Force Spectroscopy of Tip Link Proteins: A Study of Inner-Ear Biophysics

Abstract

Hair cells in the inner ear convert mechanical stimuli, in the vestibular and auditory systems, into electrical signals which can be processed by the brain. Hair cells are highly polarized with a unique elaboration of modified microvilli at their apical surface known as stereo cilia. Sensory stimuli such as sound waves of a particular frequency will result in oscillations of the stereociliary bundle at that frequency. Movements of this bundle couple mechanically to the channel. This force acts to gate the channel in turn resulting in a mechano-electrical transduction. The force is relayed to the channel by filaments known as tip links. Tip links are made of two atypical cadherins, protocadherin-15 and cadherin-23 at the lower and upper ends respectively. The tip link is held together by a non-covalent interaction between two anti-parallel pairs of EC domains. Although much has been discerned about the biophysics of this system the lack of adequate technology has prevented the direct measurement of many of these properties. We have developed molecular tools to facilitate the single molecule study of these properties. Self-assembled DNA nanoswitches are functionalized with protocadherin 15 and cadherin 23 fragments using the enzyme sortase. In order to preserve protein function, protein-DNA coupling is performed under physiological conditions. In this two-step process, a small synthetic peptide is first coupled to a DNA oligo. Next, utilizing the enzyme sortase, the protein is coupled to the DNA-peptide chimera under physiological conditions. This strategy frontloads all of the protein-incompatible chemistry so that it is performed on an oligo and a synthetic peptide, which are far more tolerant of non-physiological conditions. Once assembled, the nanoswitches are then used in an optical tweezer system to measure the rupture forces of the tip link under different conditions.