Force Dependent Unbinding of Mammalian Inner Ear Tip Link Proteins

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Project Summary/Abstract The tip link is a critical component of the transduction complex in vertebrate hair cells, which converts vestibular and auditory stimuli into an electrical signal. The tip link is composed of parallel dimers of PCDH15 and CDH23, bonded at their N-termini through a unique interface, the structure of which was recently resolved by X-ray crystallography. However, little is known about the biophysics of the hair cell tip link and how it responds to mechanical force. In this project, we will determine the biophysical characteristics of the tip link bond both statically and under force in order to understand the consequences of deafness mutations that occur at the bond interface. We will first synthesize fusion proteins containing the first two EC domains of PCDH15 and CDH23, and use dynamic force spectroscopy to describe the strength of a single bond, both in the presence and absence of calcium. Next, we will generate a series of double stranded tip link proteins, artificially dimerized by an antibody Fc domain, and measure both their strength under force and the consequence of lateral constraint on their kinetics. This will answer why the tip link has evolved to be double stranded, and how the tip link is able to faithfully transduce mechanical stimuli, yet still able to unbind at damaging sound levels. Finally, we will generate fusion proteins containing known deafness mutations which are thought to compromise bond integrity. We will then test the consequence of these deafness mutations by dynamic force spectroscopy. Elucidation of the biophysical characteristics of the tip link bond under force will inform why the tip link has evolved to be double stranded, how the transduction apparatus is able to survive for days or weeks, and how certain types of deafness mutations might be treated in the future.