PROJECT SUMMARY/ABSTRACT Deafness at birth frequently originates from defects in the development of sensory cells in the inner ear. Likewise, hearing degradation during life frequently follows damage sustained by these cells after normal development. In each case, a particularly susceptible cellular compartment is the hair bundle, a specialized structure in each sensory cell that detects and relays sound-borne vibrations. The hair bundle is an array of actin-based membrane protrusions, or stereocilia, precisely organized in rows of graded heights. Although the tiered architecture of the hair bundle is fundamental for sensory function, the molecular machinery required for its assembly during development and for the maintenance of its exact dimensions during life remains obscure. To address these open questions, we propose to exploit knowledge gained from our ongoing investigations of the GPSM2-Gαi protein complex in mouse. Absence of the scaffold protein GPSM2 or inhibitory G proteins (Gαi) result in defective hair bundle assembly, a likely etiology for congenital hearing loss in Chudley-McCullough syndrome. The GPSM2-Gαi protein complex is first enriched on one side of the nascent hair bundle only (the bare zone), and then enriched at the tip of stereocilia in the adjacent first row, a distribution required for proper stereocilia placement and elongation, respectively. We showed that the Myosin-15A motor transports GPSM2-Gαi to stereocilia tips, where in turn GPSM2-Gαi increases Myosin-15A amounts compared to other rows to define the tallest identity of the first row. Based on detailed preliminary data, we hypothesize that, 1) As yet unstudied Gαi regulators act as upstream cues to selectively enrich the GPSM2-Gαi complex only in the bare zone region of the apical membrane and only in a single row of stereocilia. 2) Prior GPSM2-Gαi enrichment on one side of the nascent hair bundle is the mechanism by which GPSM2-Gαi becomes restricted to abutting stereocilia in the first row, giving the hair bundle its tiered architecture. 3) Continued enrichment of GPSM2-Gαi at stereocilia tips after development has a role in maintenance of proper stereocilia height and girth in adult hair bundles. To test these hypotheses, we will: 1) Characterize the role of a negative Gαi protein regulator that we already established as a new deafness gene critical for GPSM2-Gαi complex localization and hair bundle morphogenesis. 2) Use a new chemical protein labeling technology and new reporter mouse models to track discrete pools of GPSM2- Gαi and follow their dynamic trafficking to the bare zone and stereocilia tips in time. 3) Inactivate GPSM2-Gαi function in structurally and functionally normal hair bundles in adults, and monitor stereocilia dimensions, stereocilia actin dynamics and mouse auditory function. A thorough understanding of the mechanisms that shape and preserve hair bundles will help interpret and design treatments for sensory cell dysfunction, the principal...