Genetic Control of Kinetochore-Driven Microtubule Growth in <i>Drosophila</i> Mitosis
Centrosome-containing cells assemble their spindles exploiting three main classes of microtubules (MTs): MTs nucleated by the centrosomes, MTs generated near the chromosomes/kinetochores, and MTs nucleated within the spindle by the augmin-dependent pathway. Mammalian and <i>Drosophila</i>...
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MDPI AG
2022-07-01
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| Online Access: | https://www.mdpi.com/2073-4409/11/14/2127 |
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| author | Julia V. Popova Gera A. Pavlova Alyona V. Razuvaeva Lyubov A. Yarinich Evgeniya N. Andreyeva Alina F. Anders Yuliya A. Galimova Fioranna Renda Maria Patrizia Somma Alexey V. Pindyurin Maurizio Gatti |
| author_facet | Julia V. Popova Gera A. Pavlova Alyona V. Razuvaeva Lyubov A. Yarinich Evgeniya N. Andreyeva Alina F. Anders Yuliya A. Galimova Fioranna Renda Maria Patrizia Somma Alexey V. Pindyurin Maurizio Gatti |
| author_sort | Julia V. Popova |
| collection | DOAJ |
| description | Centrosome-containing cells assemble their spindles exploiting three main classes of microtubules (MTs): MTs nucleated by the centrosomes, MTs generated near the chromosomes/kinetochores, and MTs nucleated within the spindle by the augmin-dependent pathway. Mammalian and <i>Drosophila</i> cells lacking the centrosomes generate MTs at kinetochores and eventually form functional bipolar spindles. However, the mechanisms underlying kinetochore-driven MT formation are poorly understood. One of the ways to elucidate these mechanisms is the analysis of spindle reassembly following MT depolymerization. Here, we used an RNA interference (RNAi)-based reverse genetics approach to dissect the process of kinetochore-driven MT regrowth (KDMTR) after colcemid-induced MT depolymerization. This MT depolymerization procedure allows a clear assessment of KDMTR, as colcemid disrupts centrosome-driven MT regrowth but not KDMTR. We examined KDMTR in normal <i>Drosophila</i> S2 cells and in S2 cells subjected to RNAi against conserved genes involved in mitotic spindle assembly: <i>mast</i>/<i>orbit</i>/<i>chb</i> (<i>CLASP1</i>), <i>mei-38</i> (<i>TPX2</i>), <i>mars</i> (<i>HURP</i>), <i>dgt6</i> (<i>HAUS6</i>), <i>Eb1</i> (<i>MAPRE1/EB1</i>), <i>Patronin</i> (<i>CAMSAP2</i>), <i>asp</i> (<i>ASPM</i>), and <i>Klp10A</i> (<i>KIF2A</i>). RNAi-mediated depletion of Mast/Orbit, Mei-38, Mars, Dgt6, and Eb1 caused a significant delay in KDMTR, while loss of Patronin had a milder negative effect on this process. In contrast, Asp or Klp10A deficiency increased the rate of KDMTR. These results coupled with the analysis of GFP-tagged proteins (Mast/Orbit, Mei-38, Mars, Eb1, Patronin, and Asp) localization during KDMTR suggested a model for kinetochore-dependent spindle reassembly. We propose that kinetochores capture the plus ends of MTs nucleated in their vicinity and that these MTs elongate at kinetochores through the action of Mast/Orbit. The Asp protein binds the MT minus ends since the beginning of KDMTR, preventing excessive and disorganized MT regrowth. Mei-38, Mars, Dgt6, Eb1, and Patronin positively regulate polymerization, bundling, and stabilization of regrowing MTs until a bipolar spindle is reformed. |
| first_indexed | 2024-03-09T10:21:12Z |
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| language | English |
| last_indexed | 2024-03-09T10:21:12Z |
| publishDate | 2022-07-01 |
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| series | Cells |
| spelling | doaj.art-5a0a4ff4291744c1991cd9ad4ca7b7bf2023-12-01T22:00:33ZengMDPI AGCells2073-44092022-07-011114212710.3390/cells11142127Genetic Control of Kinetochore-Driven Microtubule Growth in <i>Drosophila</i> MitosisJulia V. Popova0Gera A. Pavlova1Alyona V. Razuvaeva2Lyubov A. Yarinich3Evgeniya N. Andreyeva4Alina F. Anders5Yuliya A. Galimova6Fioranna Renda7Maria Patrizia Somma8Alexey V. Pindyurin9Maurizio Gatti10Institute of Molecular and Cellular Biology, Siberian Branch of Russian Academy of Sciences, 630090 Novosibirsk, RussiaInstitute of Molecular and Cellular Biology, Siberian Branch of Russian Academy of Sciences, 630090 Novosibirsk, RussiaInstitute of Molecular and Cellular Biology, Siberian Branch of Russian Academy of Sciences, 630090 Novosibirsk, RussiaInstitute of Molecular and Cellular Biology, Siberian Branch of Russian Academy of Sciences, 630090 Novosibirsk, RussiaInstitute of Molecular and Cellular Biology, Siberian Branch of Russian Academy of Sciences, 630090 Novosibirsk, RussiaInstitute of Molecular and Cellular Biology, Siberian Branch of Russian Academy of Sciences, 630090 Novosibirsk, RussiaInstitute of Molecular and Cellular Biology, Siberian Branch of Russian Academy of Sciences, 630090 Novosibirsk, RussiaInstitute of Molecular Biology and Pathology (IBPM), National Research Council (CNR), c/o Department of Biology and Biotechnology, Sapienza University of Rome, 00185 Rome, ItalyInstitute of Molecular Biology and Pathology (IBPM), National Research Council (CNR), c/o Department of Biology and Biotechnology, Sapienza University of Rome, 00185 Rome, ItalyInstitute of Molecular and Cellular Biology, Siberian Branch of Russian Academy of Sciences, 630090 Novosibirsk, RussiaInstitute of Molecular and Cellular Biology, Siberian Branch of Russian Academy of Sciences, 630090 Novosibirsk, RussiaCentrosome-containing cells assemble their spindles exploiting three main classes of microtubules (MTs): MTs nucleated by the centrosomes, MTs generated near the chromosomes/kinetochores, and MTs nucleated within the spindle by the augmin-dependent pathway. Mammalian and <i>Drosophila</i> cells lacking the centrosomes generate MTs at kinetochores and eventually form functional bipolar spindles. However, the mechanisms underlying kinetochore-driven MT formation are poorly understood. One of the ways to elucidate these mechanisms is the analysis of spindle reassembly following MT depolymerization. Here, we used an RNA interference (RNAi)-based reverse genetics approach to dissect the process of kinetochore-driven MT regrowth (KDMTR) after colcemid-induced MT depolymerization. This MT depolymerization procedure allows a clear assessment of KDMTR, as colcemid disrupts centrosome-driven MT regrowth but not KDMTR. We examined KDMTR in normal <i>Drosophila</i> S2 cells and in S2 cells subjected to RNAi against conserved genes involved in mitotic spindle assembly: <i>mast</i>/<i>orbit</i>/<i>chb</i> (<i>CLASP1</i>), <i>mei-38</i> (<i>TPX2</i>), <i>mars</i> (<i>HURP</i>), <i>dgt6</i> (<i>HAUS6</i>), <i>Eb1</i> (<i>MAPRE1/EB1</i>), <i>Patronin</i> (<i>CAMSAP2</i>), <i>asp</i> (<i>ASPM</i>), and <i>Klp10A</i> (<i>KIF2A</i>). RNAi-mediated depletion of Mast/Orbit, Mei-38, Mars, Dgt6, and Eb1 caused a significant delay in KDMTR, while loss of Patronin had a milder negative effect on this process. In contrast, Asp or Klp10A deficiency increased the rate of KDMTR. These results coupled with the analysis of GFP-tagged proteins (Mast/Orbit, Mei-38, Mars, Eb1, Patronin, and Asp) localization during KDMTR suggested a model for kinetochore-dependent spindle reassembly. We propose that kinetochores capture the plus ends of MTs nucleated in their vicinity and that these MTs elongate at kinetochores through the action of Mast/Orbit. The Asp protein binds the MT minus ends since the beginning of KDMTR, preventing excessive and disorganized MT regrowth. Mei-38, Mars, Dgt6, Eb1, and Patronin positively regulate polymerization, bundling, and stabilization of regrowing MTs until a bipolar spindle is reformed.https://www.mdpi.com/2073-4409/11/14/2127mitosisS2 cells<i>Drosophila</i>microtubule regrowthmicrotubule depolymerizationcolcemid |
| spellingShingle | Julia V. Popova Gera A. Pavlova Alyona V. Razuvaeva Lyubov A. Yarinich Evgeniya N. Andreyeva Alina F. Anders Yuliya A. Galimova Fioranna Renda Maria Patrizia Somma Alexey V. Pindyurin Maurizio Gatti Genetic Control of Kinetochore-Driven Microtubule Growth in <i>Drosophila</i> Mitosis Cells mitosis S2 cells <i>Drosophila</i> microtubule regrowth microtubule depolymerization colcemid |
| title | Genetic Control of Kinetochore-Driven Microtubule Growth in <i>Drosophila</i> Mitosis |
| title_full | Genetic Control of Kinetochore-Driven Microtubule Growth in <i>Drosophila</i> Mitosis |
| title_fullStr | Genetic Control of Kinetochore-Driven Microtubule Growth in <i>Drosophila</i> Mitosis |
| title_full_unstemmed | Genetic Control of Kinetochore-Driven Microtubule Growth in <i>Drosophila</i> Mitosis |
| title_short | Genetic Control of Kinetochore-Driven Microtubule Growth in <i>Drosophila</i> Mitosis |
| title_sort | genetic control of kinetochore driven microtubule growth in i drosophila i mitosis |
| topic | mitosis S2 cells <i>Drosophila</i> microtubule regrowth microtubule depolymerization colcemid |
| url | https://www.mdpi.com/2073-4409/11/14/2127 |
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