Structural and Molecular Basis of Transduction in Auditory Sensory Organs

National Institute On Deafness And Other Communication Disorders

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Abstract

The hair-cell mechanosensory organelle — the hair bundle — consists of a bundle of stereocilia and a single kinocilium and is essential for hearing and balance. Stereocilia contain a densely packed actin core, capped at the tip and tapered at the base, where a rootlet anchors them to the cuticular plate; disruptions to these structures contribute to both genetic and age-related hearing loss. In contrast, the kinocilium is a microtubule-based organelle that plays critical roles in hair bundle development and planar cell polarity, structurally resembling both motile and primary cilia. A central aim of our ongoing work is to generate high-resolution structural data from both stereocilia and kinocilia using cryo-electron tomography (cryo-ET) and to integrate these findings with transcriptomic and proteomic analyses to define the molecular architecture of the hair bundle. A comprehensive molecular blueprint of the hair bundle will advance our understanding of the mechanisms underlying pathologies that cause hearing and vestibular disorders and support the development of targeted therapeutic strategies. This year, we expanded the molecular profile of vestibular kinocilia by confirming the expression of TEKT1, DNAH5, and DNAH6 through antibody-labeling. These findings support the presence of motility-related proteins beyond previous reports. Integrating transcriptomic data with high-resolution structural frameworks from mammalian motile cilia, we modeled the 96-nm axonemal repeat of vestibular kinocilia. Most axonemal complexes were present, including dynein arms, radial spokes, N-DRC components, and MIPs, while DNAH3, DNAH12, and PIERCE1/2 were absent—possibly explaining the unique dynamic properties of kinocilia. To test for dynamic behavior, we performed live vestibular culture imaging and detected subtle spontaneous kinocilia motion. Further photodiode-based experiments in collaboration with David He (Creighton University) using acute mouse ampullary sensory epithelia revealed distinct small-amplitude kinocilia oscillations. These findings provide the first direct evidence of motility in mammalian vestibular kinocilia and suggest distinct molecular adaptations across species. Comparative transcriptomics among hair cells from various species indicated that zebrafish kinocilia retain DNAH3 and DNAH12, supporting a model of evolutionary divergence, where zebrafish preserve a more complete motility program than mammals. Building on our cryo-ET pipeline developed last year, we improved the quality of datasets acquired from vestibular kinocilia and initiated template matching, which enabled the detection of periodic axonemal structures, including outer dynein arms. These results provide a foundation for generating high-resolution density maps using single-particle cryo-EM approaches. While kinocilia cryo-ET data are amenable to cryo-EM single-particle approaches due to their regular architecture, obtaining high-resolution structural information for stereocilia remains a significant challenge. These actin-rich organelles exhibit considerable structural complexity, characterized by densely packed filaments and crosslinkers that vary across hair bundle rows. To address this gap, we combined and curated datasets generated in our laboratory with an additional archival dataset (EMPIAR-10898). From approximately 600 tilt-series acquired from mouse and rat vestibular tissues, 39 were selected for tomographic reconstruction and analysis. Sub volume averaging and template matching of actin filament segments enabled the generation of 3D models revealing multiple structural classes, ranging from hexagonal to orthogonal filament packing geometries. This heterogeneity suggests an inherent, biologically meaningful flexibility and structural variability in stereocilia organization, likely facilitating dynamic regulation of their shape and length. We hypothesize that hexagonal packing is mediated by actin crosslinkers such as espin, fascin, and plastin, while orthogonal packing is primarily supported by α-actinin. Additionally, we incorporated cryo-FIB milling, which improved lamella preparation and enhanced the visibility of actin filament packing. These advances represent important progress toward resolving the three-dimensional architecture of stereocilia and kinocilia in situ. We also examined stereocilia tip morphology, a critical site for both mechanotransduction and actin regulation. Using cryo-ET, we observed structural variability: in some stereocilia, actin filaments extended to the submembrane complex, while in others, they terminated more proximally. These differences are consistent with localized control of actin dynamics. Recent studies have identified several proteins involved in this regulation, including BAIAP2L2, an I-BAR domain-containing protein, as a key modulator of stereocilia tip architecture. Our data indicate that BAIAP2L2 colocalizes with Espin-1, supporting the view that these proteins interact to regulate stereocilia tip morphology and length, likely through specific binding interactions and the formation of protein condensates via phase separation. Due to molecular crowding at the tips, direct visualization of individual complexes remains challenging; however, denoising algorithms are improving the visibility of structural features. We anticipate that increasing the number of tomograms, combined with class averaging, will enhance molecular complex identification. In parallel, we are applying advanced imaging techniques to investigate the membrane compartments involved in protein and lipid trafficking and sorting in hair cells and model cell cultures. Using cryo-ET, we discovered a novel vesicular organelle—the hemifusome—in cultured mammalian cells. Hemifusomes represent an innovative paradigm in organelle biology. Their identification through in situ cryo-ET has revealed a previously unrecognized mechanism of vesicle formation that operates independently of the canonical ESCRT machinery. Hemifusomes are distinguished by unique structural features, including an extended hemifusion diaphragm and a conspicuously associated 42-nm proteolipid nanodroplet. These features support a functional role in vesicular biogenesis, where hemifusomes contribute to the formation of MVBs. Our initial dataset of ~300 tomograms across four cell lines has expanded to over 800, now incorporating tomograms from an inner ear–derived line. Notably, ribosome association is frequently observed, with ribosomes aligned in a configuration resembling endoplasmic reticulum (ER)-associated ribosomes, further implicating a functional or structural link to the ER. In summary, hemifusome-derived MVBs differ from canonical endosome-derived MVBs in three key ways: they do not incorporate endocytic tracers, suggesting they bypass the endolysosomal pathway; they lack ESCRT coats, which are characteristic of conventional MVBs; and they exhibit ribosome decoration, implying an ER origin or direct ER association. These findings support the existence of an alternative, ER-linked pathway for MVB biogenesis. Hemifusomes appear relatively abundant, constituting approximately 1–10% of vesicular organelles, offering new insight into non-canonical membrane remodeling and potentially localized protein synthesis. Additionally, cryo-ET imaging of peripheral cellular regions revealed lattice-like coats on late endosomes, positioned ~25 nm from the limiting membrane and extending up to 200 nm. These flat, rod-like arrays differ structurally from clathrin coats and may stabilize planar endosomal domains. Their dimensions are consistent with septin filaments, and we typically observed one or two such arrays per endosome. Ongoing studies using marker co-labeling aim to identify their molecular composition. Despite these scientific advances, lab productivity continues to be impacted by unequal and unprecedented staffing restrictions imposed specifically on our lab by NIDCD leadership. The Board of Scientific Counselors reviewed the lab this year and noted: “Dr. Kachar has refocused his efforts and heavily invested in the development of cryo-EM, Cryo-FIB, and cryo-ET technologies, which require significant time. He has made impressive progress in developing these cutting-edge technologies, which are expected to generate impactful results.” “There are no other teams better trained to perform such high-quality work.” “The program has had low productivity as a result of restrictions enforced upon Dr. Kachar, including hiring restrictions and loss of resources”. “It is difficult to imagine how this extensive research plan can be executed, not only because of the lack of ‘hands’ in the lab, but also of ‘brains’ to discuss the projects and develop ideas.”