Hearing and vestibular function depend critically on the existence of precise variations in stereocilium dimensions according to position in the mechanosensory hair bundle and hair cell position within the cochlea or vestibular system. At the core of the stereocilium is a specialized cytoskeletal element, the parallel actin bundle, which displays the hallmarks of a supramolecular scaffold that determines the dimensions, placement and physical properties of stereocilia. Parallel actin bundles are held together by actin-bundling proteins, which cross-link neighboring actin filaments and affect bundle properties. We discovered and are characterizing a novel family of actin-bundling proteins - the espins - which are enriched in stereocilia and play critical roles in stereocilium length and width regulation and integrity. Encoded by a single gene, the espins come in a variety of isoforms that differ significantly in their N-terminal peptides and appear to accumulate in different hair cell types and at different times during stereociliogenesis. The espins display two major biological activities, which are mediated through their defining 116-amino acid C-terminal """"""""actin-bundling module"""""""": they cross-link actin filaments in a Ca2+ resistant fashion to produce parallel actin bundles like those in stereocilia, and they cause a dramatic elongation of parallel actin bundles in cells. We discovered that the espin gene is the target of the jerker mutation in mice, which in homozygotes results in a lack of espin proteins, stereocilia that are abnormally short and thin, and deafness and vestibular dysfunction. Recently, a number of additional mutations associated with deafness have been ascribed to the espin gene of humans. We will use in-vitro binding assays, electron microscopy, small-angle x-ray scattering and confocal fluorescence-photobleaching assays to determine how espins with deafness mutations and wild-type espin isoforms differ in their actin-binding, actin-bundling and dynamics and in their effects on actin bundle structure. We will use scanning and transmission electron microscopy and immunocytochemistry to determine how the jerker deafness mutation in the mouse espin gene affects the dimensions, actin bundle ultrastructure and key protein components of hair cell stereocilia at selected stages of development in the improved congenic jerker mice we have prepared in the CBA/CaJ genetic background. As an outgrowth of our work on the espins, we have developed an advantageous cell culture model for examining the targeting, activities, dynamics and interactions of stereocilium proteins that makes use of transfected LLC-PK1-CL4 epithelial cells. We will use this CL4 cell model to examine the targeting and interactions of myosin XVa and whirlin, two other proteins strongly implicated in stereocilium elongation, and to examine the effects of deafness mutations. Hearing and balance depend upon hair cells in the inner ear, specifically on their small fingerlike extensions called stereocilia. We have discovered and are characterizing a family of stereocilia proteins called the espins, which are the target of deafness mutations in mice and humans and are required to form and maintain stereocilia. In addition, we have developed and are using a model cell culture system to better understand the biological roles of espins and other stereocilia proteins.
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