The mechanical properties of chick (Gallus domesticus) sensory hair bundles: relative contributions of structures sensitive to calcium chelation and subtilisin treatment.

Up to four link types are found between the stereocilia of chick vestibular hair bundles: tip links, horizontal top connectors, shaft connectors and ankle links. A fifth type, the kinocilial link, couples the hair bundle to the kinocilium. Brownian-motion microinterferometry was used to study the mechanical properties of the hair bundle and investigate changes caused by removing different links with the calcium chelator BAPTA or the protease subtilisin. Immunofluorescence with an antibody to the hair-cell antigen (HCA) and electron microscopy were used to verify destruction of the links. The root mean square displacement and the corresponding absolute stiffness of untreated hair bundles were 4.3 nm and 0.9 mN m(-1), respectively. The ratio of Brownian-motion spectra before and after treatment was calculated and processed using a single oscillator model to obtain relative stiffness. Treatment with BAPTA, which cleaves tip, kinocilial and ankle links, reduces hair-bundle stiffness by 43%, whilst subtilisin treatment, which breaks ankle links and shaft connectors, reduces stiffness by 48%. No changes were detected in viscous damping following either treatment. The time course of the subtilisin-induced stiffness change was close to that of HCA loss, but not to the disappearance of the ankle links, suggesting that shaft connectors make a more significant contribution to hair-bundle stiffness. Sequential treatments of the hair bundles with BAPTA and subtilisin show that the effects are additive. The implication of complete additivity is that structures resistant to both agents (e.g. top connectors and stereocilia pivots) are responsible for approximately 9% of the overall bundle stiffness.