Studies on cytoplasmic microtubule organizing activity of Centrobin and CEP215 during glial differentiation of P19 cells

Abstract

Microtubule is a cytoskeleton which plays a role in cell morphology and intracellular transport. In mitotic cells, microtubules become spindles to pull a set of chromosomes into daughter cells. The centrosome is the principal organizer of microtubules in most animal cells. However, microtubule organization outside the centrosome is also observed in specialized cells, such as neurons and polarized epithelial cells. The γ-Tubulin ring complex (γ-TuRC) is a key structure for microtubule organization. Subcellular localization and activity of γ-TuRC should be tightly regulated in accord to physiological status of the cell. Here, I studied two centrosomal proteins, centrobin and CEP215, which are essential for microtubule organization outside the centrosome. In chapter 1, I observed that specific cytoplasmic centrobin is associated with stable microtubules. Centrobin is a daughter centriole-specific protein which is critical for centriole duplication. In hippocampal cells, centrobin formed cytoplasmic dots in addition to the localization at both centrosomes with the mother and daughter centrioles. Such specific localization pattern suggests that cytoplasmic centrobin is not just a reserved pool for centrosomal localization but also has a specific role in the cytoplasm. In fact, centrobin enhanced microtubule formation outside as well as inside the centrosome. These results propose specific roles of the cytoplasmic centrobin for noncentrosomal microtubule formation in specific cell types and during the cell cycle. In chapter 2, I observed specific localization of CEP215 along the processes of astrocytes in cultured mouse hippocampal cells and differentiated P19 embryonic carcinoma cells. GFAP expression and process formation were suppressed in CEP215-deleted P19 cells. The phenotypes of CEP215 deletion were rescued by ectopic Flag-CEP215, but not by Flag-CEP215ΔPCNT and Flag-CEP215F75A, which do not interact with pericentrin and γ-tubulin, respectively. I observed reduction of the centrosomal γ-tubulin levels in CEP215-deleted P19 cells. Based on the results, I propose that the microtubule nucleating function of CEP215 is essential for glial differentiation. Taken together, these results showed the importance of microtubule organizing function of centrobin and CEP215 outside the centrosome. My work is the first report of a centrosomal protein for gliogenesis and process formation.