Biology subjects

Kachar, B.

Publications and source records attributed to Kachar, B..

9 recordsLinked to original sources

bioRxiv2025

Kinocilia of Vestibular Hair Cells: Bridging Structural and Functional Traits of Primary and Motile Cilia

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.

neuroscience↗

bioRxiv2025

Pcdh20 is a POU2F3 target gene required for proper tuft cell microvillus formation

BACKGROUND & AIMSTuft cells play protective roles in infection, inflammation, and tumorigenesis through the secretion of cytokines and eicosanoids. Tuft cells are known for their tall, blunt microvilli, thought to be analogous to mechanosensory hair cell stereocilia; however, a functional role for the microvillar apparatus has not been identified. POU2F3 is the master regulator transcription factor for tuft cells, yet how POU2F3 drives formation of this unique structure is unknown. Here, we aimed to identify POU2F3 target genes and commonalities between tuft and hair cells to better understand this unique structure. METHODSPOU2F3 ChIP-seq was performed on tuft cells and compared to the hair cell transcriptome. Tuft cell RNA-seq datasets were interrogated for hair cell structural and mechanosensory genes; expression was validated. Intestinal and gallbladder tuft cells were examined using multiple light and electron microscopy (EM) modalities. PCDH20 was knocked down in mouse models and ultrastructural analyses were performed. The tuft cell cytoskeleton was modeled using AlphaFold3 prediction. RESULTSGenes encoding structural and mechanosensory proteins common to both tuft and hair cells, including Pcdh20, were identified. Imaging localized PCDH20 to tuft cell microvilli and hair cell stereocilia. Genetic ablation of Pcdh20 in mice resulted in structural defects in tuft cell microvilli, including loss of rigidity and organization. Molecular modeling suggests PCDH20 homodimers link adjacent microvilli. CONCLUSIONSPcdh20 is a POU2F3 target gene in tuft cells, critical to maintain the rigid microvillar apparatus. These findings, together with the shared expression of mechanosensory components like TMC1, support the hypothesis that tuft cells could have mechanosensory capabilities analogous to cochlear hair cells.

cell biology↗

bioRxiv2025

Single-cell Transcriptomics Unravel Stereocilia Degeneration as a Key Contributor to Age-related Vestibular Dysfunction in Mice and Humans

Age-related vestibular dysfunction (ARVD) is a prevalent, debilitating condition in the elderly. The etiology and molecular mechanisms are poorly understood. We focused on mechanosensitive hair cells (HCs) as they are vulnerable to aging. Using single-cell RNA-seq transcriptomes of young and old mouse vestibular HCs, we show that aging vestibular HCs display both universal molecular signatures, such as genomic instability, mitochondrial dysfunction, and impaired proteostasis, and cell type-specific changes associated with deterioration of hair bundles and mechanotransduction. In alignment with transcriptomic findings, imaging and electrophysiological recordings from aged vestibular sensory epithelia confirmed the degeneration of the hair bundles and a reduction in mechanotransduction. Importantly, this deterioration of hair bundles and vestibular function precedes HC loss, highlighting impaired mechanotransduction as a key contributor to ARVD. Furthermore, molecular and cellular changes associated with aging signatures are less pronounced in vestibular HCs than in cochlear HCs, underscoring tissue-specific age-related differences between the two sensory epithelia in the inner ear.

neuroscience↗

bioRxiv2024

Placozoan secretory cell types implicated in feeding, innate immunity and regulation of behavior

Placozoa are millimeter-sized, flat, irregularly shaped ciliated animals that crawl on surfaces in warm oceans feeding on biofilms, which they digest externally. They stand out from other animals due to their simple body plans. They lack organs, body cavities, muscles and a nervous system and have only seven broadly defined morphological cell types, each with a unique distribution. Analyses of single cell transcriptomes of four species of placozoans revealed greater diversity of secretory cell types than evident from morphological studies, but the locations of many of these new cell types were unknown and it was unclear which morphological cell types they represent. Furthermore, there were contradictions between the conclusions of previous studies and the single cell RNAseq studies. To address these issues, we used mRNA probes for genes encoding secretory products expressed in different metacells in Trichoplax adhaerens to localize cells in whole mounts and in dissociated cell cultures, where their morphological features could be visualized and identified. The nature and functions of their secretory granules were further investigated with electron microscopic techniques and by imaging secretion in live animals during feeding episodes. We found that two cell types participate in disintegrating prey, one resembling a lytic cell type in mammals and another combining features of zymogen gland cells and enterocytes. We identified secretory epithelial cells expressing glycoproteins or short peptides implicated in defense. We located seven peptidergic cell types and two types of mucocytes. Our findings reveal mechanisms that placozoans use to feed and protect themselves from pathogens and clues about neuropeptidergic signaling. We compare placozoan secretory cell types with cell types in other animal phyla to gain insight about general evolutionary trends in cell type diversification, as well as pathways leading to the emergence of synapomorphies.

evolutionary biology↗

bioRxiv2024

Hemifusomes and Interacting Proteolipid Nanodroplets: Formation of a Novel Cellular Organelle Complex

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.

cell biology↗

bioRxiv2024

Transmembrane channel-like 4 and 5 proteins at microvillar tips are potential ion channels and lipid scramblases

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.

cell biology↗

bioRxiv2024

Spontaneous calcium transients in hair cell 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. 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 functionally 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.

cell biology↗

bioRxiv2022

Selective binding and transport of protocadherin 15 isoforms by stereocilia unconventional myosins in a heterologous expression system

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.

cell biology↗

bioRxiv2019

Dynamic coupling of cochlear inner hair cell intrinsic Ca2+ action potentials to Ca2+ signaling of non-sensory cells

The relationship between Ca2+ action potential (AP) activity in immature inner hair cells (IHCs) and the spontaneous ATP-dependent intercellular Ca2+ signaling in cochlear non-sensory cells (NSCs) of the greater epithelial ridge (GER) is unclear. Here, we determined that IHCs fired asynchronous Ca2+ APs also in the absence of Ca2+ activity in the GER. Patch clamp recordings from IHCs isolated from the rest of the sensory epithelium confirmed that this firing activity is an intrinsic property of immature IHCs. However, frequency, correlation index and burst duration of IHC APs increased significantly during Ca2+ wave propagation in NSCs, and depended on wave extension in the GER. Furthermore, IHC depolarization under whole cell patch clamp conditions triggered Ca2+ signals in nearby NSCs with a delay that was proportional to the distance from the stimulated IHC. Thus the immature mammalian cochlea supports bidirectional exchange of Ca2+ signals between IHCs and NSCs.\n\nIMPACT STATEMENTIn inner hair cells of the developing mammalian cochlea, Ca2+ action potentials are both intrinsic and bidirectionally coupled to the ATP-dependent Ca2+ signaling of the surrounding non-sensory cells.

developmental biology↗