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      Katanin p60 Contributes to Microtubule Instability around the Midbody and Facilitates Cytokinesis in Rat Cells

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          Abstract

          The completion of cytokinesis is crucial for mitotic cell division. Cleavage furrow ingression is followed by the breaking and resealing of the intercellular bridge, but the detailed mechanism underlying this phenomenon remains unknown. Katanin is a microtubule-severing protein comprised of an AAA ATPase subunit and an accessory subunit designated as p60 and p80, respectively. Localization of katanin p60 was observed at the midzone to midbody from anaphase to cytokinesis in rat cells, and showed a ring-shaped distribution in the gap between the inside of the contractile ring and the central spindle bundle in telophase. Katanin p60 did not bind with p80 at the midzone or midbody, and localization was shown to be dependent on microtubules. At the central spindle and the midbody, no microtubule growth plus termini were seen with katanin p60, and microtubule density was inversely correlated with katanin p60 density in the region of katanin p60 localization that seemed to lead to microtubule destabilization at the midbody. Inhibition of katanin p60 resulted in incomplete cytokinesis by regression and thus caused the appearance of binucleate cells. These results suggest that katanin p60 contributes to microtubule instability at the midzone and midbody and facilitates cytokinesis in rat cells.

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          Most cited references40

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          From polyploidy to aneuploidy, genome instability and cancer.

          Polyploidy is a frequent phenomenon in the eukaryotic world, but the biological properties of polyploid cells are not well understood. During evolution, polyploidy is thought to be an important mechanism that contributes to speciation. Polyploid, usually non-dividing, cells are formed during development in otherwise diploid organisms. A growing amount of evidence indicates that polyploid cells also arise during a variety of pathological conditions. Genetic instability in these cells might provide a route to aneuploidy and thereby contribute to the development of cancer.
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            Genes required for mitotic spindle assembly in Drosophila S2 cells.

            The formation of a metaphase spindle, a bipolar microtubule array with centrally aligned chromosomes, is a prerequisite for the faithful segregation of a cell's genetic material. Using a full-genome RNA interference screen of Drosophila S2 cells, we identified about 200 genes that contribute to spindle assembly, more than half of which were unexpected. The screen, in combination with a variety of secondary assays, led to new insights into how spindle microtubules are generated; how centrosomes are positioned; and how centrioles, centrosomes, and kinetochores are assembled.
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              Augmin: a protein complex required for centrosome-independent microtubule generation within the spindle

              Since the discovery of γ-tubulin, attention has focused on its involvement as a microtubule nucleator at the centrosome. However, mislocalization of γ-tubulin away from the centrosome does not inhibit mitotic spindle formation in Drosophila melanogaster, suggesting that a critical function for γ-tubulin might reside elsewhere. A previous RNA interference (RNAi) screen identified five genes (Dgt2–6) required for localizing γ-tubulin to spindle microtubules. We show that the Dgt proteins interact, forming a stable complex. We find that spindle microtubule generation is substantially reduced after knockdown of each Dgt protein by RNAi. Thus, the Dgt complex that we name “augmin” functions to increase microtubule number. Reduced spindle microtubule generation after augmin RNAi, particularly in the absence of functional centrosomes, has dramatic consequences on mitotic spindle formation and function, leading to reduced kinetochore fiber formation, chromosome misalignment, and spindle bipolarity defects. We also identify a functional human homologue of Dgt6. Our results suggest that an important mitotic function for γ-tubulin may lie within the spindle, where augmin and γ-tubulin function cooperatively to amplify the number of microtubules.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2013
                26 November 2013
                : 8
                : 11
                : e80392
                Affiliations
                [1 ]Department of Biomolecular Science, Faculty of Science, Toho University, Funabashi, Chiba, Japan
                [2 ]Link Genomics Co., Ltd., Tokyo, Japan
                [3 ]Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University, Tokyo, Japan
                [4 ]Proteome Analysis Center, Faculty of Science, Toho University, Funabashi, Chiba, Japan
                University of Connecticut, Storrs, United States of America
                Author notes

                Competing Interests: The following authors have an affiliation with commercial funders of this research, Link Genomics, Inc.: Takashi Kasama, Yukie Hata, Kazuya Arai, Yasutaka Makino and Shin-ichiro Niwa. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

                Conceived and designed the experiments: MM T. Kishimoto. Performed the experiments: MM TS T. Kasama YH AE MO KA. Analyzed the data: MM TS YM SN HS T. Kishimoto. Contributed reagents/materials/analysis tools: MM YH SN HS KA. Wrote the manuscript: MM T. Kishimoto. Coordination of collaboration: SN HS T. Kishimoto.

                Article
                PONE-D-13-21262
                10.1371/journal.pone.0080392
                3841192
                24303010
                f4fb7334-1b4e-46c1-a85c-13093b58672b
                Copyright @ 2013

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 23 May 2013
                : 2 October 2013
                Funding
                This research is partly supported by funding from Link Genomics, Inc. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding was received for this study.
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