Journal articles on the topic 'Nuclear mitotic apparatus'
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1
Yang, C. H., and M. Snyder. "The nuclear-mitotic apparatus protein is important in the establishment and maintenance of the bipolar mitotic spindle apparatus." Molecular Biology of the Cell 3, no. 11 (November 1992): 1259–67. http://dx.doi.org/10.1091/mbc.3.11.1259.
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The formation and maintenance of the bipolar mitotic spindle apparatus require a complex and balanced interplay of several mechanisms, including the stabilization and separation of polar microtubules and the action of various microtubule motors. Nonmicrotubule elements are also present throughout the spindle apparatus and have been proposed to provide a structural support for the spindle. The Nuclear-Mitotic Apparatus protein (NuMA) is an abundant 240 kD protein that is present in the nucleus of interphase cells and concentrates in the polar regions of the spindle apparatus during mitosis. Sequence analysis indicates that NuMA possesses an unusually long alpha-helical central region characteristic of many filament forming proteins. In this report we demonstrate that microinjection of anti-NuMA antibodies into interphase and prophase cells results in a failure to form a mitotic spindle apparatus. Furthermore, injection of metaphase cells results in the collapse of the spindle apparatus into a monopolar microtubule array. These results identify for the first time a nontubulin component important for both the establishment and stabilization of the mitotic spindle apparatus in multicellular organisms. We suggest that nonmicrotubule structural components may be important for these processes.
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Shima, David T., Noemí Cabrera-Poch, Rainer Pepperkok, and Graham Warren. "An Ordered Inheritance Strategy for the Golgi Apparatus: Visualization of Mitotic Disassembly Reveals a Role for the Mitotic Spindle." Journal of Cell Biology 141, no. 4 (May 18, 1998): 955–66. http://dx.doi.org/10.1083/jcb.141.4.955.
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During mitosis, the ribbon of the Golgi apparatus is transformed into dispersed tubulo-vesicular membranes, proposed to facilitate stochastic inheritance of this low copy number organelle at cytokinesis. Here, we have analyzed the mitotic disassembly of the Golgi apparatus in living cells and provide evidence that inheritance is accomplished through an ordered partitioning mechanism. Using a Sar1p dominant inhibitor of cargo exit from the endoplasmic reticulum (ER), we found that the disassembly of the Golgi observed during mitosis or microtubule disruption did not appear to involve retrograde transport of Golgi residents to the ER and subsequent reorganization of Golgi membrane fragments at ER exit sites, as has been suggested. Instead, direct visualization of a green fluorescent protein (GFP)-tagged Golgi resident through mitosis showed that the Golgi ribbon slowly reorganized into 1–3-μm fragments during G2/early prophase. A second stage of fragmentation occurred coincident with nuclear envelope breakdown and was accompanied by the bulk of mitotic Golgi redistribution. By metaphase, mitotic Golgi dynamics appeared to cease. Surprisingly, the disassembly of mitotic Golgi fragments was not a random event, but involved the reorganization of mitotic Golgi by microtubules, suggesting that analogous to chromosomes, the Golgi apparatus uses the mitotic spindle to ensure more accurate partitioning during cytokinesis.
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Price, Carolyn M., and David E. Pettijohn. "Redistribution of the nuclear mitotic apparatus protein (NuMA) during mitosis and nuclear assembly." Experimental Cell Research 166, no. 2 (October 1986): 295–311. http://dx.doi.org/10.1016/0014-4827(86)90478-7.
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Jones, J. C., A. E. Goldman, H. Y. Yang, and R. D. Goldman. "The organizational fate of intermediate filament networks in two epithelial cell types during mitosis." Journal of Cell Biology 100, no. 1 (January 1, 1985): 93–102. http://dx.doi.org/10.1083/jcb.100.1.93.
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Intermediate filaments (IF) appear to be attached to the nuclear envelope in various mammalian cell types. The nucleus of mouse keratinocytes is enveloped by a cagelike network of keratin-containing bundles of IF (IFB). This network appears to be continuous with the cytoplasmic IFB system that extends to the cell surface. Electron microscopy reveals that the IFB appear to terminate at the level of the nuclear envelope, frequently in association with nuclear pore complexes (Jones, J. C .R., A. E. Goldman, P. Steinert, S. Yuspa, and R. D. Goldman, 1982, Cell Motility, 2:197-213). Based on these observations of nuclear-IF associations, it is of interest to determine the fate and organizational states of IF during mitosis, a period in the cell cycle when the nuclear envelope disassembles. Immunofluorescence microscopy using a monoclonal keratin antibody and electron microscopy of thin and thick sections of mitotic mouse keratinocytes revealed that the IFB system remained intact as the cells entered mitosis and surrounded the developing mitotic spindle. IFB were close to chromosomes and often associated with chromosome arms. In contrast, in HeLa, a human epithelial cell, keratin-containing IFB appear to dissemble as cells enter mitosis (Franke, W. W., E. Schmid, C. Grund, and B. Geiger, 1982, Cell, 30:103-113). The keratin IFB in mitotic HeLa cells appeared to form amorphous nonfilamentous bodies as determined by electron microscopy. However, in HeLa, another IF system composed primarily of a 55,000-mol-wt protein (frequently termed vimentin) appears to remain morphologically intact throughout mitosis in close association with the mitotic apparatus (Celis, J.E., P.M. Larsen, S.J. Fey, and A. Celis, 1983, J. Cell Biol., 97:1429-34). We propose that the mitotic apparatus in both mouse epidermal cells and in HeLa cells is supported and centered within the cell by IFB networks.
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Johnston, J. A., and R. D. Sloboda. "A 62-kD protein required for mitotic progression is associated with the mitotic apparatus during M-phase and with the nucleus during interphase." Journal of Cell Biology 119, no. 4 (November 15, 1992): 843–54. http://dx.doi.org/10.1083/jcb.119.4.843.
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A protein of 62 kD is a substrate of a calcium/calmodulin-dependent protein kinase, and both proteins copurify with isolated mitotic apparatuses (Dinsmore, J. H., and R. D. Sloboda. 1988. Cell. 53:769-780). Phosphorylation of the 62-kD protein increases after fertilization; maximum incorporation of phosphate occurs during late metaphase and anaphase and correlates directly with microtubule disassembly as determined by in vitro experiments with isolated mitotic apparatuses. Because 62-kD protein phosphorylation occurs in a pattern similar to the accumulation of the mitotic cyclin proteins, experiments were performed to determine the relationship between cyclin and the 62-kD protein. Continuous labeling of marine embryos with [35S]methionine, as well as immunoblots of marine embryo proteins using specific antibodies, were used to identify both cyclin and the 62-kD protein. These results clearly demonstrate that the 62-kD protein is distinct from cyclin and, unlike cyclin, is a constant member of the cellular protein pool during the first two cell cycles in sea urchin and surf clam embryos. Similar results were obtained using immunofluorescence microscopy of intact eggs and embryos. In addition, immunogold electron microscopy reveals that the 62-kD protein associates with the microtubules of the mitotic apparatus in dividing cells. Interestingly, the protein changes its subcellular distribution with respect to microtubules during the cell cycle. Specifically, during mitosis the 62-kD protein associates with the mitotic apparatus; before nuclear envelope breakdown, however, the 62-kD protein is confined to the nucleus. After anaphase, the 62-kD protein returns to the nucleus, where it resides until nuclear envelope disassembly of the next cell cycle.
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Tang, T. K., C. J. Tang, Y. J. Chao, and C. W. Wu. "Nuclear mitotic apparatus protein (NuMA): spindle association, nuclear targeting and differential subcellular localization of various NuMA isoforms." Journal of Cell Science 107, no. 6 (June 1, 1994): 1389–402. http://dx.doi.org/10.1242/jcs.107.6.1389.
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We have recently shown that the nuclear mitotic apparatus protein (NuMA) is composed of at least three isoforms that differ mainly at the carboxy terminus, and are generated by alternative splicing of a common mRNA precursor from a single NuMA gene (J. Cell Sci. (1993) 104, 249–260). Transient expression of human NuMA-1 isoform (T33/p230) in Chinese hamster ovary polyoma (CHOP) cells showed that NuMA-1 was present in interphase nuclei and was concentrated at the polar regions of the spindle apparatus in mitotic cells. However, expression of two other isoforms (NuMA-m and -s) revealed a distinct subcellular localization. NuMA-m (U4/p195) and NuMA-s (U6/p194) were present in the interphase cytosol and appeared to be mainly located at the centrosomal region. When cells entered into mitosis, however, NuMA-m and -s moved to the mitotic spindle pole. Analysis of a series of linker scanning-mutants and NuMA/beta-galactosidase chimeric proteins showed that residues 1972–2007 of NuMA-1 constitute a novel nuclear localization signal (NLS) and residues 1538–2115 are necessary and sufficient for spindle association. Further analysis of the NLS by site-specific mutagenesis indicated that Lys1988 is essential for nuclear targeting, whereas Arg1984 is not. These results have allowed us tentatively to assign specific biological activities to distinct structural domains of the NuMA polypeptide.
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Paul, E. C., and A. Quaroni. "Identification of a 102 kDa protein (cytocentrin) immunologically related to keratin 19, which is a cytoplasmically derived component of the mitotic spindle pole." Journal of Cell Science 106, no. 3 (November 1, 1993): 967–81. http://dx.doi.org/10.1242/jcs.106.3.967.
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The mAb RK7, previously shown to recognize keratin 19, was also found to cross-react with a biologically unrelated 102 kDa protein, which becomes associated with the poles of the mitotic apparatus. This newly identified protein, called cytocentrin, is a stable cellular component, may be at least in part phosphorylated, and displays a cell cycle-dependent cellular localization. In interphase cells, it is diffusely distributed in the cytosol and shows no affinity for cytoplasmic microtubules. It becomes localized to the centrosome in early prophase, prior to nuclear envelope breakdown, separation of replicated centrosomes, and nucleation of mitotic apparatus microtubules. During metaphase, cytocentrin is located predominately at the mitotic poles, often appearing as an aggregate of small globular sub-components; it also associates with some polar microtubules. In late anaphase/early telophase cytocentrin dissociates entirely from the mitotic apparatus and becomes temporarily localized with microtubules in the midbody, from which it disappears by late telophase. In taxol-treated cells cytocentrin was associated with the center of the miniasters but also showed affinity for some cytoplasmic microtubules. Studies employing G2-synchronized cells and nocodazole demonstrated that cytocentrin can become associated with mitotic centrosomes independently of tubulin polymerization and that microtubules regrow from antigen-containing foci. We interpret these results to suggest that cytocentrin is a cytoplasmic protein that becomes specifically activated or modified at the onset of mitosis so that it can affiliate with the mitotic poles where it may provide a link between the pericentriolar material and other components of the mitotic apparatus.
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Chu, Xiaogang, Xuanyu Chen, Qingwen Wan, Zhen Zheng, and Quansheng Du. "Nuclear Mitotic Apparatus (NuMA) Interacts with and Regulates Astrin at the Mitotic Spindle." Journal of Biological Chemistry 291, no. 38 (July 26, 2016): 20055–67. http://dx.doi.org/10.1074/jbc.m116.724831.
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Nguyen, Christine L., and Karl Münger. "Human Papillomavirus E7 Protein Deregulates Mitosis via an Association with Nuclear Mitotic Apparatus Protein 1." Journal of Virology 83, no. 4 (December 3, 2008): 1700–1707. http://dx.doi.org/10.1128/jvi.01971-08.
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ABSTRACT We previously observed that high-risk human papillomavirus type 16 (HPV16) E7 expression leads to the delocalization of dynein from mitotic spindles (C. L. Nguyen, M. E. McLaughlin-Drubin, and K. Munger, Cancer Res. 68:8715-8722, 2008). Here, we show that HPV16 E7 associates with nuclear mitotic apparatus protein 1 (NuMA) and that NuMA binding and the ability to induce dynein delocalization map to similar carboxyl-terminal sequences of E7. Additionally, we show that the delocalization of dynein from mitotic spindles by HPV16 E7 and the interaction between HPV16 E7 and NuMA correlate with the induction of defects in chromosome alignment during prometaphase even in cells with normal centrosome numbers. Furthermore, low-risk HPV6b and HPV11 E7s also associate with NuMA and also induce a similar mitotic defect. It is possible that the disruption of mitotic events by HPV E7, via targeting of the NuMA/dynein complex and potentially other NuMA-containing complexes, contributes to viral maintenance and propagation potentially through abrogating the differentiation program of the infected epithelium. Furthermore, in concert with activities specific to high-risk HPV E6 and E7, such as the inactivation of the p53 and pRB tumor suppressors, respectively, the disruption of the NuMA/dynein network may result in mitotic errors that would make an infected cell more prone to the accumulation of aneuploidy even in the absence of supernumerary centrosomes.
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Wozniak, Richard, Brian Burke, and Valérie Doye. "Nuclear transport and the mitotic apparatus: an evolving relationship." Cellular and Molecular Life Sciences 67, no. 13 (April 8, 2010): 2215–30. http://dx.doi.org/10.1007/s00018-010-0325-7.
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Sparks, C. A., E. G. Fey, C. A. Vidair, and S. J. Doxsey. "Phosphorylation of NUMA occurs during nuclear breakdown and not mitotic spindle assembly." Journal of Cell Science 108, no. 11 (November 1, 1995): 3389–96. http://dx.doi.org/10.1242/jcs.108.11.3389.
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NuMA, the nuclear mitotic apparatus protein, is a component of the nuclear matrix at interphase that redistributes to the spindle poles at mitosis. While the function of NuMA is not known, it has been implicated in spindle organization during mitosis and nuclear reformation. Phosphorylation is thought to play a regulatory role in NuMA function. In this study, NuMA phosphorylation was examined through the cell cycle using highly synchronized cells. In intact cells labeled with 32P-orthophosphate, NuMA appeared as a 250 kDa phosphoprotein in interphase that shifted to a higher apparent molecular mass in mitosis. The shift was due to phosphorylation as shown by reduction of the shifted band to interphase mobility by phosphatase treatment. This phosphorylation event occurred roughly at the G2/M transition at the time of NuMA's release from the nucleus and its redistribution to the mitotic spindle. However, mitotic phosphorylation did not require spindle formation since the phosphorylated species was detected in nocodazole-treated cells lacking microtubule spindles. Dephosphorylation of NuMA occurred in two distinct steps, after lamin B assembled into the nuclear lamina, in early G1 and at the end of G1. Based on the timing of the phosphorylation and dephosphorylation observed in this study, we propose that they may play a role in nuclear events such as nuclear organization, transcription, or initiation of DNA replication at G1/S.
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Weaver, V. M., C. E. Carson, P. R. Walker, N. Chaly, B. Lach, Y. Raymond, D. L. Brown, and M. Sikorska. "Degradation of nuclear matrix and DNA cleavage in apoptotic thymocytes." Journal of Cell Science 109, no. 1 (January 1, 1996): 45–56. http://dx.doi.org/10.1242/jcs.109.1.45.
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In dexamethasone-treated thymocyte cultures an increase in nuclear proteolytic activity paralleled chromatin fragmentation and the appearance of small apoptotic cells. The elevation of nuclear proteolytic activity was accompanied by site-specific degradation of nuclear mitotic apparatus protein and lamin B, two essential components of the nuclear matrix. Nuclear mitotic apparatus protein phosphorylation and cleavage into 200 and 48 kDa fragments occurred within 30 minutes of dexamethasone treatment. Cleavage of lamin B, which generated a fragment of 46 kDa consistent with the central rod domain of the protein, was also detected after 30 minutes of exposure to the steroid hormone. The level of lamin B phosphorylation did not change as a result of the dexamethasone treatment and the lamina did not solubilize until the later stages of apoptosis. Initial DNA breaks, detected by the terminal transferase-mediated dUTP-biotin nick end labeling assay, occurred throughout the nuclei and solubilization of lamina was not required for this process to commence. The data presented in this paper support a model of apoptotic nuclear destruction brought about by the site-specific proteolysis of key structural proteins. Both the nuclear mitotic apparatus protein and lamin B were specifically targeted by protease(s) at early stages of the cell death pathway, which possibly initiate the cascade of degradative events in apoptosis.
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De Souza, Colin P., Shahr B. Hashmi, Tania Nayak, Berl Oakley, and Stephen A. Osmani. "Mlp1 Acts as a Mitotic Scaffold to Spatially Regulate Spindle Assembly Checkpoint Proteins in Aspergillus nidulans." Molecular Biology of the Cell 20, no. 8 (April 15, 2009): 2146–59. http://dx.doi.org/10.1091/mbc.e08-08-0878.
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During open mitosis several nuclear pore complex (NPC) proteins have mitotic specific localizations and functions. We find that the Aspergillus nidulans Mlp1 NPC protein has previously unrealized mitotic roles involving spatial regulation of spindle assembly checkpoint (SAC) proteins. In interphase, An-Mlp1 tethers the An-Mad1 and An-Mad2 SAC proteins to NPCs. During a normal mitosis, An-Mlp1, An-Mad1, and An-Mad2 localize similarly on, and around, kinetochores until telophase when they transiently localize near the spindle but not at kinetochores. During SAC activation, An-Mlp1 remains associated with kinetochores in a manner similar to An-Mad1 and An-Mad2. Although An-Mlp1 is not required for An-Mad1 kinetochore localization during early mitosis, it is essential to maintain An-Mad1 in the extended region around kinetochores in early mitosis and near the spindle in telophase. Our data are consistent with An-Mlp1 being part of a mitotic spindle matrix similar to its Drosophila orthologue and demonstrate that this matrix localizes SAC proteins. By maintaining SAC proteins near the mitotic apparatus, An-Mlp1 may help monitor mitotic progression and coordinate efficient mitotic exit. Consistent with this possibility, An-Mad1 and An-Mlp1 redistribute from the telophase matrix and associate with segregated kinetochores when mitotic exit is prevented by expression of nondegradable cyclin B.
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Mattagajasingh, Subhendra N., Shu-Ching Huang, Julia S. Hartenstein, Michael Snyder, Vincent T. Marchesi, and Edward J. Benz. "A Nonerythroid Isoform of Protein 4.1R Interacts with the Nuclear Mitotic Apparatus (NuMA) Protein." Journal of Cell Biology 145, no. 1 (April 5, 1999): 29–43. http://dx.doi.org/10.1083/jcb.145.1.29.
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Red blood cell protein 4.1 (4.1R) is an 80- kD erythrocyte phosphoprotein that stabilizes the spectrin/actin cytoskeleton. In nonerythroid cells, multiple 4.1R isoforms arise from a single gene by alternative splicing and predominantly code for a 135-kD isoform. This isoform contains a 209 amino acid extension at its NH2 terminus (head piece; HP). Immunoreactive epitopes specific for HP have been detected within the cell nucleus, nuclear matrix, centrosomes, and parts of the mitotic apparatus in dividing cells. Using a yeast two-hybrid system, in vitro binding assays, coimmunolocalization, and coimmunoprecipitation studies, we show that a 135-kD 4.1R isoform specifically interacts with the nuclear mitotic apparatus (NuMA) protein. NuMA and 4.1R partially colocalize in the interphase nucleus of MDCK cells and redistribute to the spindle poles early in mitosis. Protein 4.1R associates with NuMA in the interphase nucleus and forms a complex with spindle pole organizing proteins, NuMA, dynein, and dynactin during cell division. Overexpression of a 135-kD isoform of 4.1R alters the normal distribution of NuMA in the interphase nucleus. The minimal sequence sufficient for this interaction has been mapped to the amino acids encoded by exons 20 and 21 of 4.1R and residues 1788–1810 of NuMA. Our results not only suggest that 4.1R could, possibly, play an important role in organizing the nuclear architecture, mitotic spindle, and spindle poles, but also could define a novel role for its 22–24-kD domain.
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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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Terasaki, Mark. "Dynamics of the Endoplasmic Reticulum and Golgi Apparatus during Early Sea Urchin Development." Molecular Biology of the Cell 11, no. 3 (March 2000): 897–914. http://dx.doi.org/10.1091/mbc.11.3.897.
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The endoplasmic reticulum (ER) and Golgi were labeled by green fluorescent protein chimeras and observed by time-lapse confocal microscopy during the rapid cell cycles of sea urchin embryos. The ER undergoes a cyclical microtubule-dependent accumulation at the mitotic poles and by photobleaching experiments remains continuous through the cell cycle. Finger-like indentations of the nuclear envelope near the mitotic poles appear 2–3 min before the permeability barrier of the nuclear envelope begins to change. This permeability change in turn is ∼30 s before nuclear envelope breakdown. During interphase, there are many scattered, disconnected Golgi stacks throughout the cytoplasm, which appear as 1- to 2-μm fluorescent spots. The number of Golgi spots begins to decline soon after nuclear envelope breakdown, reaches a minimum soon after cytokinesis, and then rapidly increases. At higher magnification, smaller spots are seen, along with increased fluorescence in the ER. Quantitative measurements, along with nocodazole and photobleaching experiments, are consistent with a redistribution of some of the Golgi to the ER during mitosis. The scattered Golgi coalesce into a single large aggregate during the interphase after the ninth embryonic cleavage; this is likely to be preparatory for secretion of the hatching enzyme during the following cleavage cycle.
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Feeney, Katherine M., and Joanna L. Parish. "Targeting mitotic chromosomes: a conserved mechanism to ensure viral genome persistence." Proceedings of the Royal Society B: Biological Sciences 276, no. 1662 (January 20, 2009): 1535–44. http://dx.doi.org/10.1098/rspb.2008.1642.
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Viruses that maintain their genomes as extrachromosomal circular DNA molecules and establish infection in actively dividing cells must ensure retention of their genomes within the nuclear envelope in order to prevent genome loss. The loss of nuclear membrane integrity during mitosis dictates that paired host cell chromosomes are captured and organized by the mitotic spindle apparatus before segregation to daughter cells. This prevents inaccurate chromosomal segregation and loss of genetic material. A similar mechanism may also exist for the nuclear retention of extrachromosomal viral genomes or episomes during mitosis, particularly for genomes maintained at a low copy number in latent infections. It has been heavily debated whether such a mechanism exists and to what extent this mechanism is conserved among diverse viruses. Research over the last two decades has provided a wealth of information regarding the mechanisms by which specific tumour viruses evade mitotic and DNA damage checkpoints. Here, we discuss the similarities and differences in how specific viruses tether episomal genomes to host cell chromosomes during mitosis to ensure long-term persistence.
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Chen, Rey-Huei. "Chromosome detachment from the nuclear envelope is required for genomic stability in closed mitosis." Molecular Biology of the Cell 30, no. 13 (June 15, 2019): 1578–86. http://dx.doi.org/10.1091/mbc.e19-02-0098.
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Mitosis in metazoans involves detachment of chromosomes from the nuclear envelope (NE) and NE breakdown, whereas yeasts maintain the nuclear structure throughout mitosis. It remains unknown how chromosome attachment to the NE might affect chromosome movement in yeast. By using a rapamycin-induced dimerization system to tether a specific locus of the chromosome to the NE, I found that the tethering delays the separation and causes missegregation of the region distal to the tethered site. The phenotypes are exacerbated by mutations in kinetochore components and Aurora B kinase Ipl1. The chromosome region proximal to the centromere is less affected by the tether, but it exhibits excessive oscillation before segregation. Furthermore, the tether impacts full extension of the mitotic spindle, causing abrupt shrinkage or bending of the spindle in shortened anaphase. The study supports detachment of chromosomes from the NE being required for faithful chromosome segregation in yeast and segregation of tethered chromosomes being dependent on a fully functional mitotic apparatus.
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Prifti, Diogjena Katerina, Annie Lauzier, and Sabine Elowe. "A commercial ARHGEF17/TEM4 antibody cross-reacts with Nuclear Mitotic Apparatus protein 1 (NuMA)." PLOS ONE 17, no. 7 (July 1, 2022): e0268848. http://dx.doi.org/10.1371/journal.pone.0268848.
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The Rho family Guanine nucleotide exchange factor (GEF) ARHGEF17 (also known as TEM4) is a large protein with only 3 annotated regions: an N-terminal actin-binding domain, a Rho-specific dbl homology (DH)- pleckstrin homology (PH) type GEF domain and a seven bladed β propeller fold at the C-terminus with unknown function. TEM4 has been implicated in numerous activities that rely on regulation of the cytoskeleton including cell migration, cell-cell junction formation and the spindle assembly checkpoint during mitosis. Here we have assessed the specificity of a TEM4 polyclonal antibody that has been commonly used as a Western blotting and immunocytochemistry probe for TEM4 in mammalian cells. We find that this antibody, in addition to its intended target, cross-reacts with the Nuclear Mitotic Apparatus Protein 1 (NuMA) in Western blotting and immunoprecipitation, and detects NuMA preferentially in immunocytochemistry. This cross-reactivity, with an abundant chromatin- and mitotic spindle-associated factor, is likely to affect the interpretation of experiments that make use of this antibody probe, in particular by immunocytochemistry and immunoprecipitation.
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Kollara, Alexandra, Maurice J. Ringuette, and Theodore J. Brown. "Dynamic Distribution of Nuclear Coactivator 4 during Mitosis: Association with Mitotic Apparatus and Midbodies." PLoS ONE 6, no. 7 (July 26, 2011): e22257. http://dx.doi.org/10.1371/journal.pone.0022257.
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Lai, Kun-Yi, Matteo Rizzato, Inci Aydin, Ruth Villalonga-Planells, Hannes C. A. Drexler, and Mario Schelhaas. "A Ran-binding protein facilitates nuclear import of human papillomavirus type 16." PLOS Pathogens 17, no. 5 (May 11, 2021): e1009580. http://dx.doi.org/10.1371/journal.ppat.1009580.
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Human papillomaviruses (HPVs) utilize an atypical mode of nuclear import during cell entry. Residing in the Golgi apparatus until mitosis onset, a subviral complex composed of the minor capsid protein L2 and viral DNA (L2/vDNA) is imported into the nucleus after nuclear envelope breakdown by associating with mitotic chromatin. In this complex, L2 plays a crucial role in the interactions with cellular factors that enable delivery and ultimately tethering of the viral genome to mitotic chromatin. To date, the cellular proteins facilitating these steps remain unknown. Here, we addressed which cellular proteins may be required for this process. Using label-free mass spectrometry, biochemical assays, microscopy, and functional virological assays, we discovered that L2 engages a hitherto unknown protein complex of Ran-binding protein 10 (RanBP10), karyopherin alpha2 (KPNA2), and dynein light chain DYNLT3 to facilitate transport towards mitotic chromatin. Thus, our study not only identifies novel cellular interactors and mechanism that facilitate a poorly understood step in HPV entry, but also a novel cellular transport complex.
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Matsui, Hirotaka, Yuko Ozaki, Akiko Nagamachi, and Toshiya Inaba. "Abnormal Mitosis Due to Impairment of Centrosome Maturation by the Deletion of Candidate 7q- Responsible Genes." Blood 120, no. 21 (November 16, 2012): 1297. http://dx.doi.org/10.1182/blood.v120.21.1297.1297.
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Abstract Abstract 1297 Centrosomes acquire dense microtubule nucleation sites in the beginning of mitosis. Failure of this process (called centrosome maturation) impairs the function of mitotic centrosomes to create robust mitotic spindles, resulting in lagging and scattered chromosomes that subsequently cause abnormal nuclear morphology such as bi- tri- or multiple-nuclei with or without small nucleus, as seen routinely in MDS. We previously reported that the Miki (LOC253012) gene, located in 7q21.3, is frequently deleted in MDS patients, and that low levels of Miki are tightly associated with abnormal mitosis and nuclear morphology (BBRC 2009). Here we demonstrate that Miki plays critical roles in the formation of robust spindles required for the prompt movement of chromosomes in a poly(ADP) ribosylation (PARsylation)-dependent manner (a part of data was published in Mol. Cell 2012). While Miki was localized in the Golgi apparatus during interphase, it was relocated to centrosomes at the beggining of mitosis. Treatment of cells with Miki-specific siRNA induced ‘pseudometaphase’ condition, in which lagging chromosomes juxtaposed to, or even situated behind, spindle poles. Pseudometaphase was followed by apoptosis or abnormal exit from mitosis that creates cells with abnormal nuclear morphology. This phenotype of Miki-downregulation was caused by the reduced robustness of mitotic spindles. α -tubulin staining of siRNA-treated cells revealed curling and disorganized spindles, with an occasional chaotic centrosome at only one side of the alignment. In addition, Miki-downregulation reduced γ-tubulin signals in mitotic centrosomes and markedly inhibited microtubule nucleation, shown by the impaired accumulation of the EB1 microtubule tip-binding protein at centrosomes. In immnunoblot analysis of lysate extracted from isolated spindles/centrosomes using Miki antibody, we detected a dense 125 kDa band in addition to the expected 50 kDa band and found that the 125 kDa band represents PARsylated Miki. A recent report indicated that tankyrase-1, a PAR polymerase (PARP), is required for the progression of prometaphase. We found that the downregulation of tankyrase-1 prevents Miki from localizing to mitotic centrosomes. In addition, immunoblot analysis of immunoprecipitation revealed that Miki is a substrate for tankyrase-1. These data suggested that tankyrase-1 PARsylates and translocates Miki from the Golgi apparatus to mitotic centrosomes/spindles during the short period from late G2 to prophase. We also found that PARsylated Miki promotes CG-NAP, a major component of microtubule nucleation sites, to concentrate in mitotic centrosomes. Interestingly, the CG-NAP gene resides 1.2Mb centromeric to Miki in band 7q21, and approximately 20% of MDS patients lose one allele of both Miki and CG-NAP genes. This indicates that loss of 7q results in low expression of two crucial factors in the tankyrase-1/Miki-dependent system for centrosome maturation, and that this may cause miotic/nuclear abnormalities and chromosome instability characteristic of 7q- MDS. Disclosures: No relevant conflicts of interest to declare.
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Woodard, Geoffrey E., Ning-Na Huang, Hyeseon Cho, Toru Miki, Gregory G. Tall, and John H. Kehrl. "Ric-8A and Giα Recruit LGN, NuMA, and Dynein to the Cell Cortex To Help Orient the Mitotic Spindle." Molecular and Cellular Biology 30, no. 14 (May 17, 2010): 3519–30. http://dx.doi.org/10.1128/mcb.00394-10.
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ABSTRACT In model organisms, resistance to inhibitors of cholinesterase 8 (Ric-8), a G protein α (Gα) subunit guanine nucleotide exchange factor (GEF), functions to orient mitotic spindles during asymmetric cell divisions; however, whether Ric-8A has any role in mammalian cell division is unknown. We show here that Ric-8A and Gαi function to orient the metaphase mitotic spindle of mammalian adherent cells. During mitosis, Ric-8A localized at the cell cortex, spindle poles, centromeres, central spindle, and midbody. Pertussis toxin proved to be a useful tool in these studies since it blocked the binding of Ric-8A to Gαi, thus preventing its GEF activity for Gαi. Linking Ric-8A signaling to mammalian cell division, treatment of cells with pertussis toxin, reduction of Ric-8A expression, or decreased Gαi expression similarly affected metaphase cells. Each treatment impaired the localization of LGN (GSPM2), NuMA (microtubule binding nuclear mitotic apparatus protein), and dynein at the metaphase cell cortex and disturbed integrin-dependent mitotic spindle orientation. Live cell imaging of HeLa cells expressing green fluorescent protein-tubulin also revealed that reduced Ric-8A expression prolonged mitosis, caused occasional mitotic arrest, and decreased mitotic spindle movements. These data indicate that Ric-8A signaling leads to assembly of a cortical signaling complex that functions to orient the mitotic spindle.
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Huang, Shu-Ching, Eva S. Liu, Siu-Hong Chan, Indira D. Munagala, Heidi T. Cho, Ramasamy Jagadeeswaran, and Edward J. Benz. "Mitotic Regulation of Protein 4.1R Involves Phosphorylation by cdc2 Kinase." Molecular Biology of the Cell 16, no. 1 (January 2005): 117–27. http://dx.doi.org/10.1091/mbc.e04-05-0426.
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The nonerythrocyte isoform of the cytoskeletal protein 4.1R (4.1R) is associated with morphologically dynamic structures during cell division and has been implicated in mitotic spindle function. In this study, we define important 4.1R isoforms expressed in interphase and mitotic cells by RT-PCR and mini-cDNA library construction. Moreover, we show that 4.1R is phosphorylated by p34cdc2kinase on residues Thr60 and Ser679 in a mitosis-specific manner. Phosphorylated 4.1R135isoform(s) associate with tubulin and Nuclear Mitotic Apparatus protein (NuMA) in intact HeLa cells in vivo as well as with the microtubule-associated proteins in mitotic asters assembled in vitro. Recombinant 4.1R135is readily phosphorylated in mitotic extracts and reconstitutes mitotic aster assemblies in 4.1R-immunodepleted extracts in vitro. Furthermore, phosphorylation of these residues appears to be essential for the targeting of 4.1R to the spindle poles and for mitotic microtubule aster assembly in vitro. Phosphorylation of 4.1R also enhances its association with NuMA and tubulin. Finally, we used siRNA inhibition to deplete 4.1R from HeLa cells and provide the first direct genetic evidence that 4.1R is required to efficiently focus mitotic spindle poles. Thus, we suggest that 4.1R is a member of the suite of direct cdc2 substrates that are required for the establishment of a bipolar spindle.
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Zatsepina, O. V., A. Rousselet, P. K. Chan, M. O. Olson, E. G. Jordan, and M. Bornens. "The nucleolar phosphoprotein B23 redistributes in part to the spindle poles during mitosis." Journal of Cell Science 112, no. 4 (February 15, 1999): 455–66. http://dx.doi.org/10.1242/jcs.112.4.455.
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B23 is a major phosphoprotein in the interphasic nucleolus where it is involved in the assembly of pre-ribosomes. Using several cultured animal cells, we report that, in addition to the known redistribution of the protein during mitosis, B23 also becomes associated with mitotic spindle poles starting from early prometaphase onwards. Colocalization of B23 with the protein NuMA (Nuclear Mitotic Apparatus protein) was studied in mitotic cells and taxol-arrested cells. During the onset of mitosis, we observed that a fraction of B23 associates with, and dissociates from, the poles later than NuMA. At metaphase, both proteins are colocalized at the poles. The polar redistribution of both B23 and NuMA is mediated by microtubules. In taxol-treated cells, B23 is associated with the microtubule minus ends in the center of mitotic asters together with NuMA. Association of B23 with microtubule minus ends of mitotic asters was further confirmed with an in vitro assay, where B23 was found by western blotting to co-sediment with taxol-induced microtubule asters formed in a mitotic cell extract. Immunolabeling demonstrated that B23 and NuMA were both present at the center of the asters. Furthermore, an additional hyperphosphorylated form of B23 appeared when microtubule asters formed and associated with the asters. Immunodepletion of B23 from the mitotic extract revealed that taxol-induced microtubule asters were still observed in B23-immunodepleted mitotic extract, indicating that the presence of B23 at the poles is unlikely to be essential for spindle formation or stabilisation.
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Pines, J., and T. Hunter. "Human cyclins A and B1 are differentially located in the cell and undergo cell cycle-dependent nuclear transport." Journal of Cell Biology 115, no. 1 (October 1, 1991): 1–17. http://dx.doi.org/10.1083/jcb.115.1.1.
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We have used immunofluorescence staining to study the subcellular distribution of cyclin A and B1 during the somatic cell cycle. In both primary human fibroblasts and in epithelial tumor cells, we find that cyclin A is predominantly nuclear from S phase onwards. Cyclin A may associated with condensing chromosomes in prophase, but is not associated with condensed chromosomes in metaphase. By contrast, cyclin B1 accumulates in the cytoplasm of interphase cells and only enters the nucleus at the beginning of mitosis, before nuclear lamina breakdown. In mitotic cells, cyclin B1 associates with condensed chromosomes in prophase and metaphase, and with the mitotic apparatus. Cyclin A is degraded during metaphase and cyclin B1 is precipitously destroyed at the metaphase----anaphase transition. Cell fractionation and immunoprecipitation studies showed that both cyclin A and cyclin B1 are associated with PSTAIRE-containing proteins. The nuclear, but not the cytoplasmic form, of cyclin A is associated with a 33-kD PSTAIRE-containing protein. Cyclin B1 is associated with p34cdc2 in the cytoplasm. Thus we propose that the different localization of cyclin A and cyclin B1 in the cell cycle could be the means by which the two types of mitotic cyclin confer substrate specificity upon their associated PSTAIRE-containing protein kinase subunit.
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Yang, C. H., E. J. Lambie, and M. Snyder. "NuMA: an unusually long coiled-coil related protein in the mammalian nucleus." Journal of Cell Biology 116, no. 6 (March 15, 1992): 1303–17. http://dx.doi.org/10.1083/jcb.116.6.1303.
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A bank of 892 autoimmune sera was screened by indirect immunofluorescence on mammalian cells. Six sera were identified that recognize an antigen(s) with a cell cycle-dependent localization pattern. In interphase cells, the antibodies stained the nucleus and in mitotic cells the spindle apparatus was recognized. Immunological criteria indicate that the antigen recognized by at least one of these sera corresponds to a previously identified protein called the nuclear mitotic apparatus protein (NuMA). A cDNA which partially encodes NuMA was cloned from a lambda gt11 human placental cDNA expression library, and overlapping cDNA clones that encode the entire gene were isolated. DNA sequence analysis of the clones has identified a long open reading frame capable of encoding a protein of 238 kD. Analysis of the predicted protein sequence suggests that NuMA contains an unusually large central alpha-helical domain of 1,485 amino acids flanked by nonhelical terminal domains. The central domain is similar to coiled-coil regions in structural proteins such as myosin heavy chains, cytokeratins, and nuclear lamins which are capable of forming filaments. Double immunofluorescence experiments performed with anti-NuMA and antilamin antibodies indicate that NuMA dissociates from condensing chromosomes during early prophase, before the complete disintegration of the nuclear lamina. As mitosis progresses, NuMA reassociates with telophase chromosomes very early during nuclear reformation, before substantial accumulation of lamins on chromosomal surfaces is evident. These results indicate that the NuMA proteins may be a structural component of the nucleus and may be involved in the early steps of nuclear reformation during telophase.
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Theodoropoulos, P. A., H. Polioudaki, M. Koulentaki, E. Kouroumalis, and S. D. Georgatos. "PBC68: a nuclear pore complex protein that associates reversibly with the mitotic spindle." Journal of Cell Science 112, no. 18 (September 15, 1999): 3049–59. http://dx.doi.org/10.1242/jcs.112.18.3049.
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Using autoimmune antibodies from a patient with primary biliary cirrhosis we have identified a 68 kDa nuclear envelope protein, termed PBC68. This protein is co-precipitated with a 98 kDa and a 250 kDa polypeptide and is distinct from the nuclear lamins. Immunostaining of digitonin-permeabilized cells indicates that PBC68 is restricted to the inner (nucleoplasmic) face of the nuclear envelope, while indirect immunofluorescence and immunoelectron microscopy show that PBC68 is located on fibrillar structures emanating from the nuclear pore complex. The autoantigen is modified at early prophase and disassembles at prometaphase concurrently with the breakdown of the nuclear envelope. The disassembled material, instead of diffusing throughout the cytoplasm as other nucleoporins, is targeted to the mitotic spindle and remains stably bound to it until anaphase. At telophase PBC68 is released from the mitotic apparatus and reassembles late, after incorporation of LAP2B and B-type lamins, onto the reforming nuclear envelope. The partitioning of PBC68 in dividing cells supports the notion that subsets of nuclear envelope proteins are actively sorted during mitosis by transiently anchoring to spindle microtubules. Furthermore, the data suggest that specific constituents of pore complex are released in a stepwise fashion from their anchorage sites before becoming available for nuclear reassembly.
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Draviam, Viji Mythily, Simona Orrechia, Martin Lowe, Ruggero Pardi, and Jonathon Pines. "The Localization of Human Cyclins B1 and B2 Determines Cdk1 Substrate Specificity and Neither Enzyme Requires Mek to Disassemble the Golgi Apparatus." Journal of Cell Biology 152, no. 5 (March 5, 2001): 945–58. http://dx.doi.org/10.1083/jcb.152.5.945.
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In this paper, we show that substrate specificity is primarily conferred on human mitotic cyclin-dependent kinases (CDKs) by their subcellular localization. The difference in localization of the B-type cyclin–CDKs underlies the ability of cyclin B1–CDK1 to cause chromosome condensation, reorganization of the microtubules, and disassembly of the nuclear lamina and of the Golgi apparatus, while it restricts cyclin B2–CDK1 to disassembly of the Golgi apparatus. We identify the region of cyclin B2 responsible for its localization and show that this will direct cyclin B1 to the Golgi apparatus and confer upon it the more limited properties of cyclin B2. Equally, directing cyclin B2 to the cytoplasm with the NH2 terminus of cyclin B1 confers the broader properties of cyclin B1. Furthermore, we show that the disassembly of the Golgi apparatus initiated by either mitotic cyclin–CDK complex does not require mitogen-activated protein kinase kinase (MEK) activity.
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Hsu, H. L., and N. H. Yeh. "Dynamic changes of NuMA during the cell cycle and possible appearance of a truncated form of NuMA during apoptosis." Journal of Cell Science 109, no. 2 (February 1, 1996): 277–88. http://dx.doi.org/10.1242/jcs.109.2.277.
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We have demonstrated that dynamic redistribution of nuclear-mitotic apparatus (NuMA) protein in the cell cycle is correlated temporally and spatially with its biochemical modifications. In interphase, NuMA behaves solely as a 220 kDa nuclear matrix-associated protein. After initiation of DNA condensation during mitosis, NuMA is phosphorylated by Cdc2 kinase into a 240 kDa form which is transported quickly to the centrosomal region. Once cells have passed the metaphase-anaphase transition, the 240 kDa form of NuMA either becomes a 180 kDa truncated form which is fated to be degraded completely before mitotic exit, or returns to the 220 kDa form that relocates to the daughter nuclei and remains throughout interphase. Apparently, a proteolytic enzyme is activated during the late stages of mitosis. After induction of a 180 kDa form of NuMA in interphase HeLa cells by 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, nuclear apoptotic phenomena including chromatin condensation, DNA fragmentation, and micronucleation were observed. However, the same treatment did not induce apoptosis in mitotic phase-arrested HeLa cells. The 180 kDa form of NuMA was demonstrated to be a truncated product, at least lacking the tail domain. When HL60 cells were stimulated by diverse apoptosis inducers such as camptothecin, staurosporine, cycloheximide, and A23187, the extent of NuMA cleavage to produce a 180 kDa product was comparable with the degree of oligonucleosomal laddering. NuMA cleavage is likely to be a consequence of the onset of apoptosis. The intact 220 kDa NuMA functions in interphase cells to retain the nuclear structural integrity. Additionally, NuMA appears to act as a nuclear structural target for a death protease during apoptosis.
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Zhu, X., M. A. Mancini, K. H. Chang, C. Y. Liu, C. F. Chen, B. Shan, D. Jones, T. L. Yang-Feng, and W. H. Lee. "Characterization of a novel 350-kilodalton nuclear phosphoprotein that is specifically involved in mitotic-phase progression." Molecular and Cellular Biology 15, no. 9 (September 1995): 5017–29. http://dx.doi.org/10.1128/mcb.15.9.5017.
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A gene assigned to human chromosome 1q32-41 encodes a novel protein of 3,113 amino acids containing an internal tandem repeat of 177 amino acids. The protein, which we have named "mitosin," was identified by direct binding to purified retinoblastoma protein in vitro with a region distantly related to the retinoblastoma protein-binding site of E2F-1. Mitosin is expressed throughout S, G2, and M phases of the cell cycle but is absent in G1. Its localization is dramatically reorganized from a rather homogeneous nuclear distribution in S phase to paired dots at the kinetochore/centromere region, to the spindle apparatus, and then to the midbody during M-phase progression. This spatial reorganization coincides closely with the temporal phosphorylation patterns of mitosin. Overexpression of N-terminally truncated mutants blocks cell cycle progression mainly at G2/M. These results suggest that mitosin may play an important role in mitotic-phase progression.
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Schatten, Heide, Maureen Ripple, Ron Balczon, Meghan Taylor, and Michael Crosser. "Centrosome Proliferation in the Human Androgen-Responsive LNCaP and the Androgen-Independent DU145 Prostate Cancer Cell Lines." Microscopy and Microanalysis 4, S2 (July 1998): 1066–67. http://dx.doi.org/10.1017/s1431927600025459.
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Cancer is a disease characterized by uncontrolled cell divisions in which the molecular controls for cytoskeletal regulation are bypassed. Cell division is governed by centrosomes, microtubule-organizing cell organelles which are crucial for the organization of the mitotic apparatus during mitosis and cell division. Because centrosome abnormalities are observed in the most common human cancers, we used immunofluorescence and transmission electron microscopy to determine centrosome organization in the human androgenresponsive prostate cancer cell line LNCaP and the androgen-independent prostate cancer cell line DU145. During interphase, centrosomes are located in close vicinity to the outer nuclear membrane, duplicate during S-phase, and become separated to the mitotic poles during the transition from interphase to mitosis. Centrosome regulation is based on a number of different factors which are only partly understood. Hormones play a role during developmental regulation of prostates which might trigger the activation of centrosome proteins and consequent cell divisions in order to ensure tissue growth.
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Kong, Xiangduo, Alexander R. Ball, Eiichiro Sonoda, Jie Feng, Shunichi Takeda, Tatsuo Fukagawa, Tim J. Yen, and Kyoko Yokomori. "Cohesin Associates with Spindle Poles in a Mitosis-specific Manner and Functions in Spindle Assembly in Vertebrate Cells." Molecular Biology of the Cell 20, no. 5 (March 2009): 1289–301. http://dx.doi.org/10.1091/mbc.e08-04-0419.
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Cohesin is an essential protein complex required for sister chromatid cohesion. Cohesin associates with chromosomes and establishes sister chromatid cohesion during interphase. During metaphase, a small amount of cohesin remains at the chromosome-pairing domain, mainly at the centromeres, whereas the majority of cohesin resides in the cytoplasm, where its functions remain unclear. We describe the mitosis-specific recruitment of cohesin to the spindle poles through its association with centrosomes and interaction with nuclear mitotic apparatus protein (NuMA). Overexpression of NuMA enhances cohesin accumulation at spindle poles. Although transient cohesin depletion does not lead to visible impairment of normal spindle formation, recovery from nocodazole-induced spindle disruption was significantly impaired. Importantly, selective blocking of cohesin localization to centromeres, which disrupts centromeric sister chromatid cohesion, had no effect on this spindle reassembly process, clearly separating the roles of cohesin at kinetochores and spindle poles. In vitro, chromosome-independent spindle assembly using mitotic extracts was compromised by cohesin depletion, and it was rescued by addition of cohesin that was isolated from mitotic, but not S phase, cells. The combined results identify a novel spindle-associated role for human cohesin during mitosis, in addition to its function at the centromere/kinetochore regions.
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Rahim, Md, Ludger Klewes, Ali Zahedi-Amiri, Sabine Mai, and Kevin Coombs. "Global Interactomics Connect Nuclear Mitotic Apparatus Protein NUMA1 to Influenza Virus Maturation." Viruses 10, no. 12 (December 19, 2018): 731. http://dx.doi.org/10.3390/v10120731.
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Influenza A virus (IAV) infections remain a major human health threat. IAV has enormous genetic plasticity and can rapidly escape virus-targeted anti-viral strategies. Thus, there is increasing interest to identify host proteins and processes the virus requires for replication and maturation. The IAV non-structural protein 1 (NS1) is a critical multifunctional protein that is expressed to high levels in infected cells. Host proteins that interact with NS1 may serve as ideal targets for attenuating IAV replication. We previously developed and characterized broadly cross-reactive anti-NS1 monoclonal antibodies. For the current study, we used these mAbs to co-immunoprecipitate native IAV NS1 and interacting host proteins; 183 proteins were consistently identified in this NS1 interactome study, 124 of which have not been previously reported. RNAi screens identified 11 NS1-interacting host factors as vital for IAV replication. Knocking down one of these, nuclear mitotic apparatus protein 1 (NUMA1), dramatically reduced IAV replication. IAV genomic transcription and translation were not inhibited but transport of viral structural proteins to the cell membrane was hindered during maturation steps in NUMA1 knockdown (KD) cells.
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Endo, A., A. Moyori, A. Kobayashi, and R. W. Wong. "Nuclear mitotic apparatus protein, NuMA, modulates p53-mediated transcription in cancer cells." Cell Death & Disease 4, no. 7 (July 2013): e713-e713. http://dx.doi.org/10.1038/cddis.2013.239.
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Seo, Jae Sung, Ha Na Kim, Sun-Jick Kim, Jiyoung Bang, Eun-A. Kim, Ki Sa Sung, Hyun-Joo Yoon, Hae Yong Yoo, and Cheol Yong Choi. "Cell cycle-dependent SUMO-1 conjugation to nuclear mitotic apparatus protein (NuMA)." Biochemical and Biophysical Research Communications 443, no. 1 (January 2014): 259–65. http://dx.doi.org/10.1016/j.bbrc.2013.11.107.
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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.
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Harel, A., E. Zlotkin, S. Nainudel-Epszteyn, N. Feinstein, P. A. Fisher, and Y. Gruenbaum. "Persistence of major nuclear envelope antigens in an envelope-like structure during mitosis in Drosophila melanogaster embryos." Journal of Cell Science 94, no. 3 (November 1, 1989): 463–70. http://dx.doi.org/10.1242/jcs.94.3.463.
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Using monoclonal antibodies, we followed the fate of three different nuclear envelope proteins during mitosis in Drosophila early embryos by indirect immunofluorescence microscopy. Two of these proteins, lamin and otefin, a newly characterized nuclear envelope polypeptide with an apparent Mr of 53,000, are apparently present in an envelope-like structure that is present throughout mitosis. Immunoelectron microscopy of interphase nuclei indicates that otefin, like lamin, is not a component of nuclear pore complexes. In contrast with lamin and otefin, gp188, a putative pore complex component, was completely redistributed through the surrounding cytoplasm during prophase in comparable early embryo specimens and was present in an envelope only in interphase. Together with previous morphological studies by other workers, these data suggest that the entire mitotic apparatus including condensed chromosomes and spindle is enclosed by an envelope throughout mitosis during early embryogenesis in Drosophila. This ‘spindle envelope’, as it has been named by others, contains both lamin and otefin but probably not pore complex proteins.
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Chang, William, Jasmin N. Dynek, and Susan Smith. "NuMA is a major acceptor of poly(ADP-ribosyl)ation by tankyrase 1 in mitosis." Biochemical Journal 391, no. 2 (October 10, 2005): 177–84. http://dx.doi.org/10.1042/bj20050885.
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Tankyrase 1 is a PARP [poly(ADP-ribose) polymerase] that localizes to multiple subcellular sites, including telomeres and mitotic centrosomes. Previous studies demonstrated that cells deficient in tankyrase 1 suffered a block in resolution of sister telomeres and arrested in early anaphase [Dynek and Smith (2004) Science 304, 97–100]. This phenotype was dependent on the catalytic PARP activity of tankyrase 1. To identify critical acceptors of PARsylation [poly(ADP-ribosyl)ation] by tankyrase 1 in mitosis, tankyrase 1 immunoprecipitates were analysed for associated PARsylated proteins. We identified NuMA (nuclear mitotic apparatus protein) as a major acceptor of poly(ADP-ribose) from tankyrase 1 in mitosis. We showed by immunofluorescence and immunoprecipitation that association between tankyrase 1 and NuMA increases dramatically at the onset of mitosis, concomitant with PARsylation of NuMA. Knockdown of tankyrase 1 by siRNA (small interfering RNA) eliminates PARsylation of NuMA in mitosis, confirming tankyrase 1 as the PARP responsible for this modification. However, even in the absence of tankyrase 1 and PARsylation, NuMA localizes to spindle poles. By contrast, siRNA knockdown of NuMA results in complete loss of tankyrase 1 from spindle poles. We discuss our result in terms of a model where PARsylation of NuMA by tankyrase 1 in mitosis could play a role in sister telomere separation and/or mitotic progression.
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Moreno-Andrés, Daniel, Hideki Yokoyama, Anja Scheufen, Guillaume Holzer, Hongqi Lue, Anna Katharina Schellhaus, Marion Weberruss, Masatoshi Takagi, and Wolfram Antonin. "VPS72/YL1-Mediated H2A.Z Deposition Is Required for Nuclear Reassembly after Mitosis." Cells 9, no. 7 (July 16, 2020): 1702. http://dx.doi.org/10.3390/cells9071702.
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The eukaryotic nucleus remodels extensively during mitosis. Upon mitotic entry, the nuclear envelope breaks down and chromosomes condense into rod-shaped bodies, which are captured by the spindle apparatus and segregated during anaphase. Through telophase, chromosomes decondense and the nuclear envelope reassembles, leading to a functional interphase nucleus. While the molecular processes occurring in early mitosis are intensively investigated, our knowledge about molecular mechanisms of nuclear reassembly is rather limited. Using cell free and cellular assays, we identify the histone variant H2A.Z and its chaperone VPS72/YL1 as important factors for reassembly of a functional nucleus after mitosis. Live-cell imaging shows that siRNA-mediated downregulation of VPS72 extends the telophase in HeLa cells. In vitro, depletion of VPS72 or H2A.Z results in malformed and nonfunctional nuclei. VPS72 is part of two chromatin-remodeling complexes, SRCAP and EP400. Dissecting the mechanism of nuclear reformation using cell-free assays, we, however, show that VPS72 functions outside of the SRCAP and EP400 remodeling complexes to deposit H2A.Z, which in turn is crucial for formation of a functional nucleus.
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Ecke, Mary, Jana Prassler, and Günther Gerisch. "Genetic Instability Due to Spindle Anomalies Visualized in Mutants of Dictyostelium." Cells 10, no. 9 (August 29, 2021): 2240. http://dx.doi.org/10.3390/cells10092240.
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Aberrant centrosome activities in mutants of Dictyostelium discoideum result in anomalies of mitotic spindles that affect the reliability of chromosome segregation. Genetic instabilities caused by these deficiencies are tolerated in multinucleate cells, which can be produced by electric-pulse induced cell fusion as a source for aberrations in the mitotic apparatus of the mutant cells. Dual-color fluorescence labeling of the microtubule system and the chromosomes in live cells revealed the variability of spindle arrangements, of centrosome-nuclear interactions, and of chromosome segregation in the atypical mitoses observed.
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Auer-Grumbach, P., and M. Stangl. "Autoantibodies to Nuclear Mitotic Apparatus in a Patient with Vitiligo and Autoimmune Thyroiditis." Dermatology 186, no. 3 (1993): 229–31. http://dx.doi.org/10.1159/000247353.
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Sparks, Cynthia A., Peter L. Bangs, Gerard P. McNeil, Jeanne B. Lawrence, and Edward G. Fey. "Assignment of the Nuclear Mitotic Apparatus Protein NuMA Gene to Human Chromosome 11q13." Genomics 17, no. 1 (July 1993): 222–24. http://dx.doi.org/10.1006/geno.1993.1307.
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Machicoane, Mickael, Cristina A. de Frutos, Jenny Fink, Murielle Rocancourt, Yannis Lombardi, Sonia Garel, Matthieu Piel, and Arnaud Echard. "SLK-dependent activation of ERMs controls LGN–NuMA localization and spindle orientation." Journal of Cell Biology 205, no. 6 (June 23, 2014): 791–99. http://dx.doi.org/10.1083/jcb.201401049.
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Mitotic spindle orientation relies on a complex dialog between the spindle microtubules and the cell cortex, in which F-actin has been recently implicated. Here, we report that the membrane–actin linkers ezrin/radixin/moesin (ERMs) are strongly and directly activated by the Ste20-like kinase at mitotic entry in mammalian cells. Using microfabricated adhesive substrates to control the axis of cell division, we found that the activation of ERMs plays a key role in guiding the orientation of the mitotic spindle. Accordingly, impairing ERM activation in apical progenitors of the mouse embryonic neocortex severely disturbed spindle orientation in vivo. At the molecular level, ERM activation promotes the polarized association at the mitotic cortex of leucine-glycine-asparagine repeat protein (LGN) and nuclear mitotic apparatus (NuMA) protein, two essential factors for spindle orientation. We propose that activated ERMs, together with Gαi, are critical for the correct localization of LGN–NuMA force generator complexes and hence for proper spindle orientation.
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Si, Huaxin, Subhash C. Verma, Michael A. Lampson, Qiliang Cai, and Erle S. Robertson. "Kaposi's Sarcoma-Associated Herpesvirus-Encoded LANA Can Interact with the Nuclear Mitotic Apparatus Protein To Regulate Genome Maintenance and Segregation." Journal of Virology 82, no. 13 (April 16, 2008): 6734–46. http://dx.doi.org/10.1128/jvi.00342-08.
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ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) genomes are tethered to the host chromosomes and partitioned faithfully into daughter cells with the host chromosomes. The latency-associated nuclear antigen (LANA) is important for segregation of the newly synthesized viral genomes to the daughter nuclei. Here, we report that the nuclear mitotic apparatus protein (NuMA) and LANA can associate in KSHV-infected cells. In synchronized cells, NuMA and LANA are colocalized in interphase cells and separate during mitosis at the beginning of prophase, reassociating again at the end of telophase and cytokinesis. Silencing of NuMA expression by small interfering RNA and expression of LGN and a dominant-negative of dynactin (P150-CC1), which disrupts the association of NuMA with microtubules, resulted in the loss of KSHV terminal-repeat plasmids containing the major latent origin. Thus, NuMA is required for persistence of the KSHV episomes in daughter cells. This interaction between NuMA and LANA is critical for segregation and maintenance of the KSHV episomes through a temporally controlled mechanism of binding and release during specific phases of mitosis.
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Ciemerych, Maria A., Bernard Maro, and Jacek Z. Kubiak. "Control of duration of the first two mitoses in a mouse embryo." Zygote 7, no. 4 (November 1999): 293–300. http://dx.doi.org/10.1017/s0967199499000696.
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The duration of M-phase is largely determined by the time necessary for the formation of a functional metaphase spindle and the correct alignment of all chromosomes on the metaphase plate. The spindle assembly checkpoint prevents the exit from M-phase before the proper alignment of all chromosomes on a metaphase plate in many cell types. In the present paper we show that the first mitotic M-phase of the mouse embryo lasts about 119 min, while the second embryonic M-phase lasts only about 70 min. Histone H1 kinase is activated rapidly during nuclear envelope breakdown in both mitoses. Its maximum, however, is followed by a plateau only during the first mitosis. In the second mitosis, the inactivation of histone H1 kinase activity follows its maximum directly. Histone H1 kinase is more stable in the cytoplasts obtained from mouse embryos during the first embryonic M-phase than during the second one. The stability of histone H1 kinase is greatly increased by the presence of the mitotic apparatus in both M-phases. The mitotic spindle assembly during the first and the second mitoses differs and the first metaphase spindle is stabilised during the period of maximum histone H1 kinase activity. These data show that an unknown developmentally regulated mechanism controls the duration of the two first mitoses in the mouse embryo.
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Krauss, Sharon Wald, Jeffrey R. Spence, Shirin Bahmanyar, Angela I. M. Barth, Minjoung M. Go, Debra Czerwinski, and Adam J. Meyer. "Downregulation of Protein 4.1R, a Mature Centriole Protein, Disrupts Centrosomes, Alters Cell Cycle Progression, and Perturbs Mitotic Spindles and Anaphase." Molecular and Cellular Biology 28, no. 7 (January 22, 2008): 2283–94. http://dx.doi.org/10.1128/mcb.02021-07.
Full textAbstract:
ABSTRACT Centrosomes nucleate and organize interphase microtubules and are instrumental in mitotic bipolar spindle assembly, ensuring orderly cell cycle progression with accurate chromosome segregation. We report that the multifunctional structural protein 4.1R localizes at centrosomes to distal/subdistal regions of mature centrioles in a cell cycle-dependent pattern. Significantly, 4.1R-specific depletion mediated by RNA interference perturbs subdistal appendage proteins ninein and outer dense fiber 2/cenexin at mature centrosomes and concomitantly reduces interphase microtubule anchoring and organization. 4.1R depletion causes G1 accumulation in p53-proficient cells, similar to depletion of many other proteins that compromise centrosome integrity. In p53-deficient cells, 4.1R depletion delays S phase, but aberrant ninein distribution is not dependent on the S-phase delay. In 4.1R-depleted mitotic cells, efficient centrosome separation is reduced, resulting in monopolar spindle formation. Multipolar spindles and bipolar spindles with misaligned chromatin are also induced by 4.1R depletion. Notably, all types of defective spindles have mislocalized NuMA (nuclear mitotic apparatus protein), a 4.1R binding partner essential for spindle pole focusing. These disruptions contribute to lagging chromosomes and aberrant microtubule bridges during anaphase/telophase. Our data provide functional evidence that 4.1R makes crucial contributions to the structural integrity of centrosomes and mitotic spindles which normally enable mitosis and anaphase to proceed with the coordinated precision required to avoid pathological events.
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Waterman-Storer, Clare M., Jean M. Sanger, and Joseph W. Sanger. "Dynamics of membranous organelles in the mitotic apparatus of two cell types: A live-cell, laser-scanning confocal study." Proceedings, annual meeting, Electron Microscopy Society of America 50, no. 1 (August 1992): 696–97. http://dx.doi.org/10.1017/s042482010012388x.
Full textAbstract:
Membranous organelles comprise a large proportion of the living cell's cytoplasmic volume. In interphase, the endoplasmic reticulum (ER), mitochondria, and Golgi apparatus are characterized by a typical distribution in the cytoplasm. When cells enter M-phase, however, the cytoplasmic architecture undergoes dramatic rearrangements, as the nuclear envelope (NE) breaks down and the mitotic spindle assembles. During this process, the typical distribution of the membrane systems must be disrupted, so as to accommodate the cytoplasmic rearrangements. The role of membranous organelles within and around the mitotic apparatus has been a source of active investigation, as well as controversy. It is not known if membranes are an ubiquitous structural element of the spindle. Here, we present a laser-scanning confocal study of membrane dynamics in living mitotic cells' demonstrated by the fluorescent vital membrane dye 3,3″-dihexyloacarbocyanine iodide (DiOC6 (3)). By exploiting the thin focal planes achieved by the confocal microscope, the precise distribution of membranes within the spindle was attained in the two epithelial cell lines; PtK2 and LLC-PK. This study demonstrates that the distribution and quantity of cytoplasmic organelles within the spindle of living cells is cell-type specific.
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Warren, S. L., A. S. Landolfi, C. Curtis, and J. S. Morrow. "Cytostellin: a novel, highly conserved protein that undergoes continuous redistribution during the cell cycle." Journal of Cell Science 103, no. 2 (October 1, 1992): 381–88. http://dx.doi.org/10.1242/jcs.103.2.381.
Full textAbstract:
Cytostellin, a 240 kDa protein, has been purified from mammalian cells by immunoaffinity chromatography using monoclonal antibody H5. Immunofluorescence microscopy shows diffuse and punctate cytostellin immunoreactivity in interphase nuclei. Nuclease digestion and salt extraction are not required to expose the epitope. The onset of prophase is marked by the appearance of multiple intensely immunofluorescent cytostellin-containing ‘bodies’ within the nucleus. Nuclear disassembly is heralded by the movement of cytostellin bodies from the nucleus to multiple positions throughout the cell. Cytostellin bodies in metaphase, anaphase and telophase cells are widely dispersed, including some in cell processes far removed from the mitotic spindle apparatus. However, a distinct subset of larger, more intensely staining bodies surrounds the mitotic spindle apparatus. Cytostellin bodies remain in the cytoplasm of the daughter cells and disappear after the appearance of nascent nuclei. Cytostellin is immunologically distinct from other nuclear and cytoplasmic proteins, and it has been detected by immunoblot analysis in all species tested from yeast to humans. Based upon these findings, we postulate that cytostellin has a cell cycle-dependent function which is conserved in higher and lower eukaryotic cells.
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Compton, D. A., and D. W. Cleveland. "NuMA is required for the proper completion of mitosis." Journal of Cell Biology 120, no. 4 (February 15, 1993): 947–57. http://dx.doi.org/10.1083/jcb.120.4.947.
Full textAbstract:
NuMA is a 236-kD intranuclear protein that during mitosis is distributed into each daughter cell by association with the pericentrosomal domain of the spindle apparatus. The NuMA polypeptide consists of globular head and tail domains separated by a discontinuous 1500 amino acid coiled-coil spacer. Expression of human NuMA lacking its globular head domain results in cells that fail to undergo cytokinesis and assemble multiple small nuclei (micronuclei) in the subsequent interphase despite the appropriate localization of the truncated NuMA to both the nucleus and spindle poles. This dominant phenotype is morphologically identical to that of the tsBN2 cell line that carries a temperature-sensitive mutation in the chromatin-binding protein RCC1. At the restrictive temperature, these cells end mitosis without completing cytokinesis followed by micronucleation in the subsequent interphase. We demonstrate that the wild-type NuMA is degraded in the latest mitotic stages in these mutant cells and that NuMA is excluded from the micronuclei that assemble post-mitotically. Elevation of NuMA levels in these mutant cells by forcing the expression of wild-type NuMA is sufficient to restore post-mitotic assembly of a single normal-sized nucleus. Expression of human NuMA lacking its globular tail domain results in NuMA that fails both to target to interphase nuclei and to bind to the mitotic spindle. In the presence of this mutant, cells transit through mitosis normally, but assemble micronuclei in each daughter cell. The sum of these findings demonstrate that NuMA function is required during mitosis for the terminal phases of chromosome separation and/or nuclear reassembly.