Structural and Molecular Basis of Transduction in Auditory Sensory Organs

National Institute On Deafness And Other Communication Disorders

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Abstract

The outer hair cell (OHC) stereocilia to tectorial membrane (TM) junction (STJ) is essential for cochlear amplification. Little is known about the STJ structure, molecular composition, and plasticity. In this study we hypothesize that STJ is a significant vulnerable link in age-related hearing loss. Using scanning electron microscopy, we observed that the separation of stereocilia from the TM leaves imprints with a granular build up on both contact surfaces and is likely the principal adhesive component of the STJs. In collaboration with Uri Manor, Salk Inst., we observed that when STJs are separated, the STJ protein stereocilin (STRC) also partitions with both the TM and the stereocilia tips. We found that when pretreated with BAPTA, STRC partitions with the stereocilia tips only. This observation highlights a Ca2+- dependence of STRC interactions with the TM. Because STRC labeling is discrete and cannot make up the mass of the granular material, we investigated other TM proteins as candidate components of the STJ. TECTA (associated with DFNA8/12) and TECTB are required for TM integrity and stereocilia attachment. Our previous study had shown that TECTA immunogold labeling decorates the proteins cross bridging the collagen fibers of the TM matrix. Using high resolution confocal microscopy, we detected a TECTA labeling pattern on the underside of the TM that matches the adhesion complex. While STRC labels stereocilia imprints, TECTA labeling surrounds the imprints and is more robust. We propose that TECTA (and possibly TECTB) are adhesive components of the STJ complex. Consistent with an adhesive role, TECTA partitions with both the TM and the stereocilia tips. Remarkably, TECTA is washed away from both the TM and the stereocilia tips in the absence of Ca2+. We conclude that TECTA, STRC, and STJ integrity all depend on Ca2+. Another question we are addressing is how are STJ proteins targeted, assembled, and regulated? We first observed a tonotopic STRC enrichment that suggests co-regulation with other stereocilia tip proteins. MyoXVa and its actin regulatory cargo EPS8 are part of the tip complex and required for stereocilia elongation. Using transgenic mice lacking either MYO15A or EPS8, we observed that STRC fails to localize to the tips and TM fails to attach to the stereocilia. We also observed a decrease of TECTA at the STJs with aging in WT mice. Loss of TECTA is likely a significant factor in age-related hearing loss. Single cell RNAseq data from Dr. David He, Creighton Univ., shows that Myo15a and STRC are down-regulated with aging. Taken together our results suggest that progressive weakening of the STJ follows loss of TECTA and down-regulation of Myo15a and STRC. We also observed that in aging mice, the stereocilia imprints remain long after hair cells loss. This suggests that full bundle detachment must precede stereocilia bundle degeneration and that no remodeling of the STJ site takes place in the absence of stereocilia contact. This result is consistent with observations from other transgenic mice where the TM imprints mirror early bundle deformations, but only when there is STRC and stereocilia contact. Consistent with this possibility, whirlin (a MYO15A cargo) mutant mice show STRC expression and STJ imprints that mirror variability in stereocilia lengths and bundle shape. We conclude that: a) TECTA is a major adhesive component of the STJ complex; b) STRC and TECTA interactions are Ca2+-dependent (junctional integrity is linked to Ca2+ homeostasis); c) MYOXVA and actin regulatory cargo are implicated in STJ formation; d) Lack of STJ underly hearing loss where stereocilia fail to elongate properly; and e) STJ weakens with aging and is likely to contribute to age-related hearing loss. This raises the important question whether STJs can re-form upon rescue of hair cells and stereocilia through gene therapy? Dynamins are GTPases principally involved in the scission of vesicles from the plasma membrane or Golgi and their fusion with another compartment. In these membrane traffic processes, dynamin binds to and assembles around the neck of the endocytic vesicle, forming a helical polymer. While DNM2 is ubiquitously expressed, DNM1 and DNM3 are expressed in a cell and tissue specific manner. Dynamins have also been implicated in binding actin and other actin binding proteins that regulate the mechanical strength of actin cytoskeletal networks. In collaboration with Dr. David He, we obtained several lines of evidence suggesting that DNM1 and DNM3 play a hair cell type-specific role in proper formation and dynamic properties of the cuticular plate actin network that anchors the stereocilia bundle. Using isoform-specific antibodies we observed a distinct enrichment of DNM3 in the cuticular plate of outer hair cells while DNM1 was enriched in the cuticular plate of inner hair cells. The localization of DNM1 and DNM3 to the dense actin network of the cuticular plate was in addition to the canonical localization of these two dynamins to the sites of endocytosis around the apical surface and at the synaptic machinery of the base of the hair cells. The selective enrichment of DNM3 in OHCs is also supported by single cell transcriptome and RNAscope data. The Dnm3 knockout mice show progressive degeneration of OHC stereocilia bundles, and a striking detachment of stereocilia from the cuticular plate even in hair cells from young mice. Using confocal microscopy, we also observed a reduction in the thickness of the cuticular plate. Strikingly, measurement of the dynamic properties of the stereocilia bundle using a flexible probe showed a significant increase in bundle compliance. Hearing evaluation of mice lacking DNM3 shows progressive hearing loss with a 40-60dB SPL increased threshold at age P21. Interestingly, while the DNM3 +/- heterozygous mice show normal hearing, they are more susceptible to even low-level noise exposure. One interpretation for this noise induced hearing loss is an increased fragility of the cytoskeletal network anchoring the hair bundle rootlets to the cuticular plate. We hypothesize that DNM3 regulate the OHC cuticular plate structure and dynamic properties that contribute to the normal dynamic properties of the hair bundle. The next step in this investigation is to elucidate the interacting molecular components biochemically and in heterologous expression systems. More importantly, we plan on exploring whether the cuticular plate and stereocilia bundle dynamic properties are regulated by the GTP activity of dynamin using acute preparations as well as organ of Corti cultures. Exploring the role of dynamins in the cuticular plate will build on our previous study in collaboration with Jung-Bum Shin, UVA, that revealed a critical role of LMO7 in the regulation of cuticular plate structure and dynamic properties. In collaboration with Katie Kind, NIDCD, we extended our characterization of the spontaneous calcium activity in individual stereocilia observed in sensory organs isolated from mouse inner ears. To test whether the calcium transients in individual stereocilia occur in vivo, we imaged zebrafish lateral line hair cells. Using a transgenic line with hair-cell specific, membrane-localized GCaMP6, we observed the occurrence of spontaneous calcium transients in stereocilia at 2 days post fertilization (dpf) when the hair bundle is developing, and at 5 dpf when most hair cells are mature. We also observed transients in the zebrafish inner ear at 3 dpf. To test whether the transients originated from the MET channel, we treated the fish with 1 mM amiloride, which blocked the transients completely. Additionally, we tested whether intact tip links are needed for these transients by treating fish with 5 mM BAPTA for 15 min. Pre-treatment with BAPTA did not change the occurrence of the transients.