Structural and Molecular Basis of Transduction in Auditory Sensory Organs

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Molecular identity, structure, and elastic properties of the stereocilia tip and lateral links: Elucidating the identity, structure, and elastic properties of the tip and lateral links between stereocilia is essential for understanding their fundamental role in the process of mechanotransduction (MET). Because of their small dimensions, scarcity, and sensitivity to manipulation, the tip and lateral links are notoriously challenging to analyze morphologically. Tip and lateral links were recently examined by cryo-electron tomography (CryoET); however, they had to be splayed out and separated from their natural bundle organization to meet the freezing and imaging requirements for CryoET. We have been able to use freeze-etching combined with transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) tomography to image stereocilia links in situ, preserving both their undisturbed position and intricate conformation. In vestibular hair cells (VHCs) stereocilia bundles, we discovered an elaborate network of radial links crosslinking the hexagonally packed stereocilia within each bundle. Some of these intertwined filaments branched or made lateral contacts with neighboring filaments along the length of the stereocilia, with enrichment in shorter stereocilia. These radial links vary from 120 to 200 nm in length and show extendibility in thin sections of directly frozen and low temperature embedded tissue. Similar links were observed between the kinocilium and the neighboring stereocilia. Notably, these radial links were observed across species, including mouse, rat, and guinea pig, underscoring their significance in the dynamic properties and mechanosensitivity of hair bundles in VHCs. We are currently in the process of reconstructing the tilt series and segmenting all stereocilia links to determine their dimensions, substructure, insertion points, lateral associations, and branching patterns to elucidate their roles in bundle dynamic properties and MET. Molecular identities of components within novel vestigial kinociliary structure in inner and outer hair cells: Cilia and flagella are notable for their motile properties. By contrast, primary cilia, also known as immotile cilia, function mainly as facilitators of signal transduction. Among these various cilia, the kinocilium stands out due to its intriguing functional significance within the organ of Corti and the vestibular system. Positioned adjacent to the tallest stereocilia and connected to the stereocilia bundle, the kinocilium has sparked substantial debate concerning its dynamic properties and role in stereocilia bundle formation and MET. We have previously observed flagellar-like beating in kinocilia of frog semicircular VHCs. This observation prompted us to investigate whether the kinocilium share similarities with motile or non-motile (primary-like) cilia. While VHCs maintain kinocilia throughout life, in cochlear hair cells, kinocilia are present early postnatally but are reabsorbed as the hair cell matures, disappearing around postnatal day 12. Strikingly, using freeze-etching we observed a vestigial kinociliary structure with the ring of proteins that form the ciliary necklace. To identify kinociliary proteins, as well as novel proteins, that make up this vestigial kinociliary structure, we are comparing mRNA expression profiles of cochlear and VHCs in collaboration with David He (Creighton Univ). We have identified expression of 85 proteins associated with cilia, spanning various protein families, including CFAP, Ccdc, LRRC, WDR, DRC, NME, Dnah, Dnal, Dnnaf, IFT, TEKT, and RSPH that make up different ciliary structural features. These proteins exhibit diverse localization within different regions of the cilium, encompassing radial spoke proteins (RSP), outer and inner dynein arms (ODA and IDA), central pair complex (CPC), and Nexin dynein regulatory complex (N-DRC). Several of these proteins are distinct markers of motile cilia across a spectrum of biological systems. A specific protein, known as SPEF1 (or CLAMP), was initially discovered in our laboratory as a component of the microtubule bundle in the pillar cells of the cochlea. Our recent findings indicate a notable enrichment of this protein within VHCs relative to cochlear hair cells. Our investigative strategy revolves around leveraging comprehensive mRNA and protein databases, in combination with an exploration of the freeze-etching and CryoEM architecture of the kinocilium and the vestigial kinociliary structure in mature cochlear hair cells. Molecular mechanisms underlying membrane curvature sensing and remodeling at the stereocilia tips: The tips of stereocilia vary in shape and function depending on their ranking in the hair bundle staircase. The tallest stereocilia have oblate tips, while those in the shorter rows, where the MET channel complex is located, have prolate tips. Stereocilia prolate tips exhibit structural variability, likely reflecting local remodeling of the plasma membrane integrated into the dynamic regulation of the actin core. The molecular mechanisms by which the membrane curvature is sensed and remodeled at the stereocilia tips, however, remain unknown. Recently, the gene encoding the I-BAR protein BAIAP2L2 was associated with stereocilia shape regulation and hearing loss. We have determined that BAIAP2L2 resides in a distinct spatial compartment between the membrane and actin regulatory machinery. Further, using a heterologous co-transfection assay, we find that BAIAP2L2 self-organizes into protein condensates when it binds to components of the stereocilia MET complex and actin regulatory proteins. We propose that BAIAP2L2 forms a novel protein condensate-based scaffold that helps sculpt the membrane at stereocilia prolate tips while integrating MET and actin regulatory protein complexes. TMC4 and TMC5 in the development and function of hair cells and supporting cells: In collaboration with Drs. Jung-Bum Shin and Seham Ebrahim (UVA), we continue to investigate the role of TMC4 and TMC5, which are transiently co-expressed in hair cells and supporting cells of the developing organ of Corti and vestibular sensory epithelia. We find that when knocked out individually, these proteins do not cause any overt phenotype. We are in the process of testing whether these proteins compensate for one another.