High-resolution localization of the hair cell mechanotransduction channel components by immunogold-scanning electronic microscopy

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SUMMARY Our long-term goal is to understand at the molecular level how hair cell mechanotransduction works and why it fails in different types of hearing loss. Our strategy is to study genes that cause hearing loss in humans by modeling them in mice. A critical aspect of this research relies on localizing deafness proteins at cellular and subcellular levels in the inner ear. In the hair cell mechanosensory organelle, known as the hair bundle, high- resolution localization of proteins provides critical information for defining their functions. For example, the mechanotransduction machinery itself is organized asymmetrically along the tip link ends. Tip link nanofilaments transmit the deflection force from taller stereocilia to the tips of paired, shorter stereocilia in order to gate the channel. Therefore, a protein localized at the upper tip-link insertion complex indicates a potential role in tip-link tension maintenance. In contrast, a protein localized at the lower tip-link insertion site suggests a role with the mechano-electrical transducer (MET) channel complex. High-resolution protein localization is routinely achieved by immunogold-labeling of samples, which are subsequently embedded, sliced, and imaged using transmission electron microscopes. However, this approach is massively time-consuming and not suited for extensive quantification. An alternative approach images the surface of immunogold-labeled samples using scanning electron microscopy (SEM), however this method is limited to investigating extracellular proteins. As many proteins of the hair bundle are internal but in the vicinity of the membrane, we hypothesized that immunogold-SEM could be adapted to localize these internal proteins. Such a technical improvement would allow localization of protein distribution at the nanoscale from large numbers of hair cells, resulting in increased sample numbers for stronger quantification. In Specific Aim 1 of this proposal, we will finalize our immunogold-SEM protocols dedicated either to localizing internal proteins beneath the stereocilia membrane or proteins integral to the stereociliary membrane. Our preliminary data show that the internal actin-binding protein, EPS8, maintains its characteristic enrichment at the tip of the tallest stereocilia. At the same time, the hair bundle structure and its links are preserved. To test our membrane protein method, we will localize transmembrane protein PMCA2a. In Specific Aim 2, we will capitalize on these immunogold-SEM protocols to locate the mechanotransduction channels at nanoscale resolution. The protein distributions of TMC1, TMC2 (participating in the pore), and TMIE (required for TMCs activity), will be mapped at the tip of transducing stereocilia. The MET channel location, expected to be at the intersection between the TMCs and TMIE distributions, and its distance to the tip link will be measured. Overall, we expect to provide not only the first nanoscale map of the MET channel components, but also a seminal technique valuable to many other research fields such as cell adhesion, planar cell polarity, ciliopathies, and axon guidance among others.