Myosin III (MYO3) proteins are associated with the auditory and visual sensory systems in both invertebrates and vertebrates. In Drosophila, MYO3A (NINAC) is responsible for the transport and localization of the photo-transduction machinery in photoreceptors. In humans, mutation in MYO3A causes non-syndromic progressive hearing loss. To determine if MYO3A transports components of the mechanotransduction (MET) complex in hair cells we performed co-transfection assays in COS7 cells. Our results show that MYO3A binds and transports protocadherin 15 (PCDH15) to the tips of filopodial actin protrusions. PCDH15 is one of the components of the MET complex that makes up tip-links, the extracellular filaments that control the MET channel gating at tips of hair cell stereocilia. Three main PCDH15 isoforms have been described (PCDH15-CD1, -CD2 and -CD3). PCDH15-CD2 has been identified as the essential tip link component. Our data reveals that PCDH15-CD2, and not -CD1 or -CD3, directly interacts with MYO3A and this interaction regulates its transport and localization to the tips of actin protrusion in COS7 cells. Our evidence also suggests a role for MYO3A in the transport and localization of other components of the MET complex. MYO3A is thus potentially playing an essential role in the maintenance of the tip-link/MET channel complex. Mutations in MYO3A that affect its PCDH15-CD2 interaction and transport could explain the role MYO3A plays in human progressive hearing loss. In hair cells, stereocilia are inserted into an actin-rich cuticular plate (CP), which offers the cilia support, determines their pivoting properties, and facilitates their rebounding to resting position following deflections from the application of mechanical forces. While it has been clearly demonstrated that the stereocilia actin core tapers to about a tenth of its original diameter as it inserts into the CP, the exact three-dimensional molecular architecture at this rootlet-CP interface has yet to be determined. Based on freeze-etch electron microscopic studies, there is evidence that the stereocilia rootlets are held in place by radial cable-like fibrils that are 4-5nm in diameter. We hypothesize that αII- and βII-spectrin, both expressed in the CP, are involved in anchoring these rootlets. By employing confocal microscopy, transmission electron microscopy (TEM) of either high-pressure frozen or plunge frozen, freeze-substituted hair cell epithelia, we have investigated the precise localization of spectrin in the CP. We confirm that αII-spectrin and βII-spectrin are expressed and likely form the heterotetrameric spectrin molecules in the CP. Specifically, the two subunits are enriched at locations where the stereocilia rootlets insert into the CP. The toroid pattern as seen in confocal optical sections, parallels the freeze-etch 200nm rings, and is consistent with the distribution of radial spokes around a rootlet core. Preliminary TEM immunogold studies support this concentric arrangement of spectrin around rootlets. The MET channel is a large conductance, non-specific cation channel highly permeable to calcium. Many of its properties have been characterized in considerable detail. However, its precise localization within the stereocilia still remains controversial. Wwe employed high-resolution calcium imaging in cultured inner ear tissue isolated from mice expressing calcium indicator GCaMP3 in the plasma membrane of hair cells. We show that widespread spontaneous calcium transients occur in individual stereocilia and that these events vary in amplitude and duration. Kymographs generated from live imaging data provided evidence of frequent calcium transient fluctuations within single stereocilia suggesting quantized event amplitudes. We pinpointed non-synchronous spontaneous MET events within stereocilia to locations where they have not been reported previously. We found that, in addition to calcium transients in shorter two rows of stereocilia described before, MET remarkably also occurs at the tips of the tallest stereocilia, in the shortest, microvilli-like protrusions of immature bundles, as well as at the bases of vestibular stereocilia. Furthermore, we observed persisting activity in stereocilia under conditions when the tip link integrity was compromised, such as in splayed and disorganized stereocilia bundles. The exact origin of these calcium transients is not clear, but spontaneous openings of the MET channels due to the resting open probability may account for the calcium influx into stereocilia. Our results further show that tension applied directly to the tallest as well as shorter stereocilia membrane generates membrane tethers. Pulling these tethers evokes calcium influx exhibiting tip to base gradient. Our results demonstrate highly localized spontaneous MET in single inner ear hair cell stereocilia that occurs randomly throughout the hair bundle and we provide evidence for novel locations and previously unrecognized mechanisms of the mechano-electrical transduction. In mature mammalian inner ear, cochlear inner hair cells (IHCs) transform sound waves into electrical signals that are further conveyed via auditory nerve to the central nervous system. However, in the absence of auditory experience during early prehearing period, different mechanisms for information processing in the inner ear that are important for normal development of the auditory system have been described. Here, we investigated spontaneous activity patterns in cochlear epithelium during early development (postnatal days P1-P10). The mechanisms of spontaneous activity in the developing cochlea are still controversial and not well understood. Therefore, by employing high-resolution confocal calcium imaging in cultured organ of Corti isolated from mice expressing genetically encoded calcium indicators GCaMP3 or GCaMP6 selectively in hair cells, or both in hair cells and supporting cells, we examined the mechanisms of this complex phenomenon. Our results with calcium indicators expressed in the whole cochlear epithelium show that a large amount of spontaneous activity is present in both supporting and hair cells during early prehearing stages. Although this activity was spatially and temporally randomly distributed throughout the epithelium, we observed distinct patterns of activity. Some of the calcium transients originated in the Kolliker organ and propagated in a form of waves that, in a number of cases, triggered subsequent calcium waves in adjacent IHCs. However, a significant number of waves occurring in supporting cells did not trigger activity in adjacent hair cells. In addition, and more importantly, we observed a significant amount of spontaneous activity in individual inner and outer hair cells in a form of flickering that originated at the base of each hair cell and that was completely independent of the propagating calcium waves originating in supporting cells. Pharmacological manipulations provided evidence that propagating waves originating in supporting cells as well as waves subsequently triggered in IHCs were all eliminated by non-specific antagonists of P2 purinergic receptors. Strikingly, this manipulation did not affect the flickering in individual hair cells. Instead, additional pharmacological examination confirmed that the flickering was completely eliminated by selective blockade of L-type calcium channels. We demonstrate that widespread spontaneous activity takes place in the developing cochlea and we propose that two independent mechanisms work in concert to drive spontaneous activity in the developing organ of Corti: 1) propagating calcium waves that originate in supporting cells and are mediated by purinergic system; and 2) intrinsic, pacemaker-like activity of individual IHCs mediated by L-type calcium channels.
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