Establishment and mitotic characterization of new Drosophila acentriolar cell lines from DSas-4 mutant
Nicolas Lecland,
Alain Debec,
Audrey Delmas,
Sara Moutinho-Pereira,
Nicolas Malmanche,
Anais Bouissou,
Clémence Dupré,
Aimie Jourdan,
Brigitte Raynaud-Messina,
Helder Maiato,
Antoine Guichet
Affiliations
Nicolas Lecland
Polarity and Morphogenesis Group, Jacques Monod Institute, UMR 7592 CNRS, University Paris Diderot, 15 rue Hélène Brion, 75 205 Paris Cedex 13, France
Alain Debec
Polarity and Morphogenesis Group, Jacques Monod Institute, UMR 7592 CNRS, University Paris Diderot, 15 rue Hélène Brion, 75 205 Paris Cedex 13, France
Audrey Delmas
Centre de Biologie du Développement, Université Paul Sabatier, Bâtiment 4R3, 118 Route de Narbonne, 31062 Toulouse Cedex 9, France
Sara Moutinho-Pereira
Chromosome Instability and Dynamics Laboratory, Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal
Nicolas Malmanche
Chromosome Instability and Dynamics Laboratory, Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal
Anais Bouissou
Centre de Biologie du Développement, Université Paul Sabatier, Bâtiment 4R3, 118 Route de Narbonne, 31062 Toulouse Cedex 9, France
Clémence Dupré
Polarity and Morphogenesis Group, Jacques Monod Institute, UMR 7592 CNRS, University Paris Diderot, 15 rue Hélène Brion, 75 205 Paris Cedex 13, France
Aimie Jourdan
Polarity and Morphogenesis Group, Jacques Monod Institute, UMR 7592 CNRS, University Paris Diderot, 15 rue Hélène Brion, 75 205 Paris Cedex 13, France
Brigitte Raynaud-Messina
Centre de Biologie du Développement, Université Paul Sabatier, Bâtiment 4R3, 118 Route de Narbonne, 31062 Toulouse Cedex 9, France
Helder Maiato
Centre de Biologie du Développement, Université Paul Sabatier, Bâtiment 4R3, 118 Route de Narbonne, 31062 Toulouse Cedex 9, France
Antoine Guichet
Polarity and Morphogenesis Group, Jacques Monod Institute, UMR 7592 CNRS, University Paris Diderot, 15 rue Hélène Brion, 75 205 Paris Cedex 13, France
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.
