Regulation of planer cell polarity in the mammalian auditory sensory epithelium by microtubule-mediated processes

In the mammalian organ of Corti (OC), planar polarity of individual auditory hair cells is defined by their V-shaped hair bundle. At the tissue level, all hair cells display uniform planar polarity across the epithelium. Although it is known that tissue-level planar polarity of the OC is controlled by non-canonical Wnt/planar cell polarity (PCP) signaling, the mechanisms underlying hair cellintrinsic polarity are relatively poorly understood. Genetic evidence suggests that hair bundle polarity and orientation are dictated by the position of the microtubule-based kinocilium and its associated basal body, which also organizes the cytoplasmic microtubule array. Based on this idea, I hypothesized that microtubule-mediated processes regulate hair bundle morphology and orientation and set out to investigate this idea by focusing on two microtubule motor-associated proteins. In this work, I have uncovered a role for the plus-end directed motor molecule Kif3a in regulating hair cell planar polarization through both ciliary and non-ciliary mechanisms. Kif3a coordinates hair bundle orientation with basal body positioning through localized activation of Rac-PAK (p21-activated kinase) signaling at the cortex, leading me to speculate that microtubule capture at the hair cell cortex is integral to the polarization process. To examine microtubule-cortical interactions during hair cell polarization, I also investigated the role of the Lissencephaly 1 (Lis1) gene, a major regulator of the minus-end directed cytoplasmic dynein microtubule motor. I present evidence that Lis1 regulates localized Rac-PAK signaling in embryonic hair cells, likely through microtubule-associated Tiam1, a guanine nucleotide exchange factor for iii Rac. Lis1 ablation in postnatal hair cells significantly disrupts centrosome anchoring and the normal V-shape of hair bundles, accompanied by defects in the pericentriolar matrix and microtubule organization. Together, my results describe a novel pathway that regulates the hair cell-intrinsic polarity machinery and provide important mechanistic insights into the role of microtubules during hair cell development. iv Dedication This dissertation is dedicated to my family, whose unfailing love and support has brought me to where I am today. Also to Kevin-while you never had a chance to reach these lofty academic heights, your memory continues to inspire me along my own journey.

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