The endosomal sorting complexes required for transport (ESCRT) machinery regulate a broad range of biological processes which require similar membrane topological transformations, including intraluminal vesicle formation, viral budding, nuclear envelope resealing, cytokinetic abscission and membrane repair. These processes rely on the assembly of the main ESCRT-III membrane remodelling complex, which cooperates with AAA ATPase Vps4, to drive membrane curvature and membrane fission. Although 11 mammalian members of the ESCRT-III family have been identified, the exact mechanism by which these components are regulated to induce membrane curvature is poorly understood. Coiled-coil C2 domain containing 1A (CC2D1A) and CC2D1B are evolutionarily conserved mammalian members of the Drosophila Lethal giant disc (Lgd) family. CC2D1A and CC2D1B have been reported to directly regulate ESCRT-III-membrane deforming activity during viral budding and postmitotic nuclear envelope reformation, respectively. In addition, the function of CC2D1A has been associated with cell division but its role here is under debate. In this study, the cellular roles of both CC2D1 proteins were investigated during two biological processes: 1) the regulation of ESCRT-III dependent-EGFR trafficking at the endosome, and 2) the centrosome duplication and maturation cycle during cell cycle progression, using the simultaneous siRNA-mediated knockdown of CC2D1A/B. We report that CC2D1A localises at the early endosome and the centrosome whilst CC2D1B localises at the centrosome. Depletion of CC2D1A/B resulted in the clustering of endosomes at the nucleus but did not alter the localisation of several ESCRT-III components nor impact the rate of EGFR degradation in HeLa cells. CC2D1A/B depletion led to a variety of mitotic defects such as binucleated cells, mitotic spindles and lagging chromosomes. A novel approach was utilised to synchronise RPE-1 cells in G1 using a CDK4/6 inhibitor, palbociclib. Following palbociclib release, CC2D1A/B depletion induced G1 cell cycle arrest and reduced EdU incorporation. Synchronised CC2D1A/B-depleted cells also formed micronuclei, indicating that cells had developed chromosome segregation and stability defects. In addition, the recruitment of several centrosomal components important for centriole assembly was reduced and centriole duplication was prevented. Taken together, we reveal that CC2D1A/B is important for the correct spatial distribution of endosomes, regulating G1/S transition during cell cycle progression and mediating the localisation of several centrosomal components essential for centrosome duplication.
|Date of Award||1 Aug 2020|
- The University of Manchester
|Supervisor||Philip Woodman (Supervisor), Martin Lowe (Supervisor) & Thomas Waigh (Supervisor)|
Exploring the cellular roles of CC2D1A/B
Li, C. (Author). 1 Aug 2020
Student thesis: Phd