The studies proposed here continue our work on the biophysical mechanism of sensory transduction by hair cells. A great deal of physiological evidence acquired over the last ten years indicates that the transduction channels in vertebrate hair cells are directly activated by mechanical stimuli: that some sort of """"""""gating spring"""""""" conveys the displacement of the hair bundle to the channels, and that the stress causes channels to open. Similarly, physiological evidence suggests that adaptation of the transduction channels, most likely by a mechanical adjustment of the gating spring attachment. In this study, we will investigate the structural correlates of these physiologically-defined elements. First, we will test whether the """"""""tip links"""""""" extending between stereocilia, originally described by Pickles, are the gating springs. We will develop a method to destroy the mechanical sensitivity of single hair cells, and then find out, with scanning electron microscopy, whether the tip links are also destroyed. Second, we will test the hypothesis of Howard and Hudspeth that adaptation is mediated by the upper attachment of the point of each tip link moving along the side of the stereocilium. Bundles will be displaced by 1-2micros m, allowed to adapt, and fixed for transmission electron microscopy. The positions of the attachment points will be measured to see if their position corresponds to the expected adaptation. Further tests will be to cut the tip links to see if the attachment points move upwards, as the structural model predicts; to get the tip links and see if the bundle moves forward by a tenth of a micron, as the biophysical model predicts; and to rule out other, more macroscopic rearrangements during adaptation wit high-sensitivity video subtraction. Third, we will investigate the role of other structures associated with the stereocilia, to determine a particular what holds the stereocilia together at their hips. For this aim, we will first confirm with high-resolution video measurements that stereocilia pivot at their bases and touch (but slide) at their tips. Then we will sequentially cut each of the three linkages between stereocilia, and determine with transmission EM which are intact when the bundle remain together. This understanding of hair bundle structures associated with transduction may illuminate certain pathological conditions of the auditory system. In particular, strong evidence for the tip-links hypothesis would implicate these filaments in the temporary threshold shift caused by noise trauma. Knowledge of what holds the bundles together may help in understanding why they fall apart with noise trauma.
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