Analysis Of The Role Of Shot In Maintaining Perinuclear Microtubule Organization In Drosophila Polyploid Cells

The existence of polyploid cells in otherwise diploid organisms is widespread in nature and common in cancer cells.As part of their differentiation programs,developmentally programmed polyploid cells endoreplicate their DNA through a modified cell cycle that skips mitosis.In contrast to proliferating cells,where microtubules are organized by centrosomes,non-centrosomal microtubule organizing centers(MTOCs)govern microtubule distribution upon cell-cycle exit and differentiation.Little is known on how polyploid cells,differentiated but cycling,organize their microtubules.In this study,I took advantage of the fruit fly Drosophila melanogaster as a model organism and discovered that a perinuclear microtubule organizer(pn MTOC)is responsible for nuclear size and position in polyploid tissues.In a screening for genes involved in Collagen IV secretion,I found that knock-down of spectraplakin Shot(shot~i)in polyploid fat body adipocytes caused a matrix release defect.Further investigation showed that Shot localizes to a dense perinuclear microtubule cortex in polyploid tissues of the fly larva.Absence of Shot caused collapse of the pn MTOC into a highly condensed organizer through Kinesin-dependent microtubule sliding.Through live-imaging,I proved that both the pn MTOC and the ectopic shot~i MTOC are active microtubule organizers.Strikingly,I found that this collapsed organizer was able to trigger formation of a monopolar cytokinesis furrow,with deeply disruptive consequences for cellular organization.Knock-down of endocycle regulators Cyc E,Cdk2,or E2F reduced perinuclear microtubule concentration.Furthermore,I found polyploidyzation of normally diploid blood cells,through Cdk1 knock-down,was sufficient to induce pn MTOC formation.Additionally,other proteins involved in forming the pn MTOC are consistent with a model in which microtubule-severing protein Kat-60L1 cuts pre-existing microtubules into seedling microtubules,which are then stabilized by minus end-binding protein Patronin and anchored to the nuclear membrane through Shot,which in turn binds to nuclear transmembrane proteins to connect microtubules with the nucleus.In all,this study found a relation between polyploidy and pn MTOC,revealing critical functions of Shot and other components in establishing and maintaining perinuclear microtubule organization.