Transmembrane channel-like 1 and 2 proteins (TMC1 and TMC2) are essential for hair cell MET. In collaboration with the Griffith Lab (NIDCD) we used transgenic mice expressing TMC1-mCherry and TMC2-AcGFP to determine the localization of these proteins in stereocilia. Using high performance confocal microscopy, we observed both fluorescently-tagged TMC proteins as diffraction limited puncta along the length of stereocilia during early postnatal days, and increasingly restricted to stereociliary tips as hair cells matured. Both TMC1-mCherry and TMC2-AcGFP were excluded from the tips of the tallest row of stereocilia in the bundle, where MET activity and tip links are thought to be absent. This distribution was confirmed for native TMC1 and TMC2 by immunofluorescence using specific antibodies. Consistent with the hypothesis that TMC2 can compensate for TMC1 only during early bundle formation in cochlear hair cells, TMC2 signals at stereociliary tips rapidly disappear, first from outer hair cells and then from inner hair cells, in the early postnatal period. Conversely, TMC1 signal at the stereociliary tips remains in both outer hair cells and inner hair cells to adulthood. These data collectively establish the localization of TMC1 and TMC2 at stereocilia tips, consistent with the hypothesis that they play a local role in the MET channel complex To investigate the distinct and overlapping roles of MYO3A and MYO3B in hair cells we generated mice lacking each one of these motor proteins. No abnormalities were observed in stereocilia bundle organization and structure in either Myo3a-/- or Myo3b-/- mice. Neither mouse showed any shift in hearing thresholds (ABR) or overt vestibular dysfunction. Double knockout mice (Myo3a-/-Myo3b-/-) are not viable suggesting that MYO3A and MYO3B have essential roles during early embryonic development. At the same time, the lack of phenotype in the single knockouts indicates a degree of functional redundancy between MYO3A and MYO3B. Given that ESPN1 is a cargo protein of both MYO3A and MYO3B, we hypothesized that it may play an important role in upholding the compensatory interaction between the two paralogs in vivo. We generated a mouse lacking ESPN1 (Espn1-/-) by deleting exon 1, which contains the initiation site for translation of this isoform. The translation initiation site for the remaining, shorter, isoforms of espin is in exon 6 and is retained. Espn1-/- mice had stereocilia with a distinct reduction in their staircase slope (above), but this phenotype was restricted to extra-striolar hair cells of the otolithic organs. While this clearly associates ESPN1 with a role in stereocilia length regulation, restriction of the phenotype to only a subset of hair cells raised the possibility that an additional element is compensating for ESPN1 function. We found that espin-like (ESPNL), a structural homolog of ESPN1, also localizes to stereocilia-tips, albeit in an inverse length-dependent concentration. Heterologous expression assays show that both MYO3A and MYO3B transport ESPNL to filopodia-tips. An observed inverse correlation between ESPNL concentration and MYO3 tip localization provides evidence for a novel cargo-mediated MYO3 regulation. Biochemical analyses shows that the region of MYO3 interaction with ESPNL corresponds to the region reported for ESPN1. Our data confirm a role for ESPN1 in stereocilia-length regulation, and suggest that ESPNL is a novel member of the MYO3/ESPN complex, with both complementary and compensatory function to ESPN1, providing additional insights into mechanisms underlying stereocilia-length regulation and the late onset of DFNB30. Localization of kainate receptors in inner and outer hair cell synapses. Glutamate plays a role in hair cell afferent transmission, but the receptors that mediate neurotransmission between OHCs and type II ganglion neurons are not well defined. To determine whether kainate-type glutamate receptor (KAR) subunits are expressed in hair cell synapses, we performed an extensive immunofluorescence screen in whole-mount rat and mouse cochlea . Imunoreactivity for GLUK5 in IHC afferent synapses was postsynaptic, similar to GLUA2 (GLUR2;AMPA-type glutamate receptor (AMPAR) subunit), while GLUK2 appeared on both sides of the synapses. immunoreactivity for GLUK2 and GLUK5 was detected in OHC afferent synapses, , although GLUK2 was present only in those synapses bearing ribbons. Interestingly, GLUK1, GLUK2 and GLUK5 were also detected in OHC efferent synapses. These results indicate that AMPARs and KARs (GLUK2/GLUK5) are localized to IHC afferent synapses, while only KARs are localized to OHC afferent synapses in adults. Glutamate spillover near OHCs may act on KARs in OHC efferent terminals to modulate transmission of acoustic information and OHC electromotility. With Nir Gov (Weizmann Institute) we are studying the self-organization of rearward moving myosin motors in actin protrusions. While processive tip-directed motion leads to accumulation of myosin motors and their molecular cargo at the tips of stereocilia and other actin protrusions, it is observed that motors can also periodically form rearward moving aggregates as an essential process in the recycling of the myosins. To elucidate the underlying mechanism for this phenomenon we compared the dynamics of GFP-MYO15A and mCherry-MYO3A in filopodial protrusions in cultured cells. We tested a physical model based on a non-linear cooperative interaction between motors that showed that the spatiotemporal pattern of aggregate formation, rearward movement, and decrease in size, is directly linked to the kinetics of the motors and emerges from simple self-organization principles in the form of traveling waves and pulse trains. We helped the Tyska lab (Vanderbilt) show that intestinal microvilli are linked by protocadherins that target the microvillar tips via interactions with harmonin and MYO7B, and require harmonin in a manner analogous to what is observed in stereo cilia. These intermicrovillar links are composed of protocadherin-24 and mucin-like protocadherin, which target to microvillar tips via interactions with harmonin and Myo7b and interact to form a trans-heterophilic complex. This study also showed that mice lacking the Usher syndrome gene harmonin exhibit defects in brush border assembly. We helped Jung-Bum Shin (University of Virginia) and Peter Gillespie (Oregon Hearing Research Center) show that Xin-actin binding repeat containing 2 (XIRP2), an actin-crosslinking protein previously reported to be specifically expressed in striated muscle localizes to stereo cilia at the interface between the actin core and the membrane. Xirp2 null mice revealed high frequency hearing loss, and ultrastructural scanning electron microscopy analyses of hair cells demonstrated stereocilia degeneration in these mice. This study suggests XIRP2 is required for long-term maintenance of hair cell stereo cilia. With Mark Berryman (Ohio) we investigated the role of CLIC5 in development and maintenance of stereocilia. CLIC5-deficient jitterbug (jbg) mice revealed progressive fusion of stereocilia as early as postnatal day 10. Deafness-associated proteins that also concentrate at the stereocilia base including radix in, PTPRQ, and taperin, were mislocalized in fused stereocilia of jbg mice. PTPRQ and radixin were dispersed prior to stereocilia fusion. Biochemical assays showed an interaction of CLIC5 with radixin, taperin, and MYO6. In addition, CLIC5 and radixin failed to localize normally in fused stereocilia of MYO6 mutant mice. Based on these findings, we proposed a a model in which these proteins work together as a complex to stabilize linkages between the plasma membrane and subjacent actin cytoskeleton at and around the tapered base of the stereo cilia.
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