Single Molecule Force Spectroscopy of Hair-Cell Tip-Link Proteins

Abstract

Mechanical stimuli from sound and head movements are converted into electrical signals by hair cells of the inner ear. Key to this process are tip links, fine protein filaments that convey mechanical force to hair-cell transduction channels. Tip links are made of two atypical cadherins: protocadherin-15 and cadherin-23, positioned at the lower and upper ends respectively. The tip link is held together by a non-covalent interaction between the N-termini of these two proteins. The crystal structure of the interacting domain has been solved, and molecular dynamics simulations have provided an estimate of the force it can withstand. However the lack of adequate technology has prevented direct measurement of the unbinding force.We have developed molecular tools to facilitate single-molecule force spectroscopy of tip-link proteins, based on self-assembled DNA nanoswitches (Halvorsen et al., 2011). The tips of protocadherin-15 and cadherin-23 are enzymatically conjugated to DNA oligos, which bind in selected locations to a single-stranded M13 DNA scaffold. The remaining single-stranded M13 is hybridized to complementary DNA oligomers. Once assembled, the nanoswitches are used in an optical tweezer system to measure the rupture forces of the tip link.Measured in this way, the unbinding force is about 35 piconewtons, a value consistent with extrapolations of the molecular dynamics simulations to slower loading rates, but disconcertingly low compared to what tip links must withstand physiologically. Tip links, however, have two strands, and thus two interaction domains in close proximity. Calculations suggest that this arrangement greatly increases the average lifetime of a tip link.