| Inner-ear mechanotransduction relies on tip links, fine protein filaments made of cadherin-23 and protocadherin-15 that convey tension to mechanosensitive channels at the tips of hair-cell stereocilia. The tip-link cadherins are thought to form a heterotetrameric complex, with two cadherin-23 molecules forming the upper part of the filament and two protocadherin-15 molecules forming the lower end. The interaction between cadherin-23 and protocadherin-15 is mediated by their N-terminal tips. Missense mutations that modify the interaction interface impair binding and lead to deafness. We have developed molecular tools to perform single-molecule force spectroscopy on the tip-link bond. Self-assembling DNA nanoswitches are functionalized with the interacting tips of cadherin-23 and protocadherin-15 using the enzyme sortase under conditions that preserve protein function. These tip-link-functionalized nanoswitches are designed to provide a signature force-extension profile, which allows us to identify single-molecule rupture events that result from applying force. Using this system, we have been able to measure the cadherin-23-protocadherin-15 single-molecule force-dependent off rate, as well as the concentration-dependent on rate for a single pair of these proteins. The rates suggest that a single bond is inadequate to withstand physiological forces for physiological times, but we construct a new model for tip-link dynamics which greatly alters our understanding of tip-link function and explains the necessity for a two-filament tip link. |
