Stereocilia are actin-based cell protrusions of inner ear hair cells that play an essential role in mechano-electrical transduction (MET). Stereocilia are organized into several rows of increasing heights with the MET protein complex localized at the tips of shorter row stereocilia. At the tips of shorter row mechanotransducing stereocilia also resides a so-called "row 2 protein complex" whose dysfunction causes degeneration of the mechanotransducing stereocilia. In the present work, we show that BAIAP2L2 is localized at the tips of shorter row stereocilia in neonatal and adult mouse cochlear hair cells. Baiap2l2 inactivation causes degeneration of the mechanotransducing stereocilia, which eventually leads to profound hearing loss in mice of either sex. Consistently, electrophysiology and FM 1-43FX dye uptake results confirm that MET currents are compromised in Baiap2l2 knockout mice.Moreover, BAIAP2L2 binds to known row 2 complex components EPS8L2, TWF2, and CAPZB2, and the stereociliary tip localization of CAPZB2 is dependent on functional BAIAP2L2. Interestingly, BAIAP2L2 also binds to CIB2, a known MET complex component, and the stereociliary tip localization of BAIAP2L2 is abolished in Cib2 knockout mice. In conclusion, our present data suggest that BAIAP2L2 is a row 2 complex component, and is required for the maintenance of mechanotransducing stereocilia. Meanwhile, specific MET components such as CIB2 might play a direct role in stereocilia maintenance through binding to BAIAP2L2.

The mechanoelectrical transduction (MET) protein complex in the inner-ear hair cells is essential for hearing and balance perception. Calcium and integrin-binding protein 2 (CIB2) has been reported to be a component of MET complex, and loss of CIB2 completely abolishes MET currents in auditory hair cells, causing profound congenital hearing loss. However, loss of CIB2 does not affect MET currents in vestibular hair cells (VHCs) as well as general balance function. Here we show that CIB2 and CIB3 act redundantly to regulate MET in VHCs, as MET currents are completely abolished in the VHCs ofCib2/Cib3double knockout mice of either sex. Furthermore, we show thatCib2andCib3transcripts have complementary expression patterns in the vestibular maculae, and that they play different roles in stereocilia maintenance in VHCs.Cib2transcripts are highly expressed in the striolar region, and knockout ofCib2affects stereocilia maintenance in striolar VHCs. In contrast,Cib3transcripts are highly expressed in the extrastriolar region, and knockout ofCib3mainly affects stereocilia maintenance in extrastriolar VHCs. Simultaneous knockout ofCib2andCib3affects stereocilia maintenance of all VHCs and leads to severe balance deficits. Taken together, our present work reveals that CIB2 and CIB3 are important for stereocilia maintenance as well as MET in mouse VHCs.SIGNIFICANCE STATEMENT:CIB2 is an important component of mechanoelectrical transduction (MET) complex, and loss of CIB2 completely abolishes MET in auditory hair cells. However, MET is unaffected inCib2knockout vestibular hair cells (VHCs). In the present work, we show that CIB3 could compensate for the loss of CIB2 in VHCs, andCib2/Cib3double knockout completely abolishes MET in VHCs. Interestingly, CIB2 and CIB3 could also regulate VHC stereocilia maintenance in a non-redundant way.Cib2andCib3transcripts are highly expressed in the striolar and extrastriolar regions, respectively. Stereocilia maintenance and balance function are differently affected inCib2orCib3knockout mice. In conclusion, our data suggest that CIB2 and CIB3 are important for stereocilia maintenance and MET in mouse VHCs.

Hair cells are mechanosensitive cells in the inner ear, characterized by dozens to hundreds of actin-based stereocilia and one tubulin-based kinocilium on the apical surface of each cell. Two types of hair cells, namely cochlear hair cells and vestibular hair cells (VHCs), are responsible for the sensation of sound and balancing information, respectively. In each hair cell, the stereocilia are organized into rows of increasing heights with the mechano-electrical transduction (MET) channels localized at the tips of shorter-row stereocilia. A so-called “row 2 protein complex” also localizes at the tips of shorter-row mechanotransducing stereocilia, which plays important roles in the maintenance of mechanotransducing stereocilia. Recently, we and others identified BAIAP2L2 as a new component of row 2 complex. Baiap2l2 inactivation causes degeneration of the mechanotransducing stereocilia in cochlear hair cells, and leads to profound hearing loss in mice. In the present work, we examined the role of BAIAP2L2 in the VHC stereocilia. Confocal microscopy reveals that BAIAP2L2 immunoreactivity is localized at the tips of shorter-row stereocilia in VHCs. However, stereocilia development and maintenance are unaffected in Baiap2l2–/– VHCs. Meanwhile, MET function of VHCs as well as vestibular functions are also unaffected in Baiap2l2–/– mice. Further investigations show that the stereociliary tip localization of CAPZB2, another known row 2 complex component, is not affected in Baiap2l2–/– VHCs, consistent with the unaltered stereocilia morphology. Taken together, our present data show that BAIAP2L2 inactivation does not affect vestibular hair cell stereocilia.