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Journal articles on the topic 'Mitotic spindle'
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1
Lin, Chiou-Hong, Chi-Kuo Hu, and Hsiu-Ming Shih. "Clathrin heavy chain mediates TACC3 targeting to mitotic spindles to ensure spindle stability." Journal of Cell Biology 189, no. 7 (June 21, 2010): 1097–105. http://dx.doi.org/10.1083/jcb.200911120.
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Mitotic spindles play essential roles in chromosome congression and segregation during mitosis. Aurora A regulates spindle assembly in part via phosphorylating human TACC3 on S558, which triggers TACC3 relocalization to mitotic spindles and stabilizes microtubules (MTs). In this study, we identified clathrin heavy chain (CHC) as an adaptor protein to recruit S558-phosphorylated TACC3 onto the spindle during mitosis for MT stabilization. CHC binds phospho-S558 TACC3 via its linker domain and first CHC repeat. CHC depletion or mutation on phospho-TACC3 binding abrogates TACC3 spindle relocalization. Depletion of either or both CHC and TACC3 yields similar defective phenotypes: loss of ch-TOG on spindles, disorganized spindles, and chromosome misalignment with comparable mitotic delay. Our findings elucidate the association between aurora A phosphorylation and spindle apparatus and demonstrate that regulation from aurora A is mediated by CHC in recruiting phospho-TACC3 and subsequently ch-TOG to mitotic spindles.
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Okamoto, Curtis T., Jeana McKinney, and Young Y. Jeng. "Clathrin in mitotic spindles." American Journal of Physiology-Cell Physiology 279, no. 2 (August 1, 2000): C369—C374. http://dx.doi.org/10.1152/ajpcell.2000.279.2.c369.
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Subconfluent cultures of Madin-Darby canine kidney (MDCK) and CV-1 cells were immunostained with two monoclonal antibodies (MAbs), MAb X-22 and MAb 23, against clathrin heavy chain and with polyclonal antiserum against a conserved region of all mammalian clathrin light chains. In interphase MDCK and CV-1 cells, staining by all three antibodies resulted in the characteristic intracellular punctate vesicular and perinuclear staining pattern. In mitotic cells, all three anti-clathrin antibodies strongly stained the mitotic spindle. Staining of clathrin in the mitotic spindle was colocalized with anti-tubulin staining of microtubular arrays in the spindle. Staining of the mitotic spindle was evident in mitotic cells from prometaphase to telophase and in spindles in mitotic cells released from a thymidine-nocodazole block. In CV-1 cells, staining of clathrin in the mitotic spindle was not affected by brefeldin A. On Western blots, clathrin was detected, but not enriched, in isolated spindles. The immunodetection of clathrin in the mitotic spindle may suggest a novel role for clathrin in mitosis. Alternatively, the recruitment of clathrin to the spindle may suggest a novel regulatory mechanism for localization of clathrin in mitotic cells.
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Kim, Seul, Kyoungho Jun, Ye-Hyun Kim, and Jae Sung Kim. "Abstract 1638: Regulation of spindle pole integrity by the MASTL-ENSA-aurora a pathway during mitosis." Cancer Research 84, no. 6_Supplement (March 22, 2024): 1638. http://dx.doi.org/10.1158/1538-7445.am2024-1638.
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Abstract The preservation of spindle pole integrity is crucial for proper spindle assembly and chromosome segregation in mitosis, yet the precise mechanisms governing spindle pole integrity remain elusive. During mitosis, phosphorylated Ensa (p-ENSA) localizes to spindle poles, and the inhibition of ENSA leads to a range of mitotic defects, including misaligned chromosomes, multipolar spindles, asymmetric bipolar spindles, and centrosome aberrations, resulting in a delayed mitotic progression. Remarkably, the mitotic delay induced by ENSA inhibition is alleviated upon depletion of PP2A-B55α, but spindle pole defects persist. Significantly, we have observed an interaction between ENSA and Aurora A during mitosis, and the inhibition of ENSA diminishes Aurora A expression at the mitotic spindle poles. Intriguingly, the injection of MKI-2-sensitized tumors is associated with heightened chromosomal instability and downregulation of the MASTL-ENSA-Aurora A pathway in an orthotopic breast cancer mouse model. These findings offer new insights into the regulation of spindle pole integrity through the MASTL-ENSA-Aurora A pathway during mitosis, emphasizing the pivotal role of ENSA in recruiting Aurora A to the spindle pole, independently of PP2A-B55α. Citation Format: Seul Kim, Kyoungho Jun, Ye-Hyun Kim, Jae Sung Kim. Regulation of spindle pole integrity by the MASTL-ENSA-aurora a pathway during mitosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1638.
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Hofmann, Christian, Iain M. Cheeseman, Bruce L. Goode, Kent L. McDonald, Georjana Barnes, and David G. Drubin. "Saccharomyces cerevisiae Duo1p and Dam1p, Novel Proteins Involved in Mitotic Spindle Function." Journal of Cell Biology 143, no. 4 (November 16, 1998): 1029–40. http://dx.doi.org/10.1083/jcb.143.4.1029.
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In this paper, we describe the identification and characterization of two novel and essential mitotic spindle proteins, Duo1p and Dam1p. Duo1p was isolated because its overexpression caused defects in mitosis and a mitotic arrest. Duo1p was localized by immunofluorescence, by immunoelectron microscopy, and by tagging with green fluorescent protein (GFP), to intranuclear spindle microtubules and spindle pole bodies. Temperature-sensitive duo1 mutants arrest with short spindles. This arrest is dependent on the mitotic checkpoint. Dam1p was identified by two-hybrid analysis as a protein that binds to Duo1p. By expressing a GFP–Dam1p fusion protein in yeast, Dam1p was also shown to be associated with intranuclear spindle microtubules and spindle pole bodies in vivo. As with Duo1p, overproduction of Dam1p caused mitotic defects. Biochemical experiments demonstrated that Dam1p binds directly to microtubules with micromolar affinity. We suggest that Dam1p might localize Duo1p to intranuclear microtubules and spindle pole bodies to provide a previously unrecognized function (or functions) required for mitosis.
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Diggins-Gilicinski, M., L. Solnica-Krezel, T. G. Burland, E. C. Paul, and W. F. Dove. "The localization of the divergent beta 2-tubulin isotype in the microtubular arrays of Physarum polycephalum." Journal of Cell Science 94, no. 2 (October 1, 1989): 217–26. http://dx.doi.org/10.1242/jcs.94.2.217.
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The beta 2-tubulin isotype of Physarum polycephalum is only 83% identical in amino acid sequence with the constitutively expressed beta 1B-tubulin and the myxamoeba-specific beta 1A-tubulin isotypes. A polyclonal antibody specific for beta 2-tubulin was used to monitor the subcellular distribution of the beta 2-tubulin antigen in the mitotic spindle of the mature plasmodium - the sole microtubular array in that stage of Physarum. By immunofluorescence, the beta 2-tubulin antigen was detected throughout this anastral mitotic spindle, at all stages of mitosis. Physarum myxamoebae contain astral mitotic spindles and cytoskeletal microtubules. No beta 2-tubulin antigen was detected in the myxamoebal stage. However, as cultures of myxamoebae developed into plasmodia, the beta 2-tubulin antigen was found in the astral mitotic spindles and cytoskeletons in developing cells. Thus, the presence of the plasmodial beta 2-tubulin isotype in a mitotic spindle does not determine a closed, anastral mitosis.
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Li, Jie, Hiroki Shima, Hironari Nishizawa, Masatoshi Ikeda, Andrey Brydun, Mitsuyo Matsumoto, Hiroki Kato, et al. "Phosphorylation of BACH1 switches its function from transcription factor to mitotic chromosome regulator and promotes its interaction with HMMR." Biochemical Journal 475, no. 5 (March 15, 2018): 981–1002. http://dx.doi.org/10.1042/bcj20170520.
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The transcription repressor BACH1 performs mutually independent dual roles in transcription regulation and chromosome alignment during mitosis by supporting polar ejection force of mitotic spindle. We now found that the mitotic spindles became oblique relative to the adhesion surface following endogenous BACH1 depletion in HeLa cells. This spindle orientation rearrangement was rescued by re-expression of BACH1 depending on its interactions with HMMR and CRM1, both of which are required for the positioning of mitotic spindle, but independently of its DNA-binding activity. A mass spectrometry analysis of BACH1 complexes in interphase and M phase revealed that BACH1 lost during mitosis interactions with proteins involved in chromatin and gene expression but retained interactions with HMMR and its known partners including CHICA. By analyzing BACH1 modification using stable isotope labeling with amino acids in cell culture, mitosis-specific phosphorylations of BACH1 were observed, and mutations of these residues abolished the activity of BACH1 to restore mitotic spindle orientation in knockdown cells and to interact with HMMR. Detailed histological analysis of Bach1-deficient mice revealed lengthening of the epithelial fold structures of the intestine. These observations suggest that BACH1 performs stabilization of mitotic spindle orientation together with HMMR and CRM1 in mitosis, and that the cell cycle-specific phosphorylation switches the transcriptional and mitotic functions of BACH1.
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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.
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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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Woolner, Sarah, Lori L. O'Brien, Christiane Wiese, and William M. Bement. "Myosin-10 and actin filaments are essential for mitotic spindle function." Journal of Cell Biology 182, no. 1 (July 7, 2008): 77–88. http://dx.doi.org/10.1083/jcb.200804062.
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Mitotic spindles are microtubule-based structures responsible for chromosome partitioning during cell division. Although the roles of microtubules and microtubule-based motors in mitotic spindles are well established, whether or not actin filaments (F-actin) and F-actin–based motors (myosins) are required components of mitotic spindles has long been controversial. Based on the demonstration that myosin-10 (Myo10) is important for assembly of meiotic spindles, we assessed the role of this unconventional myosin, as well as F-actin, in mitotic spindles. We find that Myo10 localizes to mitotic spindle poles and is essential for proper spindle anchoring, normal spindle length, spindle pole integrity, and progression through metaphase. Furthermore, we show that F-actin localizes to mitotic spindles in dynamic cables that surround the spindle and extend between the spindle and the cortex. Remarkably, although proper anchoring depends on both F-actin and Myo10, the requirement for Myo10 in spindle pole integrity is F-actin independent, whereas F-actin and Myo10 actually play antagonistic roles in maintenance of spindle length.
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Compton, D. A., and C. Luo. "Mutation of the predicted p34cdc2 phosphorylation sites in NuMA impair the assembly of the mitotic spindle and block mitosis." Journal of Cell Science 108, no. 2 (February 1, 1995): 621–33. http://dx.doi.org/10.1242/jcs.108.2.621.
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NuMA is a 236 kDa intranuclear protein that is distributed into each daughter cell during mitosis through association with the pericentrosomal region of the mitotic spindle. NuMA's interaction with the microtubules of the mitotic spindle is mediated through its 45 kDa carboxyl-terminal globular tail, and there is indirect evidence suggesting that NuMA's interaction with the mitotic spindle is controlled in a mitosis-specific manner. Consistent with this evidence is the fact that all four of the predicted p34cdc2 consensus phosphorylation sites in the NuMA protein are located in the carboxyl-terminal globular domain, and we demonstrate here that NuMA is phosphorylated in a mitosis-specific fashion in vivo. To test if the predicted p34cdc2 phosphorylation sites are necessary for NuMA's mitosis-specific interaction with the mitotic spindle, we have introduced mutations into the human NuMA cDNA that convert these predicted p34cdc2 phosphorylation sites from threonine or serine residues into alanine residues, and subsequently determined the cell cycle-dependent localization of these altered NuMA proteins following their expression in tissue culture cells. While none of these specific mutations in the NuMA sequence alters the faithful targeting of the protein into the interphase nucleus, mutation of threonine residue 2040 alone or in combination with mutations in other potential p34cdc2 phosphorylation sites abolishes NuMA's ability to associate normally with the microtubules of the mitotic spindle. Instead of binding to the mitotic spindle these mutant forms of NuMA concentrate at the plasma membrane of the mitotic cell. Cells expressing these mutant forms of NuMA have disorganized mitotic spindles, fail to complete cytokinesis normally, and assemble micronuclei in the subsequent interphase. These data suggest that NuMA's interaction with the microtubules of the mitotic spindle is controlled by cell cycle-dependent phosphorylation in addition to differential subcellular compartmentalization, and the characteristics of the dominant negative phenotype induced by these mutant forms of NuMA support a role for NuMA in the organization of the mitotic spindle apparatus.
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Lacroix, Benjamin, and Julien Dumont. "Spatial and Temporal Scaling of Microtubules and Mitotic Spindles." Cells 11, no. 2 (January 12, 2022): 248. http://dx.doi.org/10.3390/cells11020248.
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During cell division, the mitotic spindle, a macromolecular structure primarily comprised of microtubules, drives chromosome alignment and partitioning between daughter cells. Mitotic spindles can sense cellular dimensions in order to adapt their length and mass to cell size. This scaling capacity is particularly remarkable during early embryo cleavage when cells divide rapidly in the absence of cell growth, thus leading to a reduction of cell volume at each division. Although mitotic spindle size scaling can occur over an order of magnitude in early embryos, in many species the duration of mitosis is relatively short, constant throughout early development and independent of cell size. Therefore, a key challenge for cells during embryo cleavage is not only to assemble a spindle of proper size, but also to do it in an appropriate time window which is compatible with embryo development. How spatial and temporal scaling of the mitotic spindle is achieved and coordinated with the duration of mitosis remains elusive. In this review, we will focus on the mechanisms that support mitotic spindle spatial and temporal scaling over a wide range of cell sizes and cellular contexts. We will present current models and propose alternative mechanisms allowing cells to spatially and temporally coordinate microtubule and mitotic spindle assembly.
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Kapoor, Tarun M., Thomas U. Mayer, Margaret L. Coughlin, and Timothy J. Mitchison. "Probing Spindle Assembly Mechanisms with Monastrol, a Small Molecule Inhibitor of the Mitotic Kinesin, Eg5." Journal of Cell Biology 150, no. 5 (September 4, 2000): 975–88. http://dx.doi.org/10.1083/jcb.150.5.975.
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Monastrol, a cell-permeable small molecule inhibitor of the mitotic kinesin, Eg5, arrests cells in mitosis with monoastral spindles. Here, we use monastrol to probe mitotic mechanisms. We find that monastrol does not inhibit progression through S and G2 phases of the cell cycle or centrosome duplication. The mitotic arrest due to monastrol is also rapidly reversible. Chromosomes in monastrol-treated cells frequently have both sister kinetochores attached to microtubules extending to the center of the monoaster (syntelic orientation). Mitotic arrest–deficient protein 2 (Mad2) localizes to a subset of kinetochores, suggesting the activation of the spindle assembly checkpoint in these cells. Mad2 localizes to some kinetochores that have attached microtubules in monastrol-treated cells, indicating that kinetochore microtubule attachment alone may not satisfy the spindle assembly checkpoint. Monastrol also inhibits bipolar spindle formation in Xenopus egg extracts. However, it does not prevent the targeting of Eg5 to the monoastral spindles that form. Imaging bipolar spindles disassembling in the presence of monastrol allowed direct observations of outward directed forces in the spindle, orthogonal to the pole-to-pole axis. Monastrol is thus a useful tool to study mitotic processes, detection and correction of chromosome malorientation, and contributions of Eg5 to spindle assembly and maintenance.
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Endow, S. A., and D. J. Komma. "Use of GFP Fusions to a Microtubule Motor Protein to Analyze Spindle Dynamics in Live Oocytes and Embryos of Drosophila." Microscopy and Microanalysis 3, S2 (August 1997): 127–28. http://dx.doi.org/10.1017/s1431927600007522.
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Ncd is a kinesin-related microtubule motor protein of Drosophila that plays essential roles in spindle assembly and function during meiosis in oocytes and mitosis in early embryos. Antibody staining experiments have localized the Ned motor protein to spindle fibers and spindle poles throughout the meiotic and early mitotic divisions, demonstrating that Ncd is a spindle motor.We have made ncd-gfp gene fusions with wild-type and S65T gfp and expressed the chimaeric genes in Drosophila to target GFP to the spindle. Transgenic Drosophila carrying the ncd-gfp gene fusions in an ncd null mutant background are wild type with respect to chromosome segregation, indicating that the Ncd-GFP fusion proteins can replace the function of wild-type Ncd. The Ncd-GFP fusion proteins in transgenic Drosophila are expressed under the regulation of the native ncd promoter.Analysis of live Drosophila oocytes and early embryos shows green fluorescent spindles, demonstrating association of Ncd-GFP with meiotic and mitotic spindles. In mitotic spindles, Ncd-GFP localizes to centrosomes (Fig. 1a) and spindle fibers (Fig. 1b).
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Yukawa, Masashi, Tomoki Kawakami, Masaki Okazaki, Kazunori Kume, Ngang Heok Tang, and Takashi Toda. "A microtubule polymerase cooperates with the kinesin-6 motor and a microtubule cross-linker to promote bipolar spindle assembly in the absence of kinesin-5 and kinesin-14 in fission yeast." Molecular Biology of the Cell 28, no. 25 (December 2017): 3647–59. http://dx.doi.org/10.1091/mbc.e17-08-0497.
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Accurate chromosome segregation relies on the bipolar mitotic spindle. In many eukaryotes, spindle formation is driven by the plus-end–directed motor kinesin-5 that generates outward force to establish spindle bipolarity. Its inhibition leads to the emergence of monopolar spindles with mitotic arrest. Intriguingly, simultaneous inactivation of the minus-end–directed motor kinesin-14 restores spindle bipolarity in many systems. Here we show that in fission yeast, three independent pathways contribute to spindle bipolarity in the absence of kinesin-5/Cut7 and kinesin-14/Pkl1. One is kinesin-6/Klp9 that engages with spindle elongation once short bipolar spindles assemble. Klp9 also ensures the medial positioning of anaphase spindles to prevent unequal chromosome segregation. Another is the Alp7/TACC-Alp14/TOG microtubule polymerase complex. Temperature-sensitive alp7cut7pkl1 mutants are arrested with either monopolar or very short spindles. Forced targeting of Alp14 to the spindle pole body is sufficient to render alp7cut7pkl1 triply deleted cells viable and promote spindle assembly, indicating that Alp14-mediated microtubule polymerization from the nuclear face of the spindle pole body could generate outward force in place of Cut7 during early mitosis. The third pathway involves the Ase1/PRC1 microtubule cross-linker that stabilizes antiparallel microtubules. Our study, therefore, unveils multifaceted interplay among kinesin-dependent and -independent pathways leading to mitotic bipolar spindle assembly.
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Encalada, Sandra E., John Willis, Rebecca Lyczak, and Bruce Bowerman. "A Spindle Checkpoint Functions during Mitosis in the Early Caenorhabditis elegans Embryo." Molecular Biology of the Cell 16, no. 3 (March 2005): 1056–70. http://dx.doi.org/10.1091/mbc.e04-08-0712.
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During mitosis, chromosome segregation is regulated by a spindle checkpoint mechanism. This checkpoint delays anaphase until all kinetochores are captured by microtubules from both spindle poles, chromosomes congress to the metaphase plate, and the tension between kinetochores and their attached microtubules is properly sensed. Although the spindle checkpoint can be activated in many different cell types, the role of this regulatory mechanism in rapidly dividing embryonic animal cells has remained controversial. Here, using time-lapse imaging of live embryonic cells, we show that chemical or mutational disruption of the mitotic spindle in early Caenorhabditis elegans embryos delays progression through mitosis. By reducing the function of conserved checkpoint genes in mutant embryos with defective mitotic spindles, we show that these delays require the spindle checkpoint. In the absence of a functional checkpoint, more severe defects in chromosome segregation are observed in mutants with abnormal mitotic spindles. We also show that the conserved kinesin CeMCAK, the CENP-F-related proteins HCP-1 and HCP-2, and the core kinetochore protein CeCENP-C all are required for this checkpoint. Our analysis indicates that spindle checkpoint mechanisms are functional in the rapidly dividing cells of an early animal embryo and that this checkpoint can prevent chromosome segregation defects during mitosis.
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Lucena, Rafael, Noah Dephoure, Steve P. Gygi, Douglas R. Kellogg, Victor A. Tallada, Rafael R. Daga, and Juan Jimenez. "Nucleocytoplasmic transport in the midzone membrane domain controls yeast mitotic spindle disassembly." Journal of Cell Biology 209, no. 3 (May 11, 2015): 387–402. http://dx.doi.org/10.1083/jcb.201412144.
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During each cell cycle, the mitotic spindle is efficiently assembled to achieve chromosome segregation and then rapidly disassembled as cells enter cytokinesis. Although much has been learned about assembly, how spindles disassemble at the end of mitosis remains unclear. Here we demonstrate that nucleocytoplasmic transport at the membrane domain surrounding the mitotic spindle midzone, here named the midzone membrane domain (MMD), is essential for spindle disassembly in Schizosaccharomyces pombe cells. We show that, during anaphase B, Imp1-mediated transport of the AAA-ATPase Cdc48 protein at the MMD allows this disassembly factor to localize at the spindle midzone, thereby promoting spindle midzone dissolution. Our findings illustrate how a separate membrane compartment supports spindle disassembly in the closed mitosis of fission yeast.
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Rahmani, Zohra, Mary E. Gagou, Christophe Lefebvre, Doruk Emre, and Roger E. Karess. "Separating the spindle, checkpoint, and timer functions of BubR1." Journal of Cell Biology 187, no. 5 (November 30, 2009): 597–605. http://dx.doi.org/10.1083/jcb.200905026.
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BubR1 performs several roles during mitosis, affecting the spindle assembly checkpoint (SAC), mitotic timing, and spindle function, but the interdependence of these functions is unclear. We have analyzed in Drosophila melanogaster the mitotic phenotypes of kinase-dead (KD) BubR1 and BubR1 lacking the N-terminal KEN box. bubR1-KD individuals have a robust SAC but abnormal spindles with thin kinetochore fibers, suggesting that the kinase activity modulates microtubule capture and/or dynamics but is relatively dispensable for SAC function. In contrast, bubR1-KEN flies have normal spindles but no SAC. Nevertheless, mitotic timing is normal as long as Mad2 is present. Thus, the SAC, timer, and spindle functions of BubR1 are substantially separable. Timing is shorter in bubR1-KEN mad2 double mutants, yet in these flies, lacking both critical SAC components, chromosomes still segregate accurately, reconfirming that in Drosophila, reliable mitosis does not need the SAC.
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Nislow, C., C. Sellitto, R. Kuriyama, and J. R. McIntosh. "A monoclonal antibody to a mitotic microtubule-associated protein blocks mitotic progression." Journal of Cell Biology 111, no. 2 (August 1, 1990): 511–22. http://dx.doi.org/10.1083/jcb.111.2.511.
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A monoclonal antibody raised against mitotic spindles isolated from CHO cells ([CHO1], Sellitto, C., and R. Kuriyama. 1988. J. Cell Biol. 106:431-439) identifies an epitope that resides on polypeptides of 95 and 105 kD and is localized in the spindles of diverse organisms. The antigen is distributed throughout the spindle at metaphase but becomes concentrated in a progressively narrower zone on either side of the spindle midplane as anaphase progresses. Microinjection of CHO1, either as an ascites fluid or as purified IgM, results in mitotic inhibition in a stage-specific and dose-dependent manner. Parallel control injections with nonimmune IgMs do not yield significant mitotic inhibition. Immunofluorescence analysis of injected cells reveals that those which complete mitosis display normal localization of CHO1, whereas arrested cells show no specific localization of the CHO1 antigen within the spindle. Immunoelectron microscopic images of such arrested cells indicate aberrant microtubule organization. The CHO1 antigen in HeLa cell extracts copurifies with taxol-stabilized microtubules. Neither of the polypeptides bearing the antigen is extracted from microtubules by ATP or GTP, but both are approximately 60% extracted with 0.5 M NaCl. Sucrose gradient analysis reveals that the antigens sediment at approximately 11S. The CHO 1 antigen appears to be a novel mitotic MAP whose proper distribution within the spindle is required for mitosis. The properties of the antigen(s) suggest that the corresponding protein(s) are part of the mechanism that holds the antiparallel microtubules of the two interdigitating half spindles together during anaphase.
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Brady, R. C., F. Cabral, and J. R. Dedman. "Identification of a 52-kD calmodulin-binding protein associated with the mitotic spindle apparatus in mammalian cells." Journal of Cell Biology 103, no. 5 (November 1, 1986): 1855–61. http://dx.doi.org/10.1083/jcb.103.5.1855.
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A pool of 10 calmodulin-binding proteins (CBPs) was isolated from Chinese hamster ovary (CHO) cells via calmodulin (CaM)-Sepharose affinity chromatography. One of these ten isolated CBPs with a molecular mass of 52 kD was also found to be present in isolated CHO cell mitotic spindles. Affinity-purified antibodies generated against this pool of isolated CBPs recognize a single 52-kD protein in isolated CHO cell mitotic spindles by immunoblot analysis. Immunofluorescence examination of CHO, 3T3, NRK, PTK-2, and HeLa cells resulted in a distinct pattern of mitotic spindle fluorescence. The localization pattern of this 52-kD CBP directly parallels that of CaM in the spindle apparatus throughout the various stages of mitosis. Interestingly, there was no association of this 52-kD CBP with cytoplasmic microtubules. As is the case with CaM, the localization pattern of the 52-kD CBP in interphase cells is diffuse within the cytoplasm and is not associated with any discrete, cellular structures. This 52-kD CBP appears to represent the first mitotic spindle-specific calmodulin-binding protein identified and represents an initial step toward the ultimate determination of CaM function in the mitotic spindle apparatus.
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Vaisberg, E. A., M. P. Koonce, and J. R. McIntosh. "Cytoplasmic dynein plays a role in mammalian mitotic spindle formation." Journal of Cell Biology 123, no. 4 (November 15, 1993): 849–58. http://dx.doi.org/10.1083/jcb.123.4.849.
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The formation and functioning of a mitotic spindle depends not only on the assembly/disassembly of microtubules but also on the action of motor enzymes. Cytoplasmic dynein has been localized to spindles, but whether or how it functions in mitotic processes is not yet known. We have cloned and expressed DNA fragments that encode the putative ATP-hydrolytic sites of the cytoplasmic dynein heavy chain from HeLa cells and from Dictyostelium. Monospecific antibodies have been raised to the resulting polypeptides, and these inhibit dynein motor activity in vitro. Their injection into mitotic mammalian cells blocks the formation of spindles in prophase or during recovery from nocodazole treatment at later stages of mitosis. Cells become arrested with unseparated centrosomes and form monopolar spindles. The injected antibodies have no detectable effect on chromosome attachment to a bipolar spindle or on motions during anaphase. These data suggest that cytoplasmic dynein plays a unique and important role in the initial events of bipolar spindle formation, while any later roles that it may play are redundant. Possible mechanisms of dynein's involvement in mitosis are discussed.
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Hatsumi, M., and S. A. Endow. "Mutants of the microtubule motor protein, nonclaret disjunctional, affect spindle structure and chromosome movement in meiosis and mitosis." Journal of Cell Science 101, no. 3 (March 1, 1992): 547–59. http://dx.doi.org/10.1242/jcs.101.3.547.
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The Drosophila microtubule motor protein, nonclaret disjunctional (ncd), is required for proper chromosome distribution in meiosis and mitosis. We have examined the meiotic and mitotic divisions in wild-type Drosophila oocytes and early embryos, and the effects of three ncd mutants (cand, ncd and ncdD) on spindle structure and chromosome movement. The ncd mutants cause abnormalities in spindle structure early in meiosis I, and abnormal chromosome configurations throughout meiosis I and II. Defective divisions continue in early embryos of the motor null mutant, cand, with abnormal early mitotic spindles. The effects of mutants on spindle structure suggest that ncd is required for proper meiotic spindle assembly, and may play a role in forming or maintaining spindle poles in meiosis. The disruption of normal meiotic and mitotic chromosome distribution by ncd mutants can be attributed to its role as a spindle motor, although a role for ncd as a chromosome-associated motor protein is not excluded. The ncd motor protein functions not only in meiosis, but also performs an active role in the early mitotic divisions of the embryo.
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Fielding, Andrew B., Iveta Dobreva, Paul C. McDonald, Leonard J. Foster, and Shoukat Dedhar. "Integrin-linked kinase localizes to the centrosome and regulates mitotic spindle organization." Journal of Cell Biology 180, no. 4 (February 18, 2008): 681–89. http://dx.doi.org/10.1083/jcb.200710074.
Full textAbstract:
Integrin-linked kinase (ILK) is a serine-threonine kinase and scaffold protein with well defined roles in focal adhesions in integrin-mediated cell adhesion, spreading, migration, and signaling. Using mass spectrometry–based proteomic approaches, we identify centrosomal and mitotic spindle proteins as interactors of ILK. α- and β-tubulin, ch-TOG (XMAP215), and RUVBL1 associate with ILK and colocalize with it to mitotic centrosomes. Inhibition of ILK activity or expression induces profound apoptosis-independent defects in the organization of the mitotic spindle and DNA segregation. ILK fails to localize to the centrosomes of abnormal spindles in RUVBL1-depleted cells. Additionally, depletion of ILK expression or inhibition of its activity inhibits Aurora A–TACC3/ch-TOG interactions, which are essential for spindle pole organization and mitosis. These data demonstrate a critical and unexpected function for ILK in the organization of centrosomal protein complexes during mitotic spindle assembly and DNA segregation.
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Zulkipli, Ihsan, Joanna Clark, Madeleine Hart, Roshan L. Shrestha, Parveen Gul, David Dang, Tami Kasichiwin, Izabela Kujawiak, Nishanth Sastry, and Viji M. Draviam. "Spindle rotation in human cells is reliant on a MARK2-mediated equatorial spindle-centering mechanism." Journal of Cell Biology 217, no. 9 (June 25, 2018): 3057–70. http://dx.doi.org/10.1083/jcb.201804166.
Full textAbstract:
The plane of cell division is defined by the final position of the mitotic spindle. The spindle is pulled and rotated to the correct position by cortical dynein. However, it is unclear how the spindle’s rotational center is maintained and what the consequences of an equatorially off centered spindle are in human cells. We analyzed spindle movements in 100s of cells exposed to protein depletions or drug treatments and uncovered a novel role for MARK2 in maintaining the spindle at the cell’s geometric center. Following MARK2 depletion, spindles glide along the cell cortex, leading to a failure in identifying the correct division plane. Surprisingly, spindle off centering in MARK2-depleted cells is not caused by excessive pull by dynein. We show that MARK2 modulates mitotic microtubule growth and length and that codepleting mitotic centromere-associated protein (MCAK), a microtubule destabilizer, rescues spindle off centering in MARK2-depleted cells. Thus, we provide the first insight into a spindle-centering mechanism needed for proper spindle rotation and, in turn, the correct division plane in human cells.
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Kwon, Mijung, Sandra Morales-Mulia, Ingrid Brust-Mascher, Gregory C. Rogers, David J. Sharp, and Jonathan M. Scholey. "The Chromokinesin, KLP3A, Drives Mitotic Spindle Pole Separation during Prometaphase and Anaphase and Facilitates Chromatid Motility." Molecular Biology of the Cell 15, no. 1 (January 2004): 219–33. http://dx.doi.org/10.1091/mbc.e03-07-0489.
Full textAbstract:
Mitosis requires the concerted activities of multiple microtubule (MT)-based motor proteins. Here we examined the contribution of the chromokinesin, KLP3A, to mitotic spindle morphogenesis and chromosome movements in Drosophila embryos and cultured S2 cells. By immunofluorescence, KLP3A associates with nonfibrous punctae that concentrate in nuclei and display MT-dependent associations with spindles. These punctae concentrate in indistinct domains associated with chromosomes and central spindles and form distinct bands associated with telophase midbodies. The functional disruption of KLP3A by antibodies or dominant negative proteins in embryos, or by RNA interference (RNAi) in S2 cells, does not block mitosis but produces defects in mitotic spindles. Time-lapse confocal observations of mitosis in living embryos reveal that KLP3A inhibition disrupts the organization of interpolar (ip) MTs and produces short spindles. Kinetic analysis suggests that KLP3A contributes to spindle pole separation during the prometaphase-to-metaphase transition (when it antagonizes Ncd) and anaphase B, to normal rates of chromatid motility during anaphase A, and to the proper spacing of daughter nuclei during telophase. We propose that KLP3A acts on MTs associated with chromosome arms and the central spindle to organize ipMT bundles, to drive spindle pole separation and to facilitate chromatid motility.
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Wilson, P. G., M. T. Fuller, and G. G. Borisy. "Monastral bipolar spindles: implications for dynamic centrosome organization." Journal of Cell Science 110, no. 4 (February 15, 1997): 451–64. http://dx.doi.org/10.1242/jcs.110.4.451.
Full textAbstract:
Implicit to all models for mitotic spindle assembly is the view that centrosomes are essentially permanent structures. Yet, immunofluorescence revealed that spindles in larval brains of urchin mutants in Drosophila were frequently monastral but bipolar; the astral pole contained a centrosome while the opposing anastral pole showed neither gamma tubulin nor a radial array of astral microtubules. Thus, mutations in the urchin gene seem to uncouple centrosome organization and spindle bipolarity in mitotic cells. Hypomorphic mutants showed a high frequency of monastral bipolar spindles but low frequencies of polyploidy, suggesting that monastral bipolar spindles might be functional. To test this hypothesis, we performed pedigree analysis of centrosome distribution and spindle structure in the four mitotic divisions of gonial cells. Prophase gonial cells showed two centrosomes, suggesting cells entered mitosis with the normal number of centrosomes and that centrosomes separated during prophase. Despite a high frequency of monastral bipolar spindles, the end products of the four mitotic divisions were equivalent in size and chromatin content. These results indicate that monastral bipolar spindles are functional and that the daughter cell derived from the anastral pole can assemble a functional bipolar spindle in the subsequent cell cycle. Cell proliferation despite high frequencies of monastral bipolar spindles can be explained if centrosome structure in mitotic cells is dynamic, allowing transient and benign disorganization of pericentriolar components. Since urchin proved to be allelic to KLP61F which encodes a kinesin related motor protein (Heck et al. (1993) J. Cell Biol. 123, 665–671), our results suggest that motors influence the dynamic organization of centrosomes.
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Gallaud, Emmanuel, Renaud Caous, Aude Pascal, Franck Bazile, Jean-Philippe Gagné, Sébastien Huet, Guy G. Poirier, Denis Chrétien, Laurent Richard-Parpaillon, and Régis Giet. "Ensconsin/Map7 promotes microtubule growth and centrosome separation in Drosophila neural stem cells." Journal of Cell Biology 204, no. 7 (March 31, 2014): 1111–21. http://dx.doi.org/10.1083/jcb.201311094.
Full textAbstract:
The mitotic spindle is crucial to achieve segregation of sister chromatids. To identify new mitotic spindle assembly regulators, we isolated 855 microtubule-associated proteins (MAPs) from Drosophila melanogaster mitotic or interphasic embryos. Using RNAi, we screened 96 poorly characterized genes in the Drosophila central nervous system to establish their possible role during spindle assembly. We found that Ensconsin/MAP7 mutant neuroblasts display shorter metaphase spindles, a defect caused by a reduced microtubule polymerization rate and enhanced by centrosome ablation. In agreement with a direct effect in regulating spindle length, Ensconsin overexpression triggered an increase in spindle length in S2 cells, whereas purified Ensconsin stimulated microtubule polymerization in vitro. Interestingly, ensc-null mutant flies also display defective centrosome separation and positioning during interphase, a phenotype also detected in kinesin-1 mutants. Collectively, our results suggest that Ensconsin cooperates with its binding partner Kinesin-1 during interphase to trigger centrosome separation. In addition, Ensconsin promotes microtubule polymerization during mitosis to control spindle length independent of Kinesin-1.
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Rieder, C. L., G. Rupp, S. P. Alexander, and R. B. Nicklas. "Three-dimensional reconstruction studies of mitotic spindle ultrastructure." Proceedings, annual meeting, Electron Microscopy Society of America 46 (1988): 32–33. http://dx.doi.org/10.1017/s0424820100102249.
Full textAbstract:
The mitotic spindle is composed chiefly of microtubules (MTs) and functions to equally distribute the replicated chromosomes to daughter cells. Spindles are generated from an interaction between the spindle poles and kinetochores. The former are responsible for generating spindle MTs while the latter act as sites for attaching the chromosomes to the MTs. As noted by McIntosh the emphasis of most mitotic investigations is structural “because the spindle is a kind of machine, and numerous structural questions are obvious as one tries to understand a device which converts chemical energy into mechanical work.”During the course of our mitosis studies it was frequently necessary to reconstruct the three-dimensional (3D) ultrastructure of spindles from serial sections. To do this we utilized the STERECON system developed at the Albany HVEM facility. Briefly, prints of serial sections are enlarged to an optimum final magnification. For each section, profiles are drawn on clear plastic sheets outlining the chromosomes, mitochondria, and spindle MTs.
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Wordeman, L., K. L. McDonald, and W. Z. Cande. "The distribution of cytoplasmic microtubules throughout the cell cycle of the centric diatom Stephanopyxis turris: their role in nuclear migration and positioning the mitotic spindle during cytokinesis." Journal of Cell Biology 102, no. 5 (May 1, 1986): 1688–98. http://dx.doi.org/10.1083/jcb.102.5.1688.
Full textAbstract:
The cell cycle of the marine centric diatom Stephanopyxis turris consists of a series of spatially and temporally well-ordered events. We have used immunofluorescence microscopy to examine the role of cytoplasmic microtubules in these events. At interphase, microtubules radiate out from the microtubule-organizing center, forming a network around the nucleus and extending much of the length and breadth of the cell. As the cell enters mitosis, this network breaks down and a highly ordered mitotic spindle is formed. Peripheral microtubule bundles radiate out from each spindle pole and swing out and away from the central spindle during anaphase. Treatment of synchronized cells with 2.5 X 10(-8) M Nocodazole reversibly inhibited nuclear migration concurrent with the disappearance of the extensive cytoplasmic microtubule arrays associated with migrating nuclei. Microtubule arrays and mitotic spindles that reformed after the drug was washed out appeared normal. In contrast, cells treated with 5.0 X 10(-8) M Nocodazole were not able to complete nuclear migration after the drug was washed out and the mitotic spindles that formed were multipolar. Normal and multipolar spindles that were displaced toward one end of the cell by the drug treatment had no effect on the plane of division during cytokinesis. The cleavage furrow always bisected the cell regardless of the position of the mitotic spindle, resulting in binucleate/anucleate daughter cells. This suggests that in S. turris, unlike animal cells, the location of the plane of division is cortically determined before mitosis.
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McNally, Karen, Anjon Audhya, Karen Oegema, and Francis J. McNally. "Katanin controls mitotic and meiotic spindle length." Journal of Cell Biology 175, no. 6 (December 18, 2006): 881–91. http://dx.doi.org/10.1083/jcb.200608117.
Full textAbstract:
Accurate control of spindle length is a conserved feature of eukaryotic cell division. Lengthening of mitotic spindles contributes to chromosome segregation and cytokinesis during mitosis in animals and fungi. In contrast, spindle shortening may contribute to conservation of egg cytoplasm during female meiosis. Katanin is a microtubule-severing enzyme that is concentrated at mitotic and meiotic spindle poles in animals. We show that inhibition of katanin slows the rate of spindle shortening in nocodazole-treated mammalian fibroblasts and in untreated Caenorhabditis elegans meiotic embryos. Wild-type C. elegans meiotic spindle shortening proceeds through an early katanin-independent phase marked by increasing microtubule density and a second, katanin-dependent phase that occurs after microtubule density stops increasing. In addition, double-mutant analysis indicated that γ-tubulin–dependent nucleation and microtubule severing may provide redundant mechanisms for increasing microtubule number during the early stages of meiotic spindle assembly.
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Chang, Paul, Margaret Coughlin, and Timothy J. Mitchison. "Interaction between Poly(ADP-ribose) and NuMA Contributes to Mitotic Spindle Pole Assembly." Molecular Biology of the Cell 20, no. 21 (November 2009): 4575–85. http://dx.doi.org/10.1091/mbc.e09-06-0477.
Full textAbstract:
Poly(ADP-ribose) (pADPr), made by PARP-5a/tankyrase-1, localizes to the poles of mitotic spindles and is required for bipolar spindle assembly, but its molecular function in the spindle is poorly understood. To investigate this, we localized pADPr at spindle poles by immuno-EM. We then developed a concentrated mitotic lysate system from HeLa cells to probe spindle pole assembly in vitro. Microtubule asters assembled in response to centrosomes and Ran-GTP in this system. Magnetic beads coated with pADPr, extended from PARP-5a, also triggered aster assembly, suggesting a functional role of the pADPr in spindle pole assembly. We found that PARP-5a is much more active in mitosis than interphase. We used mitotic PARP-5a, self-modified with pADPr chains, to capture mitosis-specific pADPr-binding proteins. Candidate binding proteins included the spindle pole protein NuMA previously shown to bind to PARP-5a directly. The rod domain of NuMA, expressed in bacteria, bound directly to pADPr. We propose that pADPr provides a dynamic cross-linking function at spindle poles by extending from covalent modification sites on PARP-5a and NuMA and binding noncovalently to NuMA and that this function helps promote assembly of exactly two poles.
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Rodionov, V. I., V. I. Gelfand, and G. G. Borisy. "Kinesin-like molecules involved in spindle formation." Journal of Cell Science 106, no. 4 (December 1, 1993): 1179–88. http://dx.doi.org/10.1242/jcs.106.4.1179.
Full textAbstract:
To study the possible involvement of kinesin-like molecules in mitosis a polyclonal antibody against the head domain of Drosophila kinesin heavy chain (HD antibody) was microinjected into PtK1 cells at the prophase-prometaphase transition. Progress of the cell through mitosis was recorded for subsequent detailed analysis. Cells injected with pre-immune IgG progressed through mitosis at rates similar to those for noninjected cells. After HD antibody injections, chromosomes failed to congress to an equatorial plane and cells failed to form a bipolar spindle. Rather, the spindle poles came together, resulting in a monopolar-like configuration with chromosomes arranged about the poles in a rosette. Sometimes the monopolar array moved to the margin of the cell in a way similar to anaphase B movement in normal cells. Antibody-injected cells progressed into the next cell cycle as evidenced by chromosome decondensation and nuclear envelope reformation. Anti-tubulin immunofluorescence confirmed the presence of a radial monopolar array of microtubules in injected cells. HD antibody stained in a punctate pattern in interphase and the spindle region in mitotic PtK1 cells. The antibody also reacted with spindle fibers of isolated mitotic CHO spindles and with kinetochores of isolated CHO chromosomes. Immunoblotting indicated that the major component recognized by the antibody is the 120 kDa kinesin heavy chain. At higher protein loads the antibody recognized also a 34 kDa polypeptide in PtK1 cell extracts, a 135 kDa polypeptide in a preparation of CHO spindles and a 300 kDa polypeptide in a preparation of CHO mitotic chromosomes. We conclude that a kinesin-like molecule is important for the formation and/or maintenance of the structure of mitotic spindle.
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Baskin, T. I., and W. Z. Cande. "Kinetic analysis of mitotic spindle elongation in vitro." Journal of Cell Science 97, no. 1 (September 1, 1990): 79–89. http://dx.doi.org/10.1242/jcs.97.1.79.
Full textAbstract:
Studies of mitotic spindle elongation in vitro using populations of diatom spindles visualized with immunofluorescence microscopy have shown that the two interdigitating half-spindles are driven apart by an ATP-dependent process that generates force in the zone of overlap between half-spindles. To characterize further the system responsible for spindle elongation, we observed spindle elongation directly with polarized light or phase-contrast video-microscopy. We report that the kinetics of spindle elongation versus time are linear. A constant rate of spindle elongation occurs despite the continuous decrease in length of the zone of overlap between half-spindles. The average rate of spindle elongation varies as a function of treatment, and rates measured match spindle elongation rates measured in vivo. When spindles elongated in the presence of polymerizing tubulin (from bovine brain), the extent of elongation was greater than the original zone of half-spindle overlap, but the rate of elongation was constant. No component of force due to tubulin polymerization was found. The total elongation observed in the presence of added tubulin could exceed a doubling of original spindle length, matching the elongation in the intact diatom. The linear rate of spindle elongation in vitro suggests that the force transducer for anaphase B is a mechanochemical ATPase, analogous to dynein or myosin, and that the force for spindle elongation does not arise from stored energy, e.g. in an elastic matrix in the midzone. Additionally, on the basis of observations described here, we conclude that the force-transduction system for spindle elongation must be able to remain in the zone of microtubule overlap during the sliding apart of half-spindles, and that the transducer can generate force between microtubules that are not strictly antiparallel.
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Green, Rebecca A., Roy Wollman, and Kenneth B. Kaplan. "APC and EB1 Function Together in Mitosis to Regulate Spindle Dynamics and Chromosome Alignment." Molecular Biology of the Cell 16, no. 10 (October 2005): 4609–22. http://dx.doi.org/10.1091/mbc.e05-03-0259.
Full textAbstract:
Recently, we have shown that a cancer causing truncation in adenomatous polyposis coli (APC) (APC1–1450) dominantly interferes with mitotic spindle function, suggesting APC regulates microtubule dynamics during mitosis. Here, we examine the possibility that APC mutants interfere with the function of EB1, a plus-end microtubule-binding protein that interacts with APC and is required for normal microtubule dynamics. We show that siRNA-mediated inhibition of APC, EB1, or APC and EB1 together give rise to similar defects in mitotic spindles and chromosome alignment without arresting cells in mitosis; in contrast inhibition of CLIP170 or LIS1 cause distinct spindle defects and mitotic arrest. We show that APC1–1450 acts as a dominant negative by forming a hetero-oligomer with the full-length APC and preventing it from interacting with EB1, which is consistent with a functional relationship between APC and EB1. Live-imaging of mitotic cells expressing EB1-GFP demonstrates that APC1–1450 compromises the dynamics of EB1-comets, increasing the frequency of EB1-GFP pausing. Together these data provide novel insight into how APC may regulate mitotic spindle function and how errors in chromosome segregation are tolerated in tumor cells.
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Wang, Xiao Min, Ye Zhai, and James E. Ferrell. "A Role for Mitogen-activated Protein Kinase in the Spindle Assembly Checkpoint in XTC Cells." Journal of Cell Biology 137, no. 2 (April 21, 1997): 433–43. http://dx.doi.org/10.1083/jcb.137.2.433.
Full textAbstract:
The spindle assembly checkpoint prevents cells whose spindles are defective or chromosomes are misaligned from initiating anaphase and leaving mitosis. Studies of Xenopus egg extracts have implicated the Erk2 mitogen-activated protein kinase (MAP kinase) in this checkpoint. Other studies have suggested that MAP kinases might be important for normal mitotic progression. Here we have investigated whether MAP kinase function is required for mitotic progression or the spindle assembly checkpoint in vivo in Xenopus tadpole cells (XTC). We determined that Erk1 and/or Erk2 are present in the mitotic spindle during prometaphase and metaphase, consistent with the idea that MAP kinase might regulate or monitor the status of the spindle. Next, we microinjected purified recombinant XCL100, a Xenopus MAP kinase phosphatase, into XTC cells in various stages of mitosis to interfere with MAP kinase activation. We found that mitotic progression was unaffected by the phosphatase. However, XCL100 rendered the cells unable to remain arrested in mitosis after treatment with nocodazole. Cells injected with phosphatase at prometaphase or metaphase exited mitosis in the presence of nocodazole—the chromosomes decondensed and the nuclear envelope re-formed—whereas cells injected with buffer or a catalytically inactive XCL100 mutant protein remained arrested in mitosis. Coinjection of constitutively active MAP kinase kinase-1, which opposes XCL100's effects on MAP kinase, antagonized the effects of XCL100. Since the only known targets of MAP kinase kinase-1 are Erk1 and Erk2, these findings argue that MAP kinase function is required for the spindle assembly checkpoint in XTC cells.
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Pfaff, Kathleen L., Christian T. Straub, Ken Chiang, Daniel M. Bear, Yi Zhou, and Leonard I. Zon. "The Zebra fish cassiopeia Mutant Reveals that SIL Is Required for Mitotic Spindle Organization." Molecular and Cellular Biology 27, no. 16 (June 18, 2007): 5887–97. http://dx.doi.org/10.1128/mcb.00175-07.
Full textAbstract:
ABSTRACT A critical step in cell division is formation of the mitotic spindle, which is a bipolar array of microtubules that mediates chromosome separation. Here, we report that the SCL-interrupting locus (SIL), a vertebrate-specific cytosolic protein, is necessary for proper mitotic spindle organization in zebrafish and human cells. A homozygous lethal zebrafish mutant, cassiopeia (csp), was identified by a genetic screen for mitotic mutant. csp mutant embryos have an increased mitotic index, have highly disorganized mitotic spindles, and often lack one or both centrosomes. These phenotypes are caused by a loss-of-function mutation in zebrafish sil. To determine if the requirement for SIL in mitotic spindle organization is conserved in mammals, we generated an antibody against human SIL, which revealed that SIL localizes to the poles of the mitotic spindle during metaphase. Furthermore, short hairpin RNA knockdown of SIL in human cells recapitulates the zebrafish csp mitotic spindle defects. These data, taken together, identify SIL as a novel, vertebrate-specific regulator of mitotic spindle assembly.
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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.
Full textAbstract:
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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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.
Full textAbstract:
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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Gergely, Zachary R., Ammon Crapo, Loren E. Hough, J. Richard McIntosh, and Meredith D. Betterton. "Kinesin-8 effects on mitotic microtubule dynamics contribute to spindle function in fission yeast." Molecular Biology of the Cell 27, no. 22 (November 7, 2016): 3490–514. http://dx.doi.org/10.1091/mbc.e15-07-0505.
Full textAbstract:
Kinesin-8 motor proteins destabilize microtubules. Their absence during cell division is associated with disorganized mitotic chromosome movements and chromosome loss. Despite recent work studying effects of kinesin-8s on microtubule dynamics, it remains unclear whether the kinesin-8 mitotic phenotypes are consequences of their effect on microtubule dynamics, their well-established motor activity, or additional, unknown functions. To better understand the role of kinesin-8 proteins in mitosis, we studied the effects of deletion of the fission yeast kinesin-8 proteins Klp5 and Klp6 on chromosome movements and spindle length dynamics. Aberrant microtubule-driven kinetochore pushing movements and tripolar mitotic spindles occurred in cells lacking Klp5 but not Klp6. Kinesin-8–deletion strains showed large fluctuations in metaphase spindle length, suggesting a disruption of spindle length stabilization. Comparison of our results from light microscopy with a mathematical model suggests that kinesin-8–induced effects on microtubule dynamics, kinetochore attachment stability, and sliding force in the spindle can explain the aberrant chromosome movements and spindle length fluctuations seen.
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Farr, Katie A., and M. Andrew Hoyt. "Bub1p Kinase Activates the Saccharomyces cerevisiae Spindle Assembly Checkpoint." Molecular and Cellular Biology 18, no. 5 (May 1, 1998): 2738–47. http://dx.doi.org/10.1128/mcb.18.5.2738.
Full textAbstract:
ABSTRACT Saccharomyces cerevisiae BUB1 encodes a protein kinase required for spindle assembly checkpoint function. In the presence of spindle damage, BUB1 is required to prevent cell cycle progression into anaphase. We have identified a dominantly actingBUB1 allele that appears to activate the spindle assembly checkpoint pathway in cells with undamaged spindles. High-level expression of BUB1-5 did not cause detectable spindle damage, yet it delayed yeast cells in mitosis at a stage following bipolar spindle assembly but prior to anaphase spindle elongation. Delayed cells possessed a G2 DNA content and elevated Clb2p mitotic cyclin levels. Unlike cells delayed in mitosis by spindle damage or MPS1 kinase overexpression, hyperphosphorylated forms of the Mad1p checkpoint protein did not accumulate. Similar to cells overexpressing MPS1, the BUB1-5 delay was dependent upon the functions of the other checkpoint genes, includingBUB2 and BUB3 and MAD1,MAD2, and MAD3. We found that the mitotic delay caused by BUB1-5 or MPS1 overexpression was interdependent upon the function of the other. This suggests that the Bub1p and Mps1p kinases act together at an early step in generating the spindle damage signal.
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Andreassen, P. R., and R. L. Margolis. "Induction of partial mitosis in BHK cells by 2-aminopurine." Journal of Cell Science 100, no. 2 (October 1, 1991): 299–310. http://dx.doi.org/10.1242/jcs.100.2.299.
Full textAbstract:
The protein kinase inhibitor 2-aminopurine (2-AP) inhibits a subset of mitotic events in BHK cells. In the presence of the drug, these cells form a bipolar spindle in mitosis, but chromatin fails to generate functioning chromosomes. Cells in 2-AP progress through a partial mitosis, in which there is no observable metaphase, anaphase or telophase events. After 12 h of exposure to 2-AP the chromatin in mitotic cells fails to condense into discrete chromosomes, and is displaced by the spindle to form ‘binucleate’ cells and cells containing abnormally shaped nuclei in the subsequent interphase. Other mitotic modifications of nuclei, such as nucleolar and nuclear lamina disassembly, occur normally. Centromeres in these nuclei do not become engaged in the spindle, but instead show either no association or a lateral arrangement around the spindle. Cells treated with 2-AP are not arrested in mitosis. Therefore, mitotic exit is not inhibited by the failure of these cells to progress through the latter stages of mitosis. Further, nocodazole-arrested cells quickly exit mitotic arrest when 2-AP is added. We conclude that 2-AP interferes with a specific subset of mitotic events, and that it allows cells to overcome check-points that require spindle function for mitotic progression.
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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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Quaroni, A., and E. C. Paul. "Cytocentrin is a Ral-binding protein involved in the assembly and function of the mitotic apparatus." Journal of Cell Science 112, no. 5 (March 1, 1999): 707–18. http://dx.doi.org/10.1242/jcs.112.5.707.
Full textAbstract:
Cytocentrin is a cytosolic protein that transiently associates with the mitotic spindle poles in early prophase, and dissociates from them after completion of mitosis. Cloning of its cDNA demonstrated a high degree of homology with three proteins known to specifically interact with an activated form of Ral. Herein we demonstrate that overexpression of cytocentrin inhibits assembly of the mitotic spindle without affecting polymerization or distribution of interphase microtubules. Conversely, loss of cytocentrin expression leads to formation of monopolar spindles. These results indicate that association of cytocentrin with the centrosome may be essential for a timely separation of the diplosomes. They also implicate Ral GTPases and their related pathways in the assembly and function of the mitotic apparatus.
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Matthews, Lisa R., Philip Carter, Danielle Thierry-Mieg, and Ken Kemphues. "ZYG-9, A Caenorhabditis elegans Protein Required for Microtubule Organization and Function, Is a Component of Meiotic and Mitotic Spindle Poles." Journal of Cell Biology 141, no. 5 (June 1, 1998): 1159–68. http://dx.doi.org/10.1083/jcb.141.5.1159.
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We describe the molecular characterization of zyg-9, a maternally acting gene essential for microtubule organization and function in early Caenorhabditis elegans embryos. Defects in zyg-9 mutants suggest that the zyg-9 product functions in the organization of the meiotic spindle and the formation of long microtubules. One-cell zyg-9 embryos exhibit both meiotic and mitotic spindle defects. Meiotic spindles are disorganized, pronuclear migration fails, and the mitotic apparatus forms at the posterior, orients incorrectly, and contains unusually short microtubules. We find that zyg-9 encodes a component of the meiotic and mitotic spindle poles. In addition to the strong staining of spindle poles, we consistently detect staining in the region of the kinetochore microtubules at metaphase and early anaphase in mitotic spindles. The ZYG-9 signal at the mitotic centrosomes is not reduced by nocodazole treatment, indicating that ZYG-9 localization to the mitotic centrosomes is not dependent upon long astral microtubules. Interestingly, in embryos lacking an organized meiotic spindle, produced either by nocodazole treatment or mutations in the mei-1 gene, ZYG-9 forms a halo around the meiotic chromosomes. The protein sequence shows partial similarity to a small set of proteins that also localize to spindle poles, suggesting a common activity of the proteins.
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Masuda, H., and T. Shibata. "Role of gamma-tubulin in mitosis-specific microtubule nucleation from the Schizosaccharomyces pombe spindle pole body." Journal of Cell Science 109, no. 1 (January 1, 1996): 165–77. http://dx.doi.org/10.1242/jcs.109.1.165.
Full textAbstract:
The ability of the Schizosacchromyces pombe spindle pole body to nucleate microtubules is activated at the onset of mitosis for forming a mitotic spindle, but it is inactivated during interphase. We have previously developed an in vitro assay for studying the molecular mechanism of spindle pole body activation using permeabilized interphase S. pombe cells and Xenopus mitotic extracts. We have shown that the interphase spindle pole body is activated indirectly by p34cdc2 protein kinase in Xenopus mitotic extracts. In this study we examined the role of gamma-tubulin, a component of both interphase and mitotic spindle pole body, in formation of the microtubule nucleating complex at the mitotic spindle pole body. A polyclonal antibody specific to S. pombe gamma-tubulin inhibited both activation of the interphase spindle pole body and microtubule nucleation from the mitotic spindle pole body. Addition of bacterially expressed S. pombe gamma-tubulin or its amino-terminal fragments to Xenopus mitotic extracts inhibited spindle pole body activation. Affinity chromatography of partially fractionated Xenopus mitotic extracts with the amino-terminal fragment of S. pombe gamma-tubulin showed that fractions bound to the fragment supported the activation. The fractions did not contain Xenopus gamma-tubulin, showing that activation of the spindle pole body is not due to recruitment of Xenopus gamma-tubulin to the spindle pole body. The spindle pole body activation occurred in extracts depleted of p34cdc2 protein kinase or MAP kinase. The activity of the fractions bound to the fragment was inhibited by a protein kinase inhibitor, staurosporine. These results suggest that S. pombe gamma-tubulin is a component of the microtubule nucleating complex, and that the function of proteins that interact with gamma-tubulin is required for activation of the spindle pole body. We present possible models for the activation that convert the immature microtubule nucleating complex at interphase into the mature microtubule nucleating complex at mitosis.
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Zhou, Amber S., John B. Tucker, Christina M. Scribano, Andrew R. Lynch, Caleb L. Carlsen, Sophia T. Pop-Vicas, Srishrika M. Pattaswamy, Mark E. Burkard, and Beth A. Weaver. "Diverse microtubule-targeted anticancer agents kill cells by inducing chromosome missegregation on multipolar spindles." PLOS Biology 21, no. 10 (October 26, 2023): e3002339. http://dx.doi.org/10.1371/journal.pbio.3002339.
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Microtubule-targeted agents are commonly used for cancer treatment, though many patients do not benefit. Microtubule-targeted drugs were assumed to elicit anticancer activity via mitotic arrest because they cause cell death following mitotic arrest in cell culture. However, we recently demonstrated that intratumoral paclitaxel concentrations are insufficient to induce mitotic arrest and rather induce chromosomal instability (CIN) via multipolar mitotic spindles. Here, we show in metastatic breast cancer and relevant human cellular models that this mechanism is conserved among clinically useful microtubule poisons. While multipolar divisions typically produce inviable progeny, multipolar spindles can be focused into near-normal bipolar spindles at any stage of mitosis. Using a novel method to quantify the rate of CIN, we demonstrate that cell death positively correlates with net loss of DNA. Spindle focusing decreases CIN and causes resistance to diverse microtubule poisons, which can be counteracted by addition of a drug that increases CIN without affecting spindle polarity. These results demonstrate conserved mechanisms of action and resistance for diverse microtubule-targeted agents. Trial registration: clinicaltrials.gov, NCT03393741.
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Solnica-Krezel, L., T. G. Burland, and W. F. Dove. "Variable pathways for developmental changes of mitosis and cytokinesis in Physarum polycephalum." Journal of Cell Biology 113, no. 3 (May 1, 1991): 591–604. http://dx.doi.org/10.1083/jcb.113.3.591.
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The development of a uninucleate ameba into a multinucleate, syncytial plasmodium in myxomycetes involves a change from the open, astral mitosis of the ameba to the intranuclear, anastral mitosis of the plasmodium, and the omission of cytokinesis from the cell cycle. We describe immunofluorescence microscopic studies of the amebal-plasmodial transition (APT) in Physarum polycephalum. We demonstrate that the reorganization of mitotic spindles commences in uninucleate cells after commitment to plasmodium formation, is completed by the binucleate stage, and occurs via different routes in individual developing cells. Most uninucleate developing cells formed mitotic spindles characteristic either of amebae or of plasmodia. However, chimeric mitotic figures exhibiting features of both amebal and plasmodial mitoses, and a novel star microtubular array were also observed. The loss of the ameba-specific alpha 3-tubulin and the accumulation of the plasmodium-specific beta 2-tubulin isotypes during development were not sufficient to explain the changes in the organization of mitotic spindles. The majority of uninucleate developing cells undergoing astral mitoses (amebal and chimeric) exhibited cytokinetic furrows, whereas cells with the anastral plasmodial mitosis exhibited no furrows. Thus, the transition from astral to anastral mitosis during the APT could be sufficient for the omission of cytokinesis from the cell cycle. However, astral mitosis may not ensure cytokinesis: some cells undergoing amebal or chimeric mitosis contained unilateral cytokinetic furrows or no furrow at all. These cells would, most probably, fail to divide. We suggest that a uninucleate committed cell undergoing amebal or chimeric mitosis can either divide or else form a binucleate cell. In contrast, a uninucleate cell with a mitotic spindle of the plasmodial type gives rise only to a binucleate cells. Further, the decision to enter mitosis after commitment to the APT is independent of the developmental changes in the organization of the mitotic spindle and cytokinesis.
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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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Hussain, Shobbir, Sandra Blanco Benavente, Elisabete Nascimento, Ilaria Dragoni, Agata Kurowski, Astrid Gillich, Peter Humphreys, and Michaela Frye. "The nucleolar RNA methyltransferase Misu (NSun2) is required for mitotic spindle stability." Journal of Cell Biology 186, no. 1 (July 13, 2009): 27–40. http://dx.doi.org/10.1083/jcb.200810180.
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Myc-induced SUN domain–containing protein (Misu or NSun2) is a nucleolar RNA methyltransferase important for c-Myc–induced proliferation in skin, but the mechanisms by which Misu contributes to cell cycle progression are unknown. In this study, we demonstrate that Misu translocates from the nucleoli in interphase to the spindle in mitosis as an RNA–protein complex that includes 18S ribosomal RNA. Functionally, depletion of Misu caused multiple mitotic defects, including formation of unstructured spindles, multipolar spindles, and chromosome missegregation, leading to aneuploidy and cell death. The presence of both RNA and Misu is required for correct spindle assembly, and this process is independent of active translation. Misu might mediate its function at the spindle by recruiting nucleolar and spindle-associated protein (NuSAP), an essential microtubule-stabilizing and bundling protein. We further identify NuSAP as a novel direct target gene of c-Myc. Collectively, our results suggest a novel mechanism by which c-Myc promotes proliferation by stabilizing the mitotic spindle in fast-dividing cells via Misu and NuSAP.
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Noree, Chalongrat, and Naraporn Sirinonthanawech. "Nuclear targeted Saccharomyces cerevisiae asparagine synthetases associate with the mitotic spindle regardless of their enzymatic activity." PLOS ONE 15, no. 12 (December 21, 2020): e0243742. http://dx.doi.org/10.1371/journal.pone.0243742.
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Recently, human asparagine synthetase has been found to be associated with the mitotic spindle. However, this event cannot be seen in yeast because yeast takes a different cell division process via closed mitosis (there is no nuclear envelope breakdown to allow the association between any cytosolic enzyme and mitotic spindle). To find out if yeast asparagine synthetase can also (but hiddenly) have this feature, the coding sequences of green fluorescent protein (GFP) and nuclear localization signal (NLS) were introduced downstream of ASN1 and ASN2, encoding asparagine synthetases Asn1p and Asn2p, respectively, in the yeast genome having mCherrry coding sequence downstream of TUB1 encoding alpha-tubulin, a building block of the mitotic spindle. The genomically engineered yeast strains showed co-localization of Asn1p-GFP-NLS (or Asn2p-GFP-NLS) and Tub1p-mCherry in dividing nuclei. In addition, an activity-disrupted mutation was introduced to ASN1 (or ASN2). The yeast mutants still exhibited co-localization between defective asparagine synthetase and mitotic spindle, indicating that the biochemical activity of asparagine synthetase is not required for its association with the mitotic spindle. Furthermore, nocodazole treatment was used to depolymerize the mitotic spindle, resulting in lack of association between the enzyme and the mitotic spindle. Although yeast cell division undergoes closed mitosis, preventing the association of its asparagine synthetase with the mitotic spindle, however, by using yeast constructs with re-localized Asn1/2p have suggested the moonlighting role of asparagine synthetase in cell division of higher eukaryotes.
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O'Regan, Laura, and Andrew M. Fry. "The Nek6 and Nek7 Protein Kinases Are Required for Robust Mitotic Spindle Formation and Cytokinesis." Molecular and Cellular Biology 29, no. 14 (May 4, 2009): 3975–90. http://dx.doi.org/10.1128/mcb.01867-08.
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ABSTRACT Nek6 and Nek7 are members of the NIMA-related serine/threonine kinase family. Previous work showed that they contribute to mitotic progression downstream of another NIMA-related kinase, Nek9, although the roles of these different kinases remain to be defined. Here, we carried out a comprehensive analysis of the regulation and function of Nek6 and Nek7 in human cells. By generating specific antibodies, we show that both Nek6 and Nek7 are activated in mitosis and that interfering with their activity by either depletion or expression of reduced-activity mutants leads to mitotic arrest and apoptosis. Interestingly, while completely inactive mutants and small interfering RNA-mediated depletion delay cells at metaphase with fragile mitotic spindles, hypomorphic mutants or RNA interference treatment combined with a spindle assembly checkpoint inhibitor delays cells at cytokinesis. Importantly, depletion of either Nek6 or Nek7 leads to defective mitotic progression, indicating that although highly similar, they are not redundant. Indeed, while both kinases localize to spindle poles, only Nek6 obviously localizes to spindle microtubules in metaphase and anaphase and to the midbody during cytokinesis. Together, these data lead us to propose that Nek6 and Nek7 play independent roles not only in robust mitotic spindle formation but also potentially in cytokinesis.