Attachment of microtubule organizing centers to intermediate filaments in epithelial cell
Date of Award
Doctor of Philosophy (Ph.D.)
Cell Biology and Anatomy
First Committee Member
Pedro J. I. Salas - Committee Chair
In simple epithelial cells, microtubule organizing centers (MTOCs) are localized to the apical domain via their attachment to the intermediate filaments (IFs). The acquisition and maintenance of this polarized orientation is essential for epithelial cells to accomplish their functions. The release of the MTOCs from the keratin scaffold at the onset of mitosis is fulfilled by cdk1 phosphorylation. In the present work, we identified the gamma-tubulin ring complex protein 6 (GCP6) as the molecule responsible for the attachment of MTOCs to IFs in simple epithelia. GCP6 was identified as a keratin-binding partner by yeast two hybrid screens. This interaction was validated by GCP6 protein binding to keratins in vitro, and pull-down with native IFs. Phosphorylation of GCP6 by cdk1 blocked the binding to keratins. GCP6 is apical in normal enterocytes but diffuse in K8-null cells. It is also apical in normal kidney proximal tubules but it becomes cytoplasmic under ischemia or in situations that mimics this conditions. GCP6 knockdown with shRNAs in CACO-2 cells resulted in alteration of the microtubule apparatus. Expression of a small fragment GCP6 (1397-1513) that competes binding to keratins in vitro displaced gamma-tubulin from the cytoskeleton and alters distribution of microtubules and microtubule-nucleation sites. Expression of a full-length S1397D mutant in the cdk1 phosphorylation site delocalized centrosomes. Conversely, expression of a S1397A mutant affected GCP6 and centrosome distribution during mitosis.The role of GCP6 phosphorylation during kidney ischemia/reperfusion was analyzed by morphological and biochemical assays.The overall goal of this dissertation is to determine the role of GCP6 in the peculiar architecture of microtubules in polarized epithelia.
Biology, Molecular; Biology, Cell
Oriolo, Andrea Susana, "Attachment of microtubule organizing centers to intermediate filaments in epithelial cell" (2007). Dissertations from ProQuest. 2572.