The actin-based stereociliary bundle (or hair bundle) on the apex of auditory hair cells serves the critical function of converting sound energy to electric signals. Its V-shaped staircase structure renders the bundle directionally sensitive to mechanical stimuli. As such, auditory hair bundles must be uniformly oriented for correct sound transduction. Abnormalities in hair bundle polarity or orientation cause deafness and hearing impairment. A long-term objective of this work is to gain a detailed understanding of the hair bundle morphogenesis programs and how genetic mutations that disrupt these programs cause sensorineural deafness. In particular, mutations in a gene named GPSM2 cause the human hereditary deafness DFNB82 and Chudley-McCullough Syndrome. However, the underlying disease mechanisms remain completely unknown. Our recent insights about a microtubule-mediated pathway in hair cells for hair bundle polarity and orientation suggest a novel testable hypothesis about GPSM2's role in this pathway. Specifically, we have uncovered a critical and previously unappreciated function of the hair cell microtubules and microtubule-based molecular motors in basal body positioning, which is critical for both hair bundle polarity and orientation. We found that hair cells deficient in either the kinesin-II subunit Kif3a or the dynein regulator Lis1 have basal body positioning defects. Consequently, both the polarized V-shape and orientation of the hair bundles are disrupted. We further demonstrate that these microtubule motors regulate an asymmetric domain of Rac GTPase-PAK signaling on the hair cell cortex to mediate basal body positioning. The major goal of this research is to further dissect the molecular components of this microtubule-mediated pathway, including the deafness gene GPSM2, and gain mechanistic insights into microtubule regulation of hair bundle polarity. Our goal will be pursued through the following specific aims. Aim 1 will test the hypothesis that the cell polarity proteins Par 3 and GPSM2 serve as cortical landmarks to tether dynein at the cortex to pull on microtubules and orient the basal body, similar to mechanisms that orient the mitotic spindle during asymmetric cell division. Aim 2 will test the hypothesis that kinesin-II mediated targeted delivery of Par3 and the Rac activator Tiam1 to the cell cortex is critical for spatial regulation of Rac signaling and basal body positioning. Aim 3 will use innovative live imaging to test the hypothesis that PAK signaling regulates both microtubule stability and cortical proteins to stabilize microtubule plus-end attachment at the cell cortex. This research will provide new avenues of investigation into hair cell development and elucidate the function of poorly understood human deafness genes. Gaining a deeper understanding of the hair bundle morphogenesis program will be essential for devising rational therapies to stimulate hair bundle repair following injury, to treat hereditary human deafness and to regenerate auditory hair cells through stem cell technologies.