Stereocilia are giant, specialized microvilli on auditory sensory cells in the inner ear. Their precise size and tiered organization are essential for function and are strictly controlled by actin-regulating proteins during postnatal development. Among these proteins, Espins (ESPNs) and Espin-like (ESPNL) proteins contribute to normal stereocilia development; however, the potential overlapping function between these families has not been explored. We found that ESPNL exists as both a long isoform and a novel short isoform. The short isoform localized differently from the long isoform and was lost from stereocilia when mechanotransduction was blocked. To understand how long isoforms contribute to stereocilia architecture, we generated mutations causing their loss while sparing short isoforms. While mutating ESPN-1 had little effect on stereocilia morphology or auditory function, loss of the long ESPNL isoform caused degeneration of shorter, mechanotransducing stereocilia rows, resulting in progressive hearing loss. Double mutant mice lacking both ESPN and ESPNL long isoforms showed a more rapid onset of the same phenotype, demonstrating that these proteins both contribute to mechanotransducing stereocilia development and maintenance.

Stereocilia, the actin-based mechanosensory protrusions of inner ear sensory hair cells, require precise dimensional control for proper mechanotransduction, yet the mechanisms governing actin assembly during development remain unclear. Their size and shape are determined by a stable core of long, parallel, unbranched filamentous (F-) actin. We find that during stereocilia widening, which is a key process for function and stability, newly expressed actin first integrates at the tip, then along the periphery of the core. To understand how actin assembles, we probe for globular (G-) actin, F-actin barbed ends, and pointed ends, and identify a tip-enriched population of short actin filaments. Overexpressing actin increases these filaments at the stereocilia periphery, suggesting a role in widening. In addition, the tip-localized myosins MYO3A/B and MYO15A, essential for normal growth, generate or stabilize short filaments. We propose that these short filaments are intermediates that mature into the long F-actin known to comprise the stereocilia core.

Myosin-7A (Myo7A) is a motor protein crucial for the organization and function of stereocilia, specialized actin-rich protrusions on the surface of inner ear hair cells that mediate hearing. Variants in Myo7A cause several forms of genetic hearing loss, including autosomal dominant DFNA11 deafness. Despite its importance, the structural elements that control Myo7A within cells are not well understood. In this study, we used cultured kidney epithelial cells to screen for mutations that activate the motor-dependent targeting of Myo7A to the tips of apical microvilli on these cells. Our findings reveal that the targeting of Myo7A is regulated by specific IQ motifs within its lever arm and that this regulation can function at least partially independent of its tail sequence. Importantly, we demonstrate that many of the DFNA11 deafness variants reported in patients activate Myo7A targeting, providing a potential explanation for the autosomal dominant genetics of this form of deafness.

Cochlear hair cells transduce sound using stereocilia, and disruption to these delicate mechanosensors is a significant cause of hearing loss. Stereocilia architecture is dependent upon the nanomotor myosin 15. A short isoform (MYO15A-2) drives stereocilia development by delivering an elongation-promoting complex (EC) to stereocilia tips, and an alternatively spliced long isoform (MYO15A-1) tunes postnatal size in shorter stereocilia, which possess mechanosensitive ion channels. Disruption of these functions causes two distinct stereocilia pathologies, which underly human autosomal recessive non-syndromic hearing loss DFNB3. Here, we characterize a new isoform, MYO15A-3, that increases expression in postnatal hair cells as the developmental MYO15A-2 isoform wanes reciprocally. We show the critical EC complex is initially delivered by MYO15A-2, followed by a postnatal handover to MYO15A-3, which continues to deliver the EC. In a Myo15a-3 mutant mouse, stereocilia develop normally with correct EC targeting, but lack the EC postnatally and do not maintain their adult architecture, leading to progressive hearing loss. We conclude MYO15A-3 delivers the EC in postnatal hair cells and that the EC and MYO15A-3 are both required to maintain stereocilia integrity. Our results add to the spectrum of stereocilia pathology underlying DFNB3 hearing loss and reveal new molecular mechanisms necessary for resilient hearing during adult life.

Mice and primary fibroblasts derived from mouse embryos completely lacking cytoplasmic β-actin, because the Actb gene was engineered to instead express γ-actin protein, have previously been found to be virtually devoid of phenotype. Here, we report the characterization of mice and mouse embryonic fibroblasts homozygous for an Actg1 allele edited to translate β-actin instead of γ-actin (Actg1-coding beta; Actg1c-b/c-b), which resulted in mice and fibroblasts that are devoid of γ-actin. We demonstrate that these Actg1c-b/c-b mice present with no measurable phenotype in survival, body mass, activity, muscle contractility, or auditory function. Primary fibroblasts derived from Actg1c-b/c-b mouse embryos were still proliferative, with several measured parameters of cell motility not different from wild type. From these and previous data, we conclude that β- and γ-actin proteins are redundant in primary embryonic fibroblasts and during normal mouse development.

Myosin-7A (Myo7A) is a motor protein crucial for the organization and function of stereocilia, specialized actin-rich protrusions on the surface of inner ear hair cells that mediate hearing. Variants in Myo7A cause several forms of genetic hearing loss, including autosomal dominant DFNA11 deafness. Despite its importance, the structural elements that control Myo7A within cells are not well understood. In this study, we used cultured kidney epithelial cells to screen for mutations that activate the motor-dependent targeting of Myo7A to the tips of apical microvilli on these cells. Our findings reveal that the targeting of Myo7A is regulated by specific IQ motifs within its lever arm and that this regulation can function at least partially independent of its tail sequence. Importantly, we demonstrate that many of the DFNA11 deafness variants reported in patients activate Myo7A targeting, providing a potential explanation for the autosomal dominant genetics of this form of deafness.

Class III myosins localize to inner ear hair cell stereocilia and are thought to be crucial for stereocilia length regulation. Mutations within the motor domain of MYO3A that disrupt its intrinsic motor properties have been associated with nonsyndromic hearing loss, suggesting that the motor properties of MYO3A are critical for its function within stereocilia. In this study, we investigated the impact of a MYO3A hearing loss mutation, H442N, using both in vitro motor assays and cell biological studies. Our results demonstrate the mutation causes a dramatic increase in intrinsic motor properties, actinactivated ATPase and in vitro actin gliding velocity, as well as an increase in actin protrusion extension velocity. We propose that both "gain of function" and "loss of function" mutations in MYO3A can impair stereocilia length regulation, which is crucial for stereocilia formation during development and normal hearing. Furthermore, we generated chimeric MYO3A constructs that replace the MYO3A motor and neck domain with the motor and neck domain of other myosins. We found that duty ratio, fraction of ATPase cycle myosin is strongly bound to actin, is a critical motor property that dictates the ability to tip localize within filopodia. In addition, in vitro actin gliding velocities correlated extremely well with filopodial extension velocities over a wide range of gliding and extension velocities. Taken together, our data suggest a model in which tip-localized myosin motors exert force that slides the membrane tip-ward, which can combat membrane tension and enhance the actin polymerization rate that ultimately drives protrusion elongation.

Auditory hair cells, which convert sound-induced vibrations in the inner ear into neural signals, depend on multiple actin populations for normal function. Stereocilia are mechanosensory protrusions formed around a core of linear, crosslinked F-actin. They are anchored in the cuticular plate, which predominantly consists of randomly oriented actin filaments. A third actin population is found near hair cell junctions, consisting of both parallel and branched filaments. Actin depolymerizing factor (ADF) and cofilin-1 (CFL1) proteins disassemble actin filaments and are required to regulate F-actin in stereocilia, but their effect on cuticular plate and junctional actin populations is unclear. Here, we show that loss of ADF and CFL1 disrupts the patterning of stereocilia into orderly bundles and that this phenotype correlates with defective development of the cuticular plate and junctional actin populations. ADF/CFL1 continue to regulate these actin populations in mature cells, which is necessary for long-term maintenance of hair cell morphology.

In the auditory and vestibular systems, stereocilia are actin-based protrusions that convert mechanical stimuli into electrical signals. During development, stereocilia elongate and widen by adding filamentous actin (F-actin), attaining their mature shape necessary for mechanosensitive function. Myosin motors, including MYO3A/B and MYO15A, are required for normal stereocilia growth, but the regulation of actin and the impact of myosins on actin assembly remain unclear. We focused on stereocilia widening, which requires the addition of new filaments to the bundle of linear F-actin comprising the initial stereocilia core. Our findings revealed that newly expressed actin incorporates at the stereocilia tip first, then along the shaft to promote stereocilia widening. The newly incorporated F-actin surrounded the existing F-actin core, suggesting that the core is stable once formed, with additional actin adding only to the periphery. To better understand the nature of incorporating actin, we used several probes to detect globular (G-) actin, F-actin barbed ends, and F-actin pointed ends. While F-actin core filaments are parallel and thought to present only barbed ends at stereocilia tips, we also detected F-actin pointed ends, indicating a previously undetected population of short actin filaments. Overexpression of actin resulted in abundant F-actin pointed and barbed ends along the periphery of the stereocilia shaft, suggesting that short actin filaments contribute to stereocilia widening. Short actin filament levels correlated with the levels of MYO3A/B and MYO15A at stereocilia tips, suggesting these myosins generate or stabilize short actin filaments essential for stereocilia widening and elongation.

Mechanosensitive hair cells in the cochlea are responsible for hearing but are vulnerable to damage by genetic mutations and environmental insults. The paucity of human cochlear tissues makes it difficult to study cochlear hair cells. Organoids offer a compelling platform to study scarce tissues in vitro; however, derivation of cochlear cell types has proven non-trivial. Here, using 3D cultures of human pluripotent stem cells, we sought to replicate key differentiation cues of cochlear specification. We found that timed modulations of Sonic Hedgehog and WNT signaling promote ventral gene expression in otic progenitors. Ventralized otic progenitors subsequently give rise to elaborately patterned epithelia containing hair cells with morphology, marker expression, and functional properties consistent with both outer and inner hair cells in the cochlea. These results suggest that early morphogenic cues are sufficient to drive cochlear induction and establish an unprecedented system to model the human auditory organ.

Assembly of the hair bundle, the sensory organelle of the inner ear, depends on differential growth of actin-based stereocilia. Separate rows of stereocilia, labeled 1 through 3 from tallest to shortest, lengthen or shorten during discrete time intervals during development. We used lattice structured illumination microscopy and surface rendering to measure dimensions of stereocilia from mouse apical inner hair cells during early postnatal development; these measurements revealed a sharp transition at postnatal day 8 between stage III (row 1 and 2 widening; row 2 shortening) and stage IV (final row 1 lengthening and widening). Tip proteins that determine row 1 lengthening did not accumulate simultaneously during stages III and IV; while the actin-bundling protein EPS8 peaked at the end of stage III, GNAI3 peaked several days later-in early stage IV-and GPSM2 peaked near the end of stage IV. To establish the contributions of key macromolecular assemblies to bundle structure, we examined mouse mutants that eliminated tip links (Cdh23v2J or Pcdh15av3J), transduction channels (TmieKO), or the row 1 tip complex (Myo15ash2). Cdh23v2J/v2J and Pcdh15av3J/av3J bundles had adjacent stereocilia in the same row that were not matched in length, revealing that a major role of these cadherins is to synchronize lengths of side-by-side stereocilia. Use of the tip-link mutants also allowed us to distinguish the role of transduction from effects of transduction proteins themselves. While levels of GNAI3 and GPSM2, which stimulate stereocilia elongation, were greatly attenuated at the tips of TmieKO/KO row 1 stereocilia, they accumulated normally in Cdh23v2J/v2J and Pcdh15av3J/av3J stereocilia. These results reinforced the suggestion that the transduction proteins themselves facilitate localization of proteins in the row 1 complex. By contrast, EPS8 concentrates at tips of all TmieKO/KO, Cdh23v2J/v2J, and Pcdh15av3J/av3J stereocilia, correlating with the less polarized distribution of stereocilia lengths in these bundles. These latter results indicated that in wild-type hair cells, the transduction complex prevents accumulation of EPS8 at the tips of shorter stereocilia, causing them to shrink (rows 2 and 3) or disappear (row 4 and microvilli). Reduced rhodamine-actin labeling at row 2 stereocilia tips of tip-link and transduction mutants suggests that transduction's role is to destabilize actin filaments there. These results suggest that regulation of stereocilia length occurs through EPS8 and that CDH23 and PCDH15 regulate stereocilia lengthening beyond their role in gating mechanotransduction channels.

Actbc–g Actg1–/– mice expressing only γ-actin from an edited Actbc–g allele have growth and survival defects despite expressing normal levels of γ-actin. Cell and auditory phenotypes associated with Actg1 KO were rescued by γ-actin expression from Actbc–g . We conclude that Actb and Actg support unique nucleotide- and protein-dependent functions.

The objectives of these studies were twofold: 1) to characterize the human choroid plexus papilloma (HIBCPP) cell line as a model of the blood-cerebrospinal fluid barrier (BCSFB) via morphology, tightness, and polarization of transporters in choroid plexus epithelia (CPe), and 2) to utilize Ussing-style electrophysiology to elucidate signaling pathways associated with the activation of the transient receptor potential vanilloid 4 (TRPV4) channel involved in cerebrospinal fluid (CSF) secretion. RT-PCR was implemented to determine gene expression of cell fate markers, junctional complex proteins, and transporters of interest. Scanning electron microscopy and confocal three-dimensional renderings of cultures grown on permeable supports were utilized to delineate the morphology of the brush border, junctional complexes, and polarization of key transporters. Electrophysiology was used to understand and explore TRPV4-mediated signaling in the HIBCPP cell line, considering both short-circuit current ( I sc ) and conductance responses. HIBCPP cells grown under optimized culture conditions exhibited minimal multilayering, developed an intermediate resistance monolayer, retained differentiation properties, and expressed, and correctly localized, junctional proteins and native transporters. We found that activation of TRPV4 resulted in a robust, multiphasic change in electrogenic ion flux and increase in conductance accompanied by substantial fluid secretion. This response appears to be modulated by a number of different effectors, implicating phospholipase C (PLC), protein kinase C (PKC), and phosphoinositide 3-kinase (PI3K) in TRPV4-mediated ion flux. The HIBCPP cell line is a representative model of the human BCSFB, which can be utilized for studies of transporter function, intracellular signaling, and regulation of CSF production.

Stereocilia on auditory sensory cells are actin-based protrusions that mechanotransduce sound into an electrical signal. These stereocilia are arranged into a bundle with three rows of increasing length to form a staircase-like morphology that is required for hearing. Stereocilia in the shorter rows, but not the tallest row, are mechanotransducing because they have force-sensitive channels localized at their tips. The onset of mechanotransduction during mouse postnatal development refines stereocilia length and width. However, it is unclear how actin is differentially regulated between stereocilia in the tallest row of the bundle and the shorter, mechanotransducing rows. Here, we show actin turnover is increased at the tips of mechanotransducing stereocilia during bundle maturation. Correspondingly, from birth to postnatal day 6, these stereocilia had increasing amounts of available actin barbed ends, where monomers can be added or lost readily, as compared with the non-mechanotransducing stereocilia in the tallest row. The increase in available barbed ends depended on both mechanotransduction and MYO15 or EPS8, which are required for the normal specification and elongation of the tallest row of stereocilia. We also found that loss of the F-actin-severing proteins ADF and cofilin-1 decreased barbed end availability at stereocilia tips. These proteins enriched at mechanotransducing stereocilia tips, and their localization was perturbed by the loss of mechanotransduction, MYO15, or EPS8. Finally, stereocilia lengths and widths were dysregulated in Adf and Cfl1 mutants. Together, these data show that actin is remodeled, likely by a severing mechanism, in response to mechanotransduction.

Fast-dissociating, specific antibodies are single-molecule imaging probes that transiently interact with their targets and are used in biological applications including image reconstruction by integrating exchangeable single-molecule localization (IRIS), a multiplexable super-resolution microscopy technique. Here, we introduce a semi-automated screen based on single-molecule total internal reflection fluorescence (TIRF) microscopy of antibody-antigen binding, which allows for identification of fast-dissociating monoclonal antibodies directly from thousands of hybridoma cultures. We develop monoclonal antibodies against three epitope tags (FLAG-tag, S-tag, and V5-tag) and two F-actin crosslinking proteins (plastin and espin). Specific antibodies show fast dissociation with half-lives ranging from 0.98 to 2.2 s. Unexpectedly, fast-dissociating yet specific antibodies are not so rare. A combination of fluorescently labeled Fab probes synthesized from these antibodies and light-sheet microscopy, such as dual-view inverted selective plane illumination microscopy (diSPIM), reveal rapid turnover of espin within long-lived F-actin cores of inner-ear sensory hair cell stereocilia, demonstrating that fast-dissociating specific antibodies can identify novel biological phenomena.

Mechanosensory bundles on auditory sensory cells are composed of stereocilia that grow in rows of decreasing height. This pattern depends on the specification of the eventual tallest row, then the assignment of distinct molecular identities to the shorter rows. Mechanotransduction refines and maintains row identity, thus instructing the form of the bundle.

Mechanosensory bundles on auditory sensory cells are composed of stereocilia that grow in rows of decreasing height. This pattern depends on the specification of the eventual tallest row, then the assignment of distinct molecular identities to the shorter rows. Mechanotransduction refines and maintains row identity, thus instructing the form of the bundle.