title: Establishment and mitotic characterization of new Drosophila acentriolar cell lines from DSas-4 mutant.
abstract: 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.
authors.affiliation: Polarity and Morphogenesis Group, Jacques Monod Institute, UMR 7592 CNRS, University Paris Diderot , 15 rue Hélène Brion, 75 205 Paris Cedex 13 , France ; Present address: Microtubule Organization Lab, Institut de Recerca Biomèdica de Barcelona, Baldiri Reixac 10-12, 08028 Barcelona, Spain.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Lecland
authors.forename: Alain
authors.initials: A
authors.lastname: Debec
authors.forename: Audrey
authors.initials: A
authors.lastname: Delmas
authors.forename: Sara
authors.initials: S
authors.lastname: Moutinho-Pereira
authors.forename: Nicolas
authors.initials: N
authors.lastname: Malmanche
authors.forename: Anais
authors.initials: A
authors.lastname: Bouissou
authors.forename: Clémence
authors.initials: C
authors.lastname: Dupré
authors.forename: Aimie
authors.initials: A
authors.lastname: Jourdan
authors.forename: Brigitte
authors.initials: B
authors.lastname: Raynaud-Messina
authors.forename: Helder
authors.initials: H
authors.lastname: Maiato
authors.forename: Antoine
authors.initials: A
authors.lastname: Guichet
keywords: Anastral; Cell lines; Centriole; Cytoskeleton; Drosophila; Mitosis; Spindle