Usher Proteins in the Inner Ear Structure and Function

Hearing loss (HL) is an etiologically heterogeneous trait that can occur at any age and degree of severity, that affects 1 in 500 newborns and >360 million people worldwide{Morton, 2006 #6;Morton, 1991 #5}. We and others previously identified the CIB2 gene encoding Calcium and Integrin-Binding protein 2 (CIB2) as the cause of HL in diverse populations{Patel, 2015 #561;Riazuddin, 2012 #68;Seco, 2016 #604;Booth, 2018 #93;Michel, 2017 #95}. CIB2 is localized in the hair cell stereocilia in rodents, and loss of CIB2 function results in profound deafness in mice with no conventional mechano-electrical transduction (MET) in the auditory hair cells, despite the presence of tip links that normally gate the MET channels{Wang, 2017 #92;Michel, 2017 #95;Giese, 2017 #97}, and despite apparently unchanged localization of mutant CIB2 in the stereocilia of knockin mice (Cib2 F91S/F91S). We also found that CIB2 binds to the MET complex components, TMC1 and TMC2, and these interactions are disrupted by deafness-causing Cib2 variants. We concluded that CIB2 is essential for the MET function{Giese, 2017 #859}. Recently, studies further confirm the role of CIB protein in MET function{Liang, 2021 #89}, however, precise mechanism remains elusive. As an auxiliary unit of the MET complex, CIB2 is particularly interesting because it may be responsible for at least some of well-known effects of Ca2+ on the hair cell MET. In this project, we will explore the precise role of CIB2 in MET function. Towards this end, we have generated a knock-in mouse strain (Cib2R186W). The p.R186W mutation does not affect CIB2 interaction with TMC1/2{Giese, 2017 #859}, and mutant mice have decreased but detectable MET currents. Our preliminary data show that activation of MET currents is slower in Cib2R186W/R186W hair cells, which indicates the impairment of force transmission to the MET channels. Here, we will test the hypothesis that CIB2 is a calcium-dependent link required for force transmission to the MET channels in mammalian auditory hair cells. Our studies also established that CIB2 deficiency results in abnormal growth of the transducing shorter row stereocilia in the hair bundle without affecting non-transducing tallest row stereocilia{Giese, 2017 #859}. Our new data suggests that CIB2 interacts with MPDZ, a scaffolding protein involves in the maintenance of stereocilia actin cytoskeleton in auditory hair cells{Jarysta, 2021 #110}. MPDZ is also required there to maintain the proper segregation of apical blueprint proteins, including GαI-GPSM2{Jarysta, 2021 #110}. We found also deficits in apical-medial targeting of GαI-GPSM2 complex proteins in Cib2 mutant mice. Here, we will test the hypothesis that CIB2 regulates stereocilia height regulation via interaction with MPDZ-GαI-GPSM2 complex. Finally, recent study has shown functional redundancy between CIB2 and CIB3 protein in regulating the MET function at least in vitro{Liang, 2021 #89}. Therefore, we will investigate the in vivo MET and stereocilia growth functional sharing between CIB2 and CIB3. The goal of this study is to determine the role of CIB2 in the auditory hair cell structure and MET function as well as the mechanisms underlying hearing impairment caused by CIB2 variants. Our specific aims are: Aim 1: To determine the role of CIB2 in hair cell mechanotransduction. Using whole cell patch clamp, we will investigate activation of MET currents in the auditory hair cells of Cib2 mutant (Cib2R186W/R186W) and control (Cib2+/+, Cib2+/R186W) mice. We will use a custom piezo-driven probe that can deflect stereocilia bundle in ~10 ms or less. Ca2+ sensitivity of MET activation will be tested by changing extracellular Ca2+ concentration and by depolarizing the cell, thereby inhibiting Ca2+ influx through the MET channels. We will also record the MET complex stiffness and overall stiffness of the hair bundle by small non-saturating stereocilia deflections with a fluid jet (controlled by simultaneous patch clamp recordings of MET currents) and high-speed camera imaging. Results of these studies will validate the role of CIB2 as an essential Ca2+-dependent mechanical link conveying the force to the MET channels. Aim 2: To elucidate the mechanisms of CIB2 involvement in regulation of stereocilia height. Here, we will: (i) examine the structural changes at the stereocilia tips in Cib2 knockout (Cib2ko/ko), and knockin (Cib2F91S& Cib2R186W) mice auditory hair cells using backscattered scanning electron microscopy combined with focus ion beam serial sectioning (FIB-SEM); (ii) further explore interactions of CIB2 (and its mutated variants) with MPDZ and other GαI-GPSM2 complex proteins, both in vitro and in vivo; (iii) investigate the targeting of MPDZ-GαI- GPSM2 complex proteins in our Cib2 knockout (Cib2ko/ko), and knockin (Cib2F91S & Cib2R186W ) mice; (iv) document MPDZ-GαI-GPSM2 complex proteins localization at the tips of stereocilia, in relation to remove of CIB2 in structurally normal stereocilia using Cib2flox and inducible Cre+ expresser mice. Finally, as an alternate reason for abberant stereocilia architecture in Cib2 mutant mice, we will investigate stereocilia membrane homeostasis, including phosphatidylserine externalization, membrane blebbing, and ectosome release in these mice.(Add ref) Aim 3: To explore the functional sharing between CIB2 and CIB3 in activation of MET complexes and stereocilia height regulation. Recent study has shown that the amplitude of MET currents in the auditory hair cells of Cib2ko/ko mice could be rescued by the expression of wildtype CIB2 and its homolog CIB3 that is expressed in slower vestibular hair cells{Jarysta, 2021 #110}. Based on our preliminary studies, we hypothesis that CIB2 confers faster activation to the MET complex compared to CIB3, and have unique role in stereocilia growth. To test this, we will (i) generate AlphaFold 2 models of TMC1 proteins alone and in complex with CIB2 and CIB3 proteins to identify key structural differences; (ii) administer wildtype purified CIB2 and CIB3 proteins (various concentrations) via the patch pipette into Cib2ko/ko hair cells and examine the activation of MET currents; (iii) perform adeno-associated virus (AAV) based CIB2 and CIB3 gene delivery in Cib2ko/ko mice at various developmental stages (P0, P3, P5, P10), and study the impact on the stereocilia height via high resolution confocal imaging and SEM. Results of these studies will help to explain why the auditory hair cells express high levels of CIB2 as compared to other members of CIB family.