Knowledge Bank

My work relates to the mechanisms of hearing and the structures that allow for the phenomenon of auditory mechanotransduction. Our perception of sound begins when sound waves going into the ear canal make contact with the eardrum. The eardrum then passes these vibrations to the middle ear, amplifying them as they travel to the cochlea. The cochlea is a fluid filled sac containing thousands of hair cells. These hair cells feature tip links, which are heterotetrameric filaments made of the non-classical cadherins cadherin-23 (CDH23) and protocadherin-15 (PCDH15), two proteins essential for hearing. These tip links connect neighboring stereocilia, which appear on top of the hair cells in a stair like formation. When vibrations created by sound waves move these stereocilia, a force will be applied to the tip links connecting them. This force will be conveyed to mechanosensitive ion channels. When the channels are opened by force, ions flow into the cell and trigger electrical signals that ultimately reach the brain for processing. My project focuses specifically on the molecular structure of the tip link with emphasis on the CDH23 component. The cadherin's that make up the tip link, like other non-classical cadherins, have smaller subunits called extracellular cadherin (EC) repeats. CDH23 has 27 EC repeats while PCDH15 has 11. Furthermore, both structures have a membrane adjacent domain (MAD), followed by transmembrane and cytoplasmic domains. The structures of all PCDH15 EC repeats have been solved, but there are still a handful of CDH23 EC repeats that have yet to be characterized. To better understand inner ear mechanotransduction at the molecular level, my project focuses on solving some of these remaining EC repeats including EC8-11, EC16-17, and EC13-15. To solve these structures I have conducted various procedures including molecular cloning, protein expression, protein purification, and x-ray crystallography. Successful experiments have allowed us to determine the molecular structures of mm CDH23 EC8-9 and mm CDH23 EC9 with mm CDH23 EC16-17 and mm CDH23 EC13-15 nearing completion. Using previously solved structures and the 3 new EC repeats (EC9, EC15, and EC16) that have not been structurally characterized before, we hope to create a complete molecular model of the tip link to better understand the structural elements mediating inner-ear mechanotransduction and to analyze the many deafness-causing mutations that can be found across the proteinaceous filament. Additionally, CDH23 is also essential for balance and proper eyesight with numerous CDH23 missense mutations affecting these processes in variable amounts with a complete deletion of the protein resulting in deafness, balance disorders, and progressive blindness (Usher Syndrome). Being able to visualize the molecular structure of this protein will allow us to learn how these certain mutations affect its overall structure and functionality.