| The microtubule network is an essential component of the eukaryotic cytoskeleton that serves as a transport infrastructure in interphase and organizes and segregates replicated chromosomes during cell division. In animal cells, these dynamic assemblies are nucleated and organized by the centrosome. The focus of this study was to identify and characterize centrosomal proteins with roles in two key processes: the recruitment of the microtubule-nucleating gamma-tubulin ring complex (gammaTuRC) to the centrosome and the organization of mitotic spindle fibers by centrosomes into the characteristic bipolar shape of the mitotic spindle.;In order to identify proteins required for the recruitment and activation of gammaTuRC at the centrosome, I used a biochemical approach and mass spectrometry to identify key elements of the microtubule-nucleating machinery. I identified several potential components of a salt-resistant PCM network that acts as a scaffold for gammaTuRC recruitment, but I was unable to biochemically identify factors that anchor gammaTuRC to this PCM scaffold. I generated a large list of substoichiometric gammaTuRC-copurifying proteins that is likely to contain gammaTuRC anchoring factors, and then screened a subset of this list for RNAi phenotypes associated with this activity in Drosophila S2 cells. However, none of the proteins appear to be required for gammauRC recruitment to the centrosome or for the formation of astral microtubules from the centrosome, although several have defective mitotic spindle formation phenotypes indicative of roles in spindle assembly.;I also used RNAi, GFP-fusion expression, immunolocalization, and fixed and live-cell microscopy to characterize the role of a new centrosomal protein, Dmob4, in mitotic spindle assembly in S2 cells. This study revealed that Dmob4 is required for centrosome separation and spindle pole organization during mitosis. Dmob4's RNAi phenotype most closely resembles that observed after abnormal spindle protein (Asp) depletion, although Asp localization is not substantially effected by Dmob4 RNAi. Expression of a Dmob4-GFP fusion protein revealed its localization to the nucleus in interphase and to spindle poles and kinetochores during mitosis. We propose that Dmob4 controls a mitotic kinase that in turn regulates downstream target proteins involved in centrosome separation and K fiber focusing at the poles. |
