Deafness is one of the most common sensory deficits in human. Since the inner ears of mice and humans are anatomically and physiologically similar, mouse mutants allow us to reveal detailed analysis of defects in the cochlea and vestibular apparatus. Loss of function mutations in transmembrane inner ear (tmie/TMIE) gene has been shown to cause deafness in mice (circling, spinner) and humans (DFNB6) which is encoded for the tmie protein. Although the tmie gene was identified a few years ago, little is known about subcellular localization and function of the tmie protein.
Aim of this present study was to produce anti-tmie polyclonal antibody and characterize the tmie protein. Polyclonal anti-tmie antibody was produced, which is recognized the tmie protein. Specificity of the anti-tmie antibody was determined by Western blot analysis. To know the expression pattern of tmie protein in postnatal developmental stages, I examined the expression of tmie protein in the liver, spleen, kidney, and lung, as well as in the cochlea. Using western blotting analysis, the expression of tmie protein in these organs has been identified in different age group of mice. These results show that tmie protein expression in the cochlea has been increased in postnatal developmental stages, indicating that tmie plays an important role in not only the development and also in the function of the cochlea.
To reveal the synthesis and subcellular localization of tmie protein, I established a stable cell line expressing tmie protein. The expression of Myc-tagged Tmie protein was confirmed by western blot analysis using an anti-Myc antibody and localization of the tmie protein was confirmed by immunostaining, using the anti- Myc antibody as well as the anti-tmie antibody.
My study demonstrates that the tmie protein is localized predominantly in the cellular membrane and to a lesser extent in the cytoplasm. These results suggest that our tmie expressing stable cell line provides a suitable in vitro model to explore Tmie synthesis and functions. Uniprot analysis explained the tmie protein has signal peptide domain, transmembrane domain, extracellular domain, cytoplasmic domain and lysine rich domain.
Establishing various delete mutant stable cell line which is expressing tmie protein will divulge the role of functional domains of the tmie protein. Using our anti-tmie antibody can identify the localization of the tmie protein in various tissues like liver, lung, kidney, spleen and cochlea. More functional studies needed in these organs to disclose the role of tmie protein. However the presence of tmie protein in the cellular membrane suggests that it may play major role in signal trafficking.