Abstract Sensory hair cells of the inner ear experience continuous mechanical and metabolic stress. The maintenance of hair cells is further challenged by damage from a variety of other ototoxic factors, including loud noise, aging, genetic defects, and ototoxic drugs. Because mammalian auditory hair cells do not regenerate, the repair of hair cell damage is important for continued auditory function. Our research program is especially interested in molecular processes involved in the maintenance of the stereocilia filamentous (F)-actin core. Recent studies have concluded that the stereocilia actin core is stable over months, implying that any structural damage must be actively repaired. The stereocilia F-actin core can sustain damage, most notably by noise exposure, which was shown to cause “gaps” in phalloidin labeling of F-actin in stereocilia. In preliminary studies, we found that these gaps are repaired in days. We therefore propose to investigate the molecular mechanisms by which the F-actin lesions are sensed and repaired. The proposed study was inspired by an emerging concept in mechanobiology, according to which F-actin possesses intrinsic mechanosensory properties. In this model, mechanical strain modulates the interaction of actin filaments with effector proteins. For a variety of actin binding proteins, their constitutive binding to F-actin is merely tuned by force. A subset of LIM domain proteins however are unique in that mechanical strain reveals previously hidden binding sites on F-actin, providing an on/off switch for downstream processes. These processes were implicated in the recruitment of actin repair substrates and in the prevention of F-actin fiber breakage. We reasoned that hair cells might employ a similar strategy to repair its F-actin-based stereocilia. In our search for molecules involved in this process, we focused on proteins that are enriched in the hair cell bundle, contain potential mechanosensor domains, and cause progressive hearing loss in human or mice with loss of function. We identified two proteins, XIRP2 (Xin Actin Binding Repeat Containing 2) and CRIP3 (cysteine rich protein 3) that fulfill these criteria. We hypothesize that XIRP2 and CRIP3 are mechanosensor proteins capable of sensing F-actin damage and recruiting additional repair factors, thus playing essential roles in hair cell stereocilia repair and maintenance. To test this, in SA1, we propose to test the hypothesized mechanosensor function of XIRP2 in fibroblasts. In preliminary studies, we discovered a novel mechanosensor domain in the C-terminus of XIRP2. We will use live cell laser ablation and cell stretch experiments to define the mechanosensor region, and investigate the mechanisms by which XIRP2’s mechanosensor function is regulated. In SA2, we propose to test the mechanosensor and repair function of XIRP2 in vivo, using a mouse model that lacks the mechanosensor domain. We will also perform ex vivo experiments to test whether fluorescently...