Bechara Kachar

Vestibular hair cells (HCs) convert gravitational and head motion cues into neural signals through mechanotransduction, mediated by the hair bundle—a mechanically integrated organelle composed of stereocilia and a kinocilium. The kinocilium, a specialized form of primary cilium, remains incompletely defined in structure, molecular composition, and function. To elucidate its characteristics, we conducted single-cell RNA sequencing of adult vestibular and cochlear HCs, uncovering a selective enrichment of primary and motile cilia-associated genes in vestibular HCs, particularly those related to the axonemal repeat complex. This enrichment of orthologous axoneme-related genes was conserved in zebrafish and human vestibular HCs, indicating a shared molecular architecture. Immunostaining validated the expression of key motile cilia markers in vestibular kinocilia. Moreover, live imaging of bullfrog and mouse HCs from crista ampullaris revealed spontaneous kinociliary motion. Together, these findings define the kinocilium as a unique organelle with molecular features of primary and motile cilia and suggest its previously unknown role as an active, force-generating element within the hair bundle.

Vestibular hair cells (HCs) convert gravitational and head motion cues into neural signals through mechanotransduction, mediated by the hair bundle—a mechanically integrated organelle composed of stereocilia and a kinocilium. The kinocilium, a specialized form of primary cilium, remains incompletely defined in structure, molecular composition, and function. To elucidate its characteristics, we conducted single-cell RNA sequencing of adult vestibular and cochlear HCs, uncovering a selective enrichment of primary and motile cilia–associated genes in vestibular HCs, particularly those related to the axonemal repeat complex. This enrichment of orthologous axonemal-related genes was conserved in zebrafish and human vestibular HCs, indicating a shared molecular architecture. Immunostaining validated the expression of key motile cilia markers in vestibular kinocilia. Moreover, live imaging of bullfrog and mouse HCs from crista ampullaris revealed spontaneous kinociliary motion. Together, these findings define the kinocilium as a unique organelle with molecular features of primary and motile cilia and suggest its previously unknown role as an active, force-generating element within the hair bundle.

The hair bundle of auditory and vestibular hair cells converts mechanical stimuli into electrical signals through mechanoelectrical transduction (MET). The MET apparatus is built around a tip link that connects neighboring stereocilia that are aligned in the direction of mechanosensitivity of the hair bundle. Upon stimulation, the MET channel complex responds to changes in tip-link tension and allows a cation influx into the cell. Ca²⁺ influx in stereocilia has been used as a signature of MET activity. Using genetically encoded Ca²⁺ sensors (GCaMP3, GCaMP6s) and high-performance fluorescence confocal microscopy, we detect spontaneous Ca²⁺ transients in individual stereocilia in developing and fully formed hair bundles. We demonstrate that this activity is abolished by MET channel blockers and thus likely originates from putative MET channels. We observe Ca²⁺ transients in the stereocilia of mice in tissue explants as well as in vivo in zebrafish hair cells, indicating this activity is evolutionarily conserved. Within stereocilia, the origin of Ca²⁺ transients is not limited to the canonical MET site at the stereocilia tip but is also present along the stereocilia length. Remarkably, we also observe these Ca²⁺ transients in the microvilli-like structures on the hair cell surface in the early stages of bundle development, prior to the onset of MET. Ca²⁺ transients are also present in the tallest rows of stereocilia in auditory hair cells, structures not traditionally thought to contain MET channels. We hypothesize that this newly described activity may reflect stochastic and spontaneous MET channel opening. Localization of these transients to other regions of the stereocilia indicates the presence of a pool of channels or channel precursors. Our work provides insights into MET channel assembly, maturation, function, and turnover. .

The pleiomorphic structure and dynamic behavior of cellular endomembrane systems have been extensively studied using classical electron microscopy. However, fixation and staining constraints limit the in situ visualization of transient interactions, such as membrane fusion, scission, and intraluminal vesicle formation, potentially overlooking intermediate structures like membrane hemifusion. Using in situ cryo-electron tomography in four mammalian cell lines, we identify heterotypic hemifused vesicles featuring an extended hemifusion diaphragm consistently associated with a 42-nanometer proteolipid nanodroplet (PND). We designate these vesicular organelle complexes as “hemifusomes.” Hemifusomes constitute up to 10% of vesicular organelles at the cell periphery but do not engage in canonical endocytic pathways. These structures exhibit diverse conformations and frequently contain intraluminal vesicles. Building on the continuum of related morphologies observed, we propose that hemifusomes serve as platforms for vesicular biogenesis, mediated by the PND. These findings provide direct in situ evidence of long-lived hemifused vesicle complexes and introduce an ESCRT-independent model for multivesicular body (MVB) formation.

Vestibular hair cells (HCs) convert gravitational and head motion cues into neural signals through mechanotransduction, mediated by the hair bundle—a mechanically integrated organelle composed of stereocilia and a kinocilium. The kinocilium, a specialized form of primary cilium, remains incompletely defined in structure, molecular composition, and function. To elucidate its characteristics, we conducted single-cell RNA sequencing of adult vestibular and cochlear HCs, uncovering a selective enrichment of primary and motile cilia–associated genes in vestibular HCs, particularly those related to the axonemal repeat complex. This enrichment of orthologous axonemal-related genes was conserved in zebrafish and human vestibular HCs, indicating a shared molecular architecture. Immunostaining validated the expression of key motile cilia markers in vestibular kinocilia. Moreover, live imaging of bullfrog and mouse HCs from crista ampullaris revealed spontaneous kinociliary motion. Together, these findings define the kinocilium as a unique organelle with molecular features of primary and motile cilia and suggest its previously unknown role as an active, force-generating element within the hair bundle.

The complex, pleiomorphic membrane structure of the vesicular components within the endolysosomal system has been appreciated through decades of classical electron microscopy. However, due to the heavy fixation and staining required in these approaches, in situ visualization of fragile intermediates between early endosomes, late endosomes and ultimately multivesicular bodies (MVBs), remains elusive, raising the likelihood that other structures may have also been overlooked. Here, using in situ cryo-electron tomography in four mammalian cell lines, we discover heterotypic hemifused vesicles that share an extended hemifusion diaphragm, associated with a 42nm proteolipid nanodroplet (PND). We term this previously undescribed vesicular organelle-complex, “hemifusome”. Hemifusomes make up approximately 10% of the organelle pool of the endolysosomal system, but do not participate directly in transferrin-mediated endocytosis. Hemifusomes exist in compound conformations and also contain intraluminal vesicles. Based on their range of morphologies, and the consistent presence of the PND at sites of compound hemifused vesicles, we propose that hemifusomes function as platforms for vesicular biogenesis mediated by the PND. These findings offer direct in situ evidence for a long-lived hemifusion diaphragm, and a new, ESCRT-independent model for the formation of late endosomes containing intraluminal vesicles and ultimately MVBs.

Within cells, vesicle fusion, scission, and the formation of intraluminal vesicles are critical processes that facilitate traffic, degradation, and recycling of cellular components, and maintenance of cellular homeostasis. Despite significant advancements in elucidating the molecular mechanisms that drive these dynamic processes, the direct in situ visualization of membrane remodeling intermediates remains challenging. Here, through the application of cryo-electron tomography in mammalian cells, we have identified a previously undescribed vesicular organelle complex with unique membrane topology: heterotypic hemifused vesicles that share extended hemifusion diaphragms (HDs) with a 42 nm proteolipid nanodroplet (PND) at their rim. We have termed these organelle complexes “hemifusomes”. The HDs of hemifusomes exhibit a range of sizes and curvatures, including the formation of lens-shaped compartments encapsulated within the membrane bilayer. The morphological diversity of the lens-shaped vesicle aligns with a step-wise process of their intraluminal budding, ultimately leading to their scission and the generation of intraluminal vesicles. We propose that hemifusomes function as versatile platforms for protein and lipid sorting and as central hubs for the biogenesis of intraluminal vesicles and the formation of multivesicular bodies.

Microvilli—membrane bound actin protrusions on the surface of epithelial cells—are sites of critical processes including absorption, secretion, and adhesion. Increasing evidence suggests microvilli are mechanosensitive, but underlying molecules and mechanisms remain unknown. Here, we localize transmembrane channel-like proteins 4 and 5 (TMC4 and 5) and calcium and integrin binding protein 3 (CIB3) to microvillar tips in intestinal epithelial cells, near glycocalyx insertion sites. We find that TMC5 colocalizes with CIB3 in cultured cells and that a TMC5 fragment forms a complex with CIB3 in vitro . Homology and AlphaFold2 models reveal a putative ion permeation pathway in TMC4 and 5, and molecular dynamics simulations predict both proteins can conduct ions and perform lipid scrambling. These findings raise the possibility that TMC4 and 5 interact with CIB3 at microvillar tips to form a mechanosensitive complex, akin to TMC1 and 2, and CIB2 and 3, within the mechanotransduction channel complex at the tips of inner ear stereocilia.

The hair bundle of auditory and vestibular hair cells converts mechanical stimuli into electrical signals through mechanoelectrical transduction (MET). The MET apparatus is built around a tip link that connects neighboring stereocilia that are aligned in the direction of mechanosensitivity of the hair bundle. Upon stimulation, the MET channel complex responds to changes in tip-link tension and allows a cation influx into the cell. Ca ²⁺ influx in stereocilia has been used as a signature of MET activity. Using genetically encoded Ca ²⁺ sensors (GCaMP3, GCaMP6s) and high-performance fluorescence confocal microscopy, we detect spontaneous Ca ²⁺ transients in individual stereocilia in developing and fully formed hair bundles. We demonstrate that this activity is abolished by MET channel blockers and thus likely originates from putative MET channels. We observe Ca ²⁺ transients in the stereocilia of mice in tissue explants as well as in vivo in zebrafish hair cells, indicating this activity is functionally conserved. Within stereocilia, the origin of Ca ²⁺ transients is not limited to the canonical MET site at the stereocilia tip but is also present along the stereocilia length. Remarkably, we also observe these Ca ²⁺ transients in the microvilli-like structures on the hair cell surface in the early stages of bundle development, prior to the onset of MET. Ca ²⁺ transients are also present in the tallest rows of stereocilia in auditory hair cells, structures not traditionally thought to contain MET channels. We hypothesize that this newly described activity may reflect stochastic and spontaneous MET channel opening. Localization of these transients to other regions of the stereocilia indicates the presence of a pool of channels or channel precursors. Our work provides insights into MET channel assembly, maturation, function, and turnover.

During hair cell development, the mechanoelectrical transduction (MET) apparatus is assembled at the stereocilia tips, where it coexists with the stereocilia actin regulatory machinery. While the myosin-based tipward transport of actin regulatory proteins is well studied, isoform complexity and built-in redundancies in the MET apparatus have limited our understanding of how MET components are transported. We used a heterologous expression system to elucidate the myosin selective transport of isoforms of protocadherin 15 (PCDH15), the protein that mechanically gates the MET apparatus. We show that MYO7A selectively transports the CD3 isoform while MYO3A and MYO3B transports the CD2 isoform. Furthermore, MYO15A showed an insignificant role in the transport of PCDH15, and none of the myosins tested transport PCDH15-CD1. Our data suggest an important role for MYO3A, MYO3B, and MYO7A in the MET apparatus formation and highlight the intricate nature of MET and actin regulation during development and functional maturation of the stereocilia bundle.