and cultured primary cells, we demonstrate that NuMA is an essential mitotic component with distinct contributions to the establishment and maintenance of focused spindle poles. When mitotic NuMA function is disrupted, centrosomes provide initial focusing activity, but continued centrosome attachment to spindle fibers under tension is defective, and the maintenance of focused kinetochore fibers at spindle poles throughout mitosis is prevented. Without centrosomes and NuMA, initial establishment of spindle microtubule focusing completely fails. Thus, NuMA is a defining feature of the mammalian spindle pole and functions as an essential tether linking bulk microtubules of the spindle to centrosomes.
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Blower, Michael D., Elma Feric, Karsten Weis, and Rebecca Heald. "Genome-wide analysis demonstrates conserved localization of messenger RNAs to mitotic microtubules." Journal of Cell Biology 179, no. 7 (December 31, 2007): 1365–73. http://dx.doi.org/10.1083/jcb.200705163.
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RNA localization is of critical importance in many fundamental cell biological and developmental processes by regulating the spatial control of gene expression. To investigate how spindle-localized RNAs might influence mitosis, we comprehensively surveyed all messenger RNAs (mRNAs) that bound to microtubules during metaphase in both Xenopus laevis egg extracts and mitotic human cell extracts. We identify conserved classes of mRNAs that are enriched on microtubules in both human and X. laevis. Active mitotic translation occurs on X. laevis meiotic spindles, and a subset of microtubule-bound mRNAs (MT-mRNAs) associate with polyribosomes. Although many MT-mRNAs associate with polyribosomes, we find that active translation is not required for mRNA localization to mitotic microtubules. Our results represent the first genome-wide survey of mRNAs localized to a specific cytoskeletal component and suggest that microtubule localization of specific mRNAs is likely to function in mitotic regulation and mRNA segregation during cell division.
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Whitehead, C. M., and J. B. Rattner. "Expanding the role of HsEg5 within the mitotic and post-mitotic phases of the cell cycle." Journal of Cell Science 111, no. 17 (September 1, 1998): 2551–61. http://dx.doi.org/10.1242/jcs.111.17.2551.
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The BimC family of kinesin like proteins are involved in spindle dynamics in a wide variety of organisms. The human member of this family, HsEg5, has been implicated in centrosome separation during prophase/prometaphase and in the organization of in vitro mitotic asters. HsEg5 displays a complex distribution during mitosis, associating with the centrosomes, spindle microtubules, specific regions of the intracellular bridge and a microtubule bundle that forms in association with the post-mitotic migration of the centrosome. In an effort to determine the function of HsEg5 during late mitotic events and refine its proposed function during early mitotic centrosome separation, we microinjected antibodies specific to HsEg5 into HeLa cells during various stages of mitosis. In the presence of HsEg5 antibodies we find that the microtubule arrays responsible for both pre- and post-mitotic centrosome movement never form. Similarly, the microtubule bundle within the intracellular bridge becomes prematurely altered following karyokinesis resulting in the loss of the microtubule array at either end of the bridge. In addition, some peri-centrosomal material at the spindle poles becomes fragmented and the distribution of the spindle protein NuMA becomes more concentrated at the minus ends of the spindle microtubules. Our study also provides direct evidence that there is a link between post-mitotic centrosome migration and Golgi complex positioning and reformation following mitosis. We conclude that HsEg5 plays a recurrent role in establishing and/or determining the stability of specific microtubule arrays that form during cell division and that this role may encompass the ability of HsEg5 to influence the distribution of other protein components associated with cell division
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Yu, Ruoying, Huihui Wu, Hazrat Ismail, Shihao Du, Jun Cao, Jianyu Wang, Tarsha Ward, et al. "Methylation of PLK1 by SET7/9 ensures accurate kinetochore–microtubule dynamics." Journal of Molecular Cell Biology 12, no. 6 (December 21, 2019): 462–76. http://dx.doi.org/10.1093/jmcb/mjz107.
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Abstract Faithful segregation of mitotic chromosomes requires bi-orientation of sister chromatids, which relies on the sensing of correct attachments between spindle microtubules and kinetochores. Although the mechanisms underlying PLK1 activation have been extensively studied, the regulatory mechanisms that couple PLK1 activity to accurate chromosome segregation are not well understood. In particular, PLK1 is implicated in stabilizing kinetochore–microtubule attachments, but how kinetochore PLK1 activity is regulated to avoid hyperstabilized kinetochore–microtubules in mitosis remains elusive. Here, we show that kinetochore PLK1 kinase activity is modulated by SET7/9 via lysine methylation during early mitosis. The SET7/9-elicited dimethylation occurs at the Lys191 of PLK1, which tunes down its activity by limiting ATP utilization. Overexpression of the non-methylatable PLK1 mutant or chemical inhibition of SET7/9 methyltransferase activity resulted in mitotic arrest due to destabilized kinetochore–microtubule attachments. These data suggest that kinetochore PLK1 is essential for stable kinetochore–microtubule attachments and methylation by SET7/9 promotes dynamic kinetochore–microtubule attachments for accurate error correction. Our findings define a novel homeostatic regulation at the kinetochore that integrates protein phosphorylation and methylation with accurate chromosome segregation for maintenance of genomic stability.
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Domnitz, Sarah B., Michael Wagenbach, Justin Decarreau, and Linda Wordeman. "MCAK activity at microtubule tips regulates spindle microtubule length to promote robust kinetochore attachment." Journal of Cell Biology 197, no. 2 (April 9, 2012): 231–37. http://dx.doi.org/10.1083/jcb.201108147.
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Mitotic centromere-associated kinesin (MCAK) is a microtubule-depolymerizing kinesin-13 member that can track with polymerizing microtubule tips (hereafter referred to as tip tracking) during both interphase and mitosis. MCAK tracks with microtubule tips by binding to end-binding proteins (EBs) through the microtubule tip localization signal SKIP, which lies N terminal to MCAK’s neck and motor domain. The functional significance of MCAK’s tip-tracking behavior during mitosis has never been explained. In this paper, we identify and define a mitotic function specific to the microtubule tip–associated population of MCAK: negative regulation of microtubule length within the assembling bipolar spindle. This function depends on MCAK’s ability to bind EBs and track with polymerizing nonkinetochore microtubule tips. Although this activity antagonizes centrosome separation during bipolarization, it ultimately benefits the dividing cell by promoting robust kinetochore attachments to the spindle microtubules.
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Kinoshita, Kazuhisa, Tim L. Noetzel, Laurence Pelletier, Karl Mechtler, David N. Drechsel, Anne Schwager, Mike Lee, Jordan W. Raff, and Anthony A. Hyman. "Aurora A phosphorylation of TACC3/maskin is required for centrosome-dependent microtubule assembly in mitosis." Journal of Cell Biology 170, no. 7 (September 19, 2005): 1047–55. http://dx.doi.org/10.1083/jcb.200503023.
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Centrosomes act as sites of microtubule growth, but little is known about how the number and stability of microtubules emanating from a centrosome are controlled during the cell cycle. We studied the role of the TACC3–XMAP215 complex in this process by using purified proteins and Xenopus laevis egg extracts. We show that TACC3 forms a one-to-one complex with and enhances the microtubule-stabilizing activity of XMAP215 in vitro. TACC3 enhances the number of microtubules emanating from mitotic centrosomes, and its targeting to centrosomes is regulated by Aurora A–dependent phosphorylation. We propose that Aurora A regulation of TACC3 activity defines a centrosome-specific mechanism for regulation of microtubule polymerization in mitosis.
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Allan, V. J., and R. D. Vale. "Cell cycle control of microtubule-based membrane transport and tubule formation in vitro." Journal of Cell Biology 113, no. 2 (April 15, 1991): 347–59. http://dx.doi.org/10.1083/jcb.113.2.347.
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When higher eukaryotic cells enter mitosis, membrane organization changes dramatically and traffic between membrane compartments is inhibited. Since membrane transport along microtubules is involved in secretion, endocytosis, and the positioning of organelles during interphase, we have explored whether the mitotic reorganization of membrane could involve a change in microtubule-based membrane transport. This question was examined by reconstituting organelle transport along microtubules in Xenopus egg extracts, which can be converted between interphase and metaphase states in vitro in the absence of protein synthesis. Interphase extracts support the microtubule-dependent formation of abundant polygonal networks of membrane tubules and the transport of small vesicles. In metaphase extracts, however, the plus end- and minus end-directed movements of vesicles along microtubules as well as the formation of tubular membrane networks are all reduced substantially. By fractionating the extracts into soluble and membrane components, we have shown that the cell cycle state of the supernatant determines the extent of microtubule-based membrane movement. Interphase but not metaphase Xenopus soluble factors also stimulate movement of membranes from a rat liver Golgi fraction. In contrast to above findings with organelle transport, the minus end-directed movements of microtubules on glass surfaces and of latex beads along microtubules are similar in interphase and metaphase extracts, suggesting that cytoplasmic dynein, the predominant soluble motor in frog extracts, retains its force-generating activity throughout the cell cycle. A change in the association of motors with membranes may therefore explain the differing levels of organelle transport activity in interphase and mitotic extracts. We propose that the regulation of organelle transport may contribute significantly to the changes in membrane structure and function observed during mitosis in living cells.
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O'donnell, Kerry. "A reevaluation of the mitotic spindle pole body cycle in Tilletia caries based on freeze-substitution techniques." Canadian Journal of Botany 72, no. 10 (October 1, 1994): 1412–23. http://dx.doi.org/10.1139/b94-174.
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Mitosis in the wheat pathogen Tilletia caries (Basidiomycota, Tilletiales) was investigated by electron microscopy of serially sectioned, fast-frozen, freeze-substituted mitotic cells called ballistospores. A duplicated spindle pole body consisting of two identical, three-layered globular elements connected by a middle piece was attached to the extranuclear face of each nucleus at interphase. During mitosis, astral and spindle microtubules radiated from the globular elements that form the poles of an intranuclear spindle. At metaphase, chromosomes were interspersed with the nonkinetochore microtubules, and they were spread along the central two-thirds of the spindle. Each chromatid was attached to a spindle pole by a single, continuous, kinetochore microtubule. Postmitotic replication of the spindle pole body occurred during late telophase to interphase. Results from this study are presented in the form of a model of the mitotic spindle pole body cycle in Tilletia, and this model is compared with the one previously reported for Tilletia and other basidiomycetes. Key words: electron microscopy, freeze substitution, mitosis, spindle pole body, Tilletia.
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Matuliene, J., R. Essner, J. Ryu, Y. Hamaguchi, P. W. Baas, T. Haraguchi, Y. Hiraoka, and R. Kuriyama. "Function of a minus-end-directed kinesin-like motor protein in mammalian cells." Journal of Cell Science 112, no. 22 (November 15, 1999): 4041–50. http://dx.doi.org/10.1242/jcs.112.22.4041.
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CHO2 is a mammalian minus-end-directed kinesin-like motor protein present in interphase centrosomes/nuclei and mitotic spindle fibers/poles. Expression of HA- or GFP-tagged subfragments in transfected CHO cells revealed the presence of the nuclear localization site at the N-terminal tail. This domain becomes associated with spindle fibers during mitosis, indicating that the tail is capable of interaction with microtubules in vivo. While the central stalk diffusely distributes in the entire cytoplasm of cells, the motor domain co-localizes with microtubules throughout the cell cycle, which is eliminated by mutation of the ATP-binding consensus motif from GKT to AAA. Overexpression of the full-length CHO2 causes mitotic arrest and spindle abnormality. The effect of protein expression was first seen around the polar region where microtubule tended to be bundled together. A higher level of protein expression induces more elongated spindles which eventually become disorganized by loosing the structural integrity between microtubule bundles. Live cell observation demonstrated that GFP-labeled microtubule bundles underwent continuous changes in their relative position to one another through repeated attachment and detachment at one end; this results in the formation of irregular number of microtubule focal points in mitotic arrested cells. Thus the primary action of CHO2 appears to cross-link microtubules and move toward the minus-end direction to maintain association of the microtubule end at the pole. In contrast to the full-length of CHO2, overexpression of neither truncated nor mutant polypeptides resulted in significant effects on mitosis and mitotic spindles, suggesting that the function of CHO2 in mammalian cells may be redundant with other motor molecules during cell division.
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Romé, Pierre, Emilie Montembault, Nathalie Franck, Aude Pascal, David M. Glover, and Régis Giet. "Aurora A contributes to p150glued phosphorylation and function during mitosis." Journal of Cell Biology 189, no. 4 (May 17, 2010): 651–59. http://dx.doi.org/10.1083/jcb.201001144.
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Aurora A is a spindle pole–associated protein kinase required for mitotic spindle assembly and chromosome segregation. In this study, we show that Drosophila melanogaster aurora A phosphorylates the dynactin subunit p150glued on sites required for its association with the mitotic spindle. Dynactin strongly accumulates on microtubules during prophase but disappears as soon as the nuclear envelope breaks down, suggesting that its spindle localization is tightly regulated. If aurora A's function is compromised, dynactin and dynein become enriched on mitotic spindle microtubules. Phosphorylation sites are localized within the conserved microtubule-binding domain (MBD) of the p150glued. Although wild-type p150glued binds weakly to spindle microtubules, a variant that can no longer be phosphorylated by aurora A remains associated with spindle microtubules and fails to rescue depletion of endogenous p150glued. Our results suggest that aurora A kinase participates in vivo to the phosphoregulation of the p150glued MBD to limit the microtubule binding of the dynein–dynactin complex and thus regulates spindle assembly.
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Tate, J. E., and J. R. Palisano. "Comparison of the Effect of the Carbamate Herbicides, Barban and Chlorpropham, on Mrc-5 and Hela Cells." Microscopy and Microanalysis 5, S2 (August 1999): 1154–55. http://dx.doi.org/10.1017/s1431927600019097.
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Barban and chlorpropham are common carbamate herbicides that disrupt mitosis by destabilizing plant microtubules. Tubulins, protein subunits of microtubules, in plant and animal cells are highly conserved through evolution. Plant and animal cells have been shown to possess similar microtubule structural proteins, microtubule binding proteins, and organizational proteins. These similarities suggest that herbicides targeting plant microtubules might also affect animal microtubules. Previous tubulin immunofluorescence microscopic studies of HeLa cells, a human cervical cancer cell line, have shown that barban is strongly cytoskeletotoxic and chlorpropham is weakly cytoskeletotoxic. Both barban and chlorpropham have been shown to disrupt mitosis and disorganize spindle apparatus formation in numerous types of mammalian cancer cells.This investigation was undertaken to examine the effect of barban and chlorpropham on MRC-5 cells, a normal human fibroblast cell line, as well as on HeLa cells. A monoclonal antibody to α-tubulin, a microtubule specific protein, was used to probe the formation of spindle apparatuses in dividing cells.
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Cross, Marie K., and Maureen A. Powers. "Nup98 regulates bipolar spindle assembly through association with microtubules and opposition of MCAK." Molecular Biology of the Cell 22, no. 5 (March 2011): 661–72. http://dx.doi.org/10.1091/mbc.e10-06-0478.
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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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Logan, Caitlin M., and A. Sue Menko. "Microtubules: Evolving roles and critical cellular interactions." Experimental Biology and Medicine 244, no. 15 (August 6, 2019): 1240–54. http://dx.doi.org/10.1177/1535370219867296.
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Microtubules are cytoskeletal elements known as drivers of directed cell migration, vesicle and organelle trafficking, and mitosis. In this review, we discuss new research in the lens that has shed light into further roles for stable microtubules in the process of development and morphogenesis. In the lens, as well as other systems, distinct roles for characteristically dynamic microtubules and stabilized populations are coming to light. Understanding the mechanisms of microtubule stabilization and the associated microtubule post-translational modifications is an evolving field of study. Appropriate cellular homeostasis relies on not only one cytoskeletal element, but also rather an interaction between cytoskeletal proteins as well as other cellular regulators. Microtubules are key integrators with actin and intermediate filaments, as well as cell–cell junctional proteins and other cellular regulators including myosin and RhoGTPases to maintain this balance. Impact statement The role of microtubules in cellular functioning is constantly expanding. In this review, we examine new and exciting fields of discovery for microtubule’s involvement in morphogenesis, highlight our evolving understanding of differential roles for stabilized versus dynamic subpopulations, and further understanding of microtubules as a cellular integrator.
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Liu, Leyuan, Rui Xie, Chaofeng Yang, and Wallace L. McKeehan. "Dual Function Microtubule- and Mitochondria-Associated Proteins Mediate Mitotic Cell Death." Analytical Cellular Pathology 31, no. 5 (January 1, 2009): 393–405. http://dx.doi.org/10.1155/2009/783817.
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Background: Survival and evolution of aneuploid cells after an asymmetric segregation of chromosomes at mitosis may be the common initiating event and underlying cause of the genetic diversity and adaptability of cancers. We hypothesize that mechanisms exist to detect impending aneuploidy and prevent it before completion of an aberrant mitosis.Methods: The distribution of isoforms of C19ORF5, an interactive partner with mitochondria-associated LRPPRC and tumor suppressor RASSF1A, state of spindle microtubules and mitochondrial aggregation was analyzed in synchronized mitotic cells and cells stalled in mitosis after treatment with paclitaxel.Results: C19ORF5 distributed broadly across the mitotic spindle and reversibly accumulated during reversible mitotic arrest. Prolonged stabilization of microtubules caused an accumulation of a C19ORF5 product with dual MAP and MtAP properties that caused irreversible aggregation of mitochondria and death of mitotic cells.Conclusions: Dual function microtubule-associated (MAP) and mitochondria-associated (MtAP) proteins generated by prolonged mitotic arrest trigger mitochondrial-induced mitotic cell death. This is a potential mechanism to prevent minimal survivable aneuploidy resulting from an aberrant cell division and cancers in general at their earliest common origin.
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Karsenti, Eric. "Severing microtubules in mitosis." Current Biology 3, no. 4 (April 1993): 208–10. http://dx.doi.org/10.1016/0960-9822(93)90334-k.
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Hause, G., B. Hause, and A. A. M. Van Lammeren. "Microtubular and actin filament configurations during microspore and pollen development in Brassica napus cv. Topas." Canadian Journal of Botany 70, no. 7 (July 1, 1992): 1369–76. http://dx.doi.org/10.1139/b92-172.
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The structures of the microtubular and microfilamental cytoskeletons were investigated during the development of microspores and pollen grains of Brassica napus L. cv. Topas. Microfilaments were observed directly with rhodamine–phalloidin and microtubules with FITC by indirect immunofluorescent staining and transmission electron microscopy. We observed microtubules in all developmental stages and noted several changes in the configuration of the microtubular cytoskeleton during microspore development, microspore mitosis, and pollen development. A preprophase band before microspore mitosis was not observed. The arrest of the microspore nucleus in an eccentric position is likely caused by microtubules as is the shape of the phragmoplast at microspore mitosis. Despite the application of various staining methods, i.e., labelling of fixed and unfixed fresh and cryosectioned microspores and pollen with rhodamine–phalloidin, microfilaments could not be observed in all developmental stages. Prominent microfilamental arrays were observed during cytokinesis of microspore mitosis and during the free generative cell stage. They mark the stages with different configurations. Key words: Brassica napus, immunolabelling, cytoskeleton, microspore and pollen development.
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Adamakis, Ioannis-Dimosthenis S., Emmanuel Panteris, and Eleftherios P. Eleftheriou. "Tubulin Acetylation Mediates Bisphenol A Effects on the Microtubule Arrays of Allium cepa and Triticum turgidum." Biomolecules 9, no. 5 (May 11, 2019): 185. http://dx.doi.org/10.3390/biom9050185.
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The effects of bisphenol A (BPA), a prevalent endocrine disruptor, on both interphase and mitotic microtubule array organization was examined by immunofluorescence microscopy in meristematic root cells of Triticum turgidum (durum wheat) and Allium cepa (onion). In interphase cells of A. cepa, BPA treatment resulted in substitution of cortical microtubules by annular/spiral tubulin structures, while in T. turgidum BPA induced cortical microtubule fragmentation. Immunolocalization of acetylated α-tubulin revealed that cortical microtubules of T. turgidum were highly acetylated, unlike those of A. cepa. In addition, elevation of tubulin acetylation by trichostatin A in A. cepa resulted in microtubule disruption similar to that observed in T. turgidum. BPA also disrupted all mitotic microtubule arrays in both species. It is also worth noting that mitotic microtubule arrays were acetylated in both plants. As assessed by BPA removal, its effects are reversible. Furthermore, taxol-stabilized microtubules were resistant to BPA, while recovery from oryzalin treatment in BPA solution resulted in the formation of ring-like tubulin conformations. Overall, these findings indicate the following: (1) BPA affects plant mitosis/cytokinesis by disrupting microtubule organization. (2) Microtubule disassembly probably results from impairment of free tubulin subunit polymerization. (3) The differences in cortical microtubule responses to BPA among the species studied are correlated to the degree of tubulin acetylation.
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Broad, Amanda J., and Jennifer G. DeLuca. "The right place at the right time: Aurora B kinase localization to centromeres and kinetochores." Essays in Biochemistry 64, no. 2 (May 14, 2020): 299–311. http://dx.doi.org/10.1042/ebc20190081.
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Abstract The fidelity of chromosome segregation during mitosis is intimately linked to the function of kinetochores, which are large protein complexes assembled at sites of centromeric heterochromatin on mitotic chromosomes. These key “orchestrators” of mitosis physically connect chromosomes to spindle microtubules and transduce forces through these connections to congress chromosomes and silence the spindle assembly checkpoint. Kinetochore-microtubule attachments are highly regulated to ensure that incorrect attachments are not prematurely stabilized, but instead released and corrected. The kinase activity of the centromeric protein Aurora B is required for kinetochore-microtubule destabilization during mitosis, but how the kinase acts on outer kinetochore substrates to selectively destabilize immature and erroneous attachments remains debated. Here, we review recent literature that sheds light on how Aurora B kinase is recruited to both centromeres and kinetochores and discuss possible mechanisms for how kinase interactions with substrates at distinct regions of mitotic chromosomes are regulated.
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Silverman-Gavrila, Rosalind V., and Andrew Wilde. "Ran Is Required before Metaphase for Spindle Assembly and Chromosome Alignment and after Metaphase for Chromosome Segregation and Spindle Midbody Organization." Molecular Biology of the Cell 17, no. 4 (April 2006): 2069–80. http://dx.doi.org/10.1091/mbc.e05-10-0991.
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The Ran pathway has been shown to have a role in spindle assembly. However, the extent of the role of the Ran pathway in mitosis in vivo is unclear. We report that perturbation of the Ran pathway disrupted multiple steps of mitosis in syncytial Drosophila embryos and uncovered new mitotic processes that are regulated by Ran. During the onset of mitosis, the Ran pathway is required for the production, organization, and targeting of centrosomally nucleated microtubules to chromosomes. However, the role of Ran is not restricted to microtubule organization, because Ran is also required for the alignment of chromosomes at the metaphase plate. In addition, the Ran pathway is required for postmetaphase events, including chromosome segregation and the assembly of the microtubule midbody. The Ran pathway mediates these mitotic events, in part, by facilitating the correct targeting of the kinase Aurora A and the kinesins KLP61F and KLP3A to spindles.
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Lee, Wei-Lih, Jessica R. Oberle, and John A. Cooper. "The role of the lissencephaly protein Pac1 during nuclear migration in budding yeast." Journal of Cell Biology 160, no. 3 (February 3, 2003): 355–64. http://dx.doi.org/10.1083/jcb.200209022.
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During mitosis in Saccharomyces cerevisiae, the mitotic spindle moves into the mother–bud neck via dynein-dependent sliding of cytoplasmic microtubules along the cortex of the bud. Here we show that Pac1, the yeast homologue of the human lissencephaly protein LIS1, plays a key role in this process. First, genetic interactions placed Pac1 in the dynein/dynactin pathway. Second, cells lacking Pac1 failed to display microtubule sliding in the bud, resulting in defective mitotic spindle movement and nuclear segregation. Third, Pac1 localized to the plus ends (distal tips) of cytoplasmic microtubules in the bud. This localization did not depend on the dynein heavy chain Dyn1. Moreover, the Pac1 fluorescence intensity at the microtubule end was enhanced in cells lacking dynactin or the cortical attachment molecule Num1. Fourth, dynein heavy chain Dyn1 also localized to the tips of cytoplasmic microtubules in wild-type cells. Dynein localization required Pac1 and, like Pac1, was enhanced in cells lacking the dynactin component Arp1 or the cortical attachment molecule Num1. Our results suggest that Pac1 targets dynein to microtubule tips, which is necessary for sliding of microtubules along the bud cortex. Dynein must remain inactive until microtubule ends interact with the bud cortex, at which time dynein and Pac1 appear to be offloaded from the microtubule to the cortex.