Caractérisation des rôles de l"Anilline durant la cytokinèse

Cytokinesis is a multi-step conserved process by which a cell physically separates into two. First, signalling between the mitotic spindle and the cortex provides cues to position the cleavage furrow. Then, an acto-myosin contractile ring (CR) is assembled and will constrict the cell at its equator. As the CR constricts the mitotic spindle’s midzone will mature to form the midbody (MB) and a stable midbody ring (MR) forms to stabilize the intercellular bridge until abscission. Defects at any of these steps can cause cytokinesis failure and generate binucleate polyploid cells, often observed in pathological conditions such as cancer. Genetic and biochemical screens have identified the key, conserved proteins that constitute the cytokinetic machinery, however, it remains unclear how CR closure is coupled to MB and MR formation and how plasma membrane anchoring is maintained at this key transition events. Anillin is a core and essential component that scaffolds many other cytokinesis proteins such as actin, myosin and septins, but it remains unclear how Anillin acts with its numerous partners in time and space to guide progress through cytokinesis. To address these questions we performed a structure-function analysis of Anillin by time‐lapse microscopy in Drosophila S2 cells. Anillin localizes to the CR and the MR and its depletion does not affect CR assembly, but rather blocks cytokinesis at a mid‐late furrowing stage. We have found that Anillin is required for the stability of the CR, formation of the MR and the maturation of the MB. Truncation analysis revealed that Anillin N-terminus connected with the actomyosin CR and supported formation of stable MR-like structures, but these could not maintain anchoring of the plasma membrane. Conversely, Anillin C-terminus failed to connect with the CR or MR but recruited the septin Peanut to the equatorial cortex and together mediated anchorage of the MR to the membrane. Next, we further characterized this CR to MR transition and show that it proceeds via a previously uncharacterized maturation process that requires opposing mechanisms of removal and retention of Anillin at the equator. The Citron kinase Sticky acts on the N-terminus of Anillin to retain it at the mature MR, whereas the septin cytoskeleton acts on the C-terminus of Anillin to locally trim away excess membrane from the nascent MR via internalization,extrusion, and shedding of the plasma membrane and associated proteins. Our structure function analysis of Anillin also revealed a requirement for Anillin in MB maturation via its C-terminus that mediates the recruitment of septins and binds to the microtubule associated protein Tumbleweed/RacGAP50C (Tum). Tum is a GTPase activating protein (GAP) required early during cytokinesis for central spindle formation and furrow initiation and later for anchorage of the MB to the plasma membrane. However, there is no consensus on the role of its GAP activity and no known functions for its interaction with Anillin. Our structure function study by real-time microscopy of Tum revealed that the GAP activity of Tum and its central domain are required for CR formation and closure. Furthermore, we found that the putative Anillin binding region of Tum contributes to Tum cortical recruitment and MB/MR maturation later during cytokinesis in an Anillin-dependant manner. Finally, we unveil a genetic interaction between Tum and Anillin that mediates furrow initiation. These results suggest that the interaction between Anillin and Tum links the CR\MR to the spindle/MB throughout cytokinesis. Together, the results presented in this thesis show that through its multiple interactions Anillin is a key protein involved throughout cytokinesis from initiation of the cleavage furrow to the formation of the CR, its stabilization and maturation to a MR as well as its anchoring to the plasma membrane. Key words: Anillin, cytokinesis, Contractile ring, Midbody ring, Midbody, Tum, Tumbleweed / RacGAP50C, septin, Peanut, Citron Kinase, Sticky, Intercellular bridge.

Keyphrases: Anillin, cytokinesis, cell division, RacGAP50C, Citron kinase