Identification and Characterization of the Novel Stereocilia Protein TMEM145 and its Interaction Partners in Cochlear Outer Hair Cells
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Background: The precision of the mammalian hearing strictly relies on the functionality of auditory sensory hair cells and its mechanoelectrical transduction of sound. Outer hair cells amplify cochlear vibrations, essentially lowering the auditory threshold. The mechanoelectrical transduction in OHCs is initiated by the deflection of the stereocilia hair bundle, mechanically coupled to the overlying tectorial membrane via the tectorial membrane attachment crowns and connected among each other via the horizontal top connectors. Both protein complexes are poorly understood and their composition incompletely characterized. All known components like tubby or stereocilin lack any transmembrane domain to interact, predicting the presence of at least one unknown transmembrane linker.
Aim of the thesis: The objectives of this thesis are (A) to clarify the expression and localization of tubby and the closely related tubby-like protein 3 within the inner ear (Paper 1) and (B) to identify & characterize the missing tectorial membrane attachment crown and horizontal top connector transmembrane linker using imaging and biochemical assays (Paper 2).
(Paper 1): We analysed and compared the cellular and subcellular localization of tubby and tubby-like protein 3 within the mouse inner ear. Using fluorescence-based microscopical methods together with confocal laser scanning microscopy, we found tubby displays a yet unknown localization to hair cell primary cilia, the kinocilia, in the cochlea of neonatal mice before it is prominently localized to outer hair cell stereocilia. Further, we localized the closely related tubby-like protein 3 to cochlear and vestibular kinocilia of neonatal hair cells. While absent in the mature vestibular system, tubby-like protein 3 localizes to microtubule-rich supporting cell phalangeal processes of the mature organ of Corti. These results show a complex spatio-temporal expression pattern of tubby and tubby-like protein 3, indicating unknown functions of the two proteins in the inner ear.
(Paper 2): We identified the novel G-protein coupled receptor-related protein TMEM145 as the missing transmembrane linker within the tectorial membrane attachment crowns and horizontal top connectors. Two independent tmem145-deficient mouse models are suffering from an early on-set hearing loss caused by dysfunction of outer hair cell cochlear amplification. TMEM145 displays the identical cellular und sub-cellular localization and a similar spatio-temporal expression pattern compared with the known tectorial membrane attachment crown and horizontal top connector components tubby and stereocilin. The absence of TMEM145 causes subsequently the loss tubby and stereocilin while TMEM145 and tubby are absent in stereocilin-deficient mice. Using scanning electron microscopy and fluorescence microscopy techniques, additional consequences caused by the lack of TMEM145 were identified, in detail a loss of the stereocilia-tectorial membrane coupling and a compromised coherence of the outer hair cell stereocilia hair bundle. We identified tubby and stereocilin as direct interaction partners of TMEM145 using computational modelling and biochemical pull-down experiments. We further showed that tubby interacts with the c-terminus of TMEM145, while its N-terminus interacts with stereocilin. In heterologous expression systems, we clarified the membrane topology of TMEM145 and found a co-expression with tubby facilitates its plasma membrane abundance. To summarize, we identified TMEM145 as a transmembrane tectorial membrane attachment crowns and horizontal top connectors component connecting tubby and stereocilin, being essential for the outer hair cell stereocilia-tectorial membrane coupling, the hair bundle integrity and the overall outer hair cell function as cochlear amplifier.
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