Hearing loss (HL) is an etiologically diverse condition that can occur at any age and severity level, affecting 1 in 500 infants and more than 360 million people globally1; 2. In numerous ethnicities, we have previously identified pathogenic variants in the CIB2 gene encoding Calcium and Integrin-Binding protein 2 (CIB2) as the etiology of HL4-6. In past funding periods, we discovered that CIB2 is expressed in the mouse hair cell stereocilia and binds to the TMC1 and TMC2 components of the hair cell MET complex, and that deafness-causing CIB2 mutations disrupt these interactions. We concluded that CIB2 is essential for the MET function7. We also generated two mouse models carrying the human deafness-related Cib2 variants (Cib2F91S and Cib2R186W knock-ins) and characterized them together with a mouse line lacking CIB2 (Cib2ko). We found that Cib2F91S and Cib2ko mouse strains are deaf and lack typical MET responses in the auditory hair cells, despite the existence of tip links that are ordinarily responsible for gating the MET channels. In contrast, the p.R186W mutation does not disrupt the interaction between CIB2 and TMC1/27, and MET currents in mutant mice are diminished but still detectable. It is particularly intriguing that CIB2's participation in the MET machinery may be responsible for at least some of the several well-known effects of Ca2+ on hair cell mechanotransduction. Here, we will investigate the precise function of CIB2 in MET. We hypothesize that CIB2 is a calcium-dependent element that regulates sensitivity of the MET channels and force transmission to these channels in the mammalian auditory hair cells of. Our findings further determined that CIB2 deficiency causes an overgrowth of transducing shorter row stereocilia in the hair bundle without altering the non-transducing tallest row stereocilia7. This observation cannot be explained solely by the loss of mechanotransduction, since blockage of the MET channels causes an opposite effect, the retraction of transducing stereocilia8; 9. Hence, CIB2 must have some role in stereocilia growth, unrelated to mechanotransduction. Here, we will investigate the involvement of CIB2 in the molecular networks implicated in the development and patterning of auditory stereocilia bundles. A recent work has demonstrated functional redundancy between CIB2 and CIB3 proteins in regulating MET function3. Therefore, using in vitro and in vivo rescue experiments, we will test the hypothesis that cochlear hair cells require CIB2 for faster mechanotransduction and specific hair bundle shape. Finally, we will explore the function of CIB3 in hearing. The expected outcomes of this study are to uncover the precise mechanism of deafness associated with CIB2 deficiency, the physiological role of CIB proteins in hair cell functions, especially in MET, and their role in stereocilia staircase patterning. The potential for positive impact is inherent; deciphering the functions of HL proteins is a critical step towar...