The mechanosensory functions of hair cells require precise variations in stereocilium length according to position in the hair bundle and according to position in the cochlea or vestibular system. At the core of the stereocilium is a parallel-actin-bundle scaffold. We have identified and characterized hair cell espin as a actin-bundling protein of this parallel actin bundle, mapped the jerker deafness mutation to a frameshift mutation in the espin gene, and shown that jerker homozygotes are unexpectedly espin deficient. Since the parallel actin bundle at the core of the stereocilium has recently been shown to undergo continuous renewal through tread milling, we reasoned that hair cell espin cross-links might increase the steady-state length of a stereocilium by affecting actin polymerization-depolymerization reactions in its core actin bundle. Accordingly, we have detected gradients in hair cell espin expression that are positively correlated with increases in stereocilium length along the cochlea, and shortening of stereocilia has been noted in the hair cells of espin-deficient jerker homozygotes. We have recently determined that hair cell espin causes up to 10-fold increases in the steady-state length of the core actin bundles of stereocilia and microvilli in transected cells and can mediate actin polymerization in vivo and in vitro. We will: (1) Elucidate the role of hair cell espin in regulating the length and dynamics of parallel actin bundles. Confocal microscopy will be used to monitor the effect of hair cell espin on stereocilium/microvillus length and to map the relevant domains. Fluorescence-recovery-after-photobleaching and biochemical assays will be used to examine the effect of hair cell espin on actin treadmilling in vivo and in vitro. A bead-based actin polymerization assay will be used to establish the direction and mechanism of espin-mediated actin polymerization. (2) Establish the nature of the defects in the hair cell stereocilia of jerker homozygotes and elucidate the molecular basis for their espin deficiency. Scanning and transmission electron microscopy will be used to monitor the changes in stereocilium length and core actin bundle ultrastructure in jerker mice. Pulse labeling, subcellular fractionation and in situ hybridization will be used to determine whether the deficiency of espin in jerker homozygotes results from accelerated protein degradation or translational inhibition and whether it is involves targeting to nuclei/nucleoli by the frameshifted C-terminal peptide of the jerker espins.
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