Cellular actin protrusions (e.g. filopodia, microvilli, and stereocilia) display a broad range of lengths and lifetimes critically related to their specific cellular function. Stereocilia, the mechanosensory organelles of hair cells, are a distinctive class of actin-based cellular protrusions with an unparalleled ability to regulate their lengths over time. Our laboratory has made advances towards elucidating the mechanisms that underlie the formation, regulation, renewal, and life span of stereocilia. Inner ear hair cell stereocilia are composed of parallel, uniformly polarized and crosslinked actin filaments. In earlier studies we have shown that in developing neonatal hair cells stereocilia actin filaments are continuously polymerizing and depolymerizing in a treadmilling process that results in a dynamic renewal process while maintaining steady-state lengths. Whether stereocilia actin filaments are dynamically turning over throughout the lifetime of the hair cell, and whether mature stereocilia have any structural plasticity has remained an important open question in the hearing research field, especially since structural plasticity in hair cell stereocilia may have implications for the development of therapeutic interventions for both inherited and acquired hearing losses. Here we report that we were successfully able to culture and transfect adult vestibular hair cells with GFP-beta- and gamma-actin. Our experiments revealed that actin filaments in mature hair cell stereocilia (P30) are continuously polymerizing, albeit at a much slower rate than developing hair cell stereocilia. Similar to what has been shown in earlier studies on neonatal tissue, the actin polymerization rate was proportional to the stereocilia length;longer stereocilia had a higher rate of actin polymerization than shorter stereocilia. Using immunogold labeling, we also show that both the beta- and gamma-actin isoforms colocalize in stereocilia actin filaments as early as embryonic day 16.5, with 40% more gamma-actin than beta-actin. In contrast, in adult mouse stereocilia, beta- and gamma-actin are homogeneously distributed in a 1:1 ratio. Interestingly, stereocilia from aging 2-year-old mice presented almost 3:1 ratio of beta- to gamma-actin, suggesting that the expression levels of each actin isoform may be modulated throughout the lifetime of the organism. Furthermore, we show that stereocilia in aging mice often show variations in size and shape, including extremely long stereocilia, revealing intrinsic plasticity in the dynamics and structure of stereocilia actin cores. Notably, these very long stereocilia are similar to the stereocilia found in various mice with mutations in stereocilia proteins such as myosin VI, gelsolin, and PTPRQ (protein tyrosine phosphatase receptor Q). Overall, our data provides evidence for continuous structural plasticity of adult hair cell stereocilia actin cores, which may lend important insight towards future attempts at therapeutic treatment of acquired and inherited hearing loss. Proper hearing and balance depend on the staircase shaped bundle of inner ear hair cell stereocilia. In comparison to other actin protrusions (e.g. microvilli), stereocilia are extraordinary in at least 2 ways: 1) Differential regulation - in each hair cell there are rows of stereocilia with lengths that increase in height by several micrometers, but stereocilia within the same row vary in height by no more than several nanometers. 2) Length normal epithelial microvilli are 500 nanometers long, while stereocilia are up to 120 micrometers long. A large number of mutations that cause deafness affect proteins involved in regulating stereocilia length. Our recent work has revealed that stereocilia are dynamic structures undergoing constant renewal and regulation via the activities of numerous myosin motor proteins and their actin-regulatory cargoes. Myosin XVa (MyoXVa) and its cargo whirlin are implicated in deafness (DFNB3 and DFNB31, respectively) and have been shown to localize at stereocilia tips and to be essential for stereocilia elongation. Given that whirlin is a scaffolding protein with no actin-regulatory activity, it remains unclear how these proteins work together to elongate stereocilia. Here we show that the actin-regulatory protein Eps8 interacts with MyoXVa and whirlin, and that mice lacking Eps8 have very short stereocilia similar to MyoXVa- and whirlin-deficient mice. We also show that Eps8 localizes to stereocilia tips in concentrations directly proportional to length, showing for the first time a relationship between the amounts of an actin-regulatory protein and stereocilia length, revealing a biochemical mechanism for differential stereocilia elongation. We show that Eps8 fails to accumulate at the tips of stereocilia in the absence of MyoXVa, that overexpression of MyoXVa results in both elongation of stereocilia and increased accumulation of Eps8 at stereocilia tips, and that the exogenous expression of MyoXVa in MyoXVa-deficient hair cells rescues Eps8 tip localization. We also found that both MyoXVa and Eps8 appear in reduced amounts at the tips of whirlin-deficient stereocilia, which suggests that whirlin plays an integral role in the efficient accumulation of the MyoXVa:Eps8 complex at stereocilia tips, perhaps via its scaffolding activity. Our data demonstrates that MyoXVa, whirlin, and Eps8 are integral components of a stereocilia tip complex, where Eps8 is a central actin-regulatory element transported by MyoXVa to stereocilia tips for elongation of the stereocilia actin core. This work provides insight towards DFNB3 and DFNB31 pathologies, and identifies EPS8 as a candidate deafness gene. In the most accepted model for hair cell mechanotransduction, a cluster of myosin motors located at the stereocilia upper tip-link density (UTLD) keeps the tip-link under tension at rest. Both myosin VIIa (MYO7A) and myosin 1c have been implicated in mechanotransduction based on functional studies. However, localization studies are conflicting, leaving open the question of which myosin localizes at the UTLD and generates the tip-link resting tension. Using immunofluorescence, we now show that MYO7A and sans, a MYO7A-interacting protein, cluster at the UTLD. Analysis of the immunofluorescence intensity indicates that eight or more MYO7A molecules are present at each UTLD, consistent with a direct role for MYO7A in maintaining tip-link tension. MYO7A and sans localization at the UTLD is confirmed by transfection of hair cells with GFP-tagged constructs for these proteins. Cotransfection studies in a heterologous system show that MYO7A, sans, and the UTLD protein harmonin-b form a tripartite complex and that each protein is capable of interacting with one another independently. We propose that MYO7A, sans, and harmonin-b form the core components of the UTLD molecular complex. In this complex, MYO7A is likely the motor element that pulls on CDH23 to exert tension on the tip-link.
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