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1. Establishment and mitotic characterization of new Drosophila acentriolar cell lines from DSas-4 mutant

Author

Nicolas Lecland, Alain Debec, Audrey Delmas, Sara Moutinho-Pereira, Nicolas Malmanche, Anais Bouissou, Clémence Dupré, Aimie Jourdan, Brigitte Raynaud-Messina, Helder Maiato, and Antoine Guichet

Subjects
Centriole, Mitosis, Spindle, Cytoskeleton, Drosophila, Anastral, Cell lines, Science, Biology (General), QH301-705.5
Abstract

Summary In animal cells the centrosome is commonly viewed as the main cellular structure driving microtubule (MT) assembly into the mitotic spindle apparatus. However, additional pathways, such as those mediated by chromatin and augmin, are involved in the establishment of functional spindles. The molecular mechanisms involved in these pathways remain poorly understood, mostly due to limitations inherent to current experimental systems available. To overcome these limitations we have developed six new Drosophila cell lines derived from Drosophila homozygous mutants for DSas-4, a protein essential for centriole biogenesis. These cells lack detectable centrosomal structures, astral MT, with dispersed pericentriolar proteins D-PLP, Centrosomin and γ-tubulin. They show poorly focused spindle poles that reach the plasma membrane. Despite being compromised for functional centrosome, these cells could successfully undergo mitosis. Live-cell imaging analysis of acentriolar spindle assembly revealed that nascent MTs are nucleated from multiple points in the vicinity of chromosomes. These nascent MTs then grow away from kinetochores allowing the expansion of fibers that will be part of the future acentriolar spindle. MT repolymerization assays illustrate that acentriolar spindle assembly occurs “inside-out” from the chromosomes. Colchicine-mediated depolymerization of MTs further revealed the presence of a functional Spindle Assembly Checkpoint (SAC) in the acentriolar cells. Finally, pilot RNAi experiments open the potential use of these cell lines for the molecular dissection of anastral pathways in spindle and centrosome assembly.

Published
2013
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2. Establishment and mitotic characterization of new Drosophila acentriolar cell lines from DSas-4 mutant

Author

Brigitte Raynaud-Messina, Aimie Jourdan, Alain Debec, Nicolas Lecland, Clémence Dupré, Sara Moutinho-Pereira, Helder Maiato, Nicolas Malmanche, Anaïs Bouissou, Antoine Guichet, Audrey Delmas, Institut Jacques Monod (IJM (UMR_7592)), Université Paris Diderot - Paris 7 (UPD7)-Centre National de la Recherche Scientifique (CNRS), Centre de biologie du développement (CBD), Centre National de la Recherche Scientifique (CNRS)-Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre de Biologie Intégrative (CBI), Université Toulouse III - Paul Sabatier (UT3), Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Chromosome Instability and Dynamics Laboratory, Universidade do Porto-Instituto de Biologia Molecular e Celular, Department of Experimental Biology, Universidade do Porto-Faculdade de Medicina - Porto - Portugal, ANR 'Blanche' grant Cymempol, Blan06-3-139786, ARC grant SL220100601358, 'Ligue Contre le Cancer' grant RS11/75-34, laboratory of Andreas Merdes, ARC (4720XP023OF), Centre National de la Recherche Scientifique, Pierre Fabre Laboratorie, the laboratory of H.M. is funded by grants PTDC/SAU-GMG/099704/2008 and PTDC/SAU-ONC/112917/2009 from Fundação para a Ciência e a Tecnologia of Portugal (COMPETE-FEDER), the Human Frontier Research Program and the 7th framework program grant PRECISE from the European Research Council, Université Fédérale Toulouse Midi-Pyrénées-Université Fédérale Toulouse Midi-Pyrénées, Universidade do Porto [Porto]-Instituto de Biologia Molecular e Celular, and Universidade do Porto [Porto]-Faculdade de Medicina - Porto - Portugal

Subjects
Spindle, Centriole, QH301-705.5, Science, Mitosis, [SDV.BC]Life Sciences [q-bio]/Cellular Biology, Biology, Bioinformatics, General Biochemistry, Genetics and Molecular Biology, Spindle pole body, 03 medical and health sciences, 0302 clinical medicine, Microtubule, [SDV.BBM]Life Sciences [q-bio]/Biochemistry, Molecular Biology, Biology (General), [SDV.BDD]Life Sciences [q-bio]/Development Biology, Cytoskeleton, 030304 developmental biology, 0303 health sciences, Kinetochore, [SDV.BDLR]Life Sciences [q-bio]/Reproductive Biology, Anastral, Chromatin, Cell biology, Spindle checkpoint, Centrosome, Cell lines, Drosophila, General Agricultural and Biological Sciences, 030217 neurology & neurosurgery, Research Article
Abstract

Summary In animal cells the centrosome is commonly viewed as the main cellular structure driving microtubule (MT) assembly into the mitotic spindle apparatus. However, additional pathways, such as those mediated by chromatin and augmin, are involved in the establishment of functional spindles. The molecular mechanisms involved in these pathways remain poorly understood, mostly due to limitations inherent to current experimental systems available. To overcome these limitations we have developed six new Drosophila cell lines derived from Drosophila homozygous mutants for DSas-4, a protein essential for centriole biogenesis. These cells lack detectable centrosomal structures, astral MT, with dispersed pericentriolar proteins D-PLP, Centrosomin and γ-tubulin. They show poorly focused spindle poles that reach the plasma membrane. Despite being compromised for functional centrosome, these cells could successfully undergo mitosis. Live-cell imaging analysis of acentriolar spindle assembly revealed that nascent MTs are nucleated from multiple points in the vicinity of chromosomes. These nascent MTs then grow away from kinetochores allowing the expansion of fibers that will be part of the future acentriolar spindle. MT repolymerization assays illustrate that acentriolar spindle assembly occurs “inside-out” from the chromosomes. Colchicine-mediated depolymerization of MTs further revealed the presence of a functional Spindle Assembly Checkpoint (SAC) in the acentriolar cells. Finally, pilot RNAi experiments open the potential use of these cell lines for the molecular dissection of anastral pathways in spindle and centrosome assembly.

Published
2013

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