Damage to the receptor cells of the inner ear, the hair cells, is a common underlying cause of hearing impairment. A complete understanding of hair-cell function requires the identification and characterization of the proteins that confer the cell?s unique properties, including proteins present in the hair bundle, the cell?s mechanosensitive organelle. Such a biochemical characterization has been impeded by the scarcity of material available for analysis. The generation of specific labeling reagents, for example monoclonal antibodies, should provide the means to overcome this obstacle: immunological tools will permit us to identify critical hair-cell and hair-bundle proteins, to study their intracellular targeting, and to pinpoint other molecules with which they interact. Recombinant antibodies displayed on the surface of filamentous bacteriophage should allow us to sidestep many of the difficulties inherent in obtaining conventional antibodies against low-abundance proteins. Production of recombinant antibodies does not in principle require immunization, large numbers of antibodies can be isolated, and sophisticated selection schemes for isolation of interesting clones can be devised. Over the past decade, substantial progress has been made in the generation and manipulation of recombinant antibodies, however their application in the study of low-abundance proteins has not been thoroughly tested. Our proposed studies will extend this methodology to the study of rare proteins - in particular, those present in hair cells and hair bundles. To obtain tools for the study of hair-cell and hair-bundle proteins, including components of the transduction apparatus, we will use a bacteriophage-displayed library of recombinant antibodies directed against inner-ear proteins. We will isolate antibody-bearing bacteriophage that recognize subclasses of saccular hair-cell proteins; such antibodies will provide the necessary reagents to characterize hair-cell and hair-bundle constituents and provide a means to further understand the molecular events that result in a functional, extraordinarily sensitive mechanoreceptive cell.
