MOLECULAR MOTORS IN RETINAL CELL MOTILITY

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The ultimate goal of our research is to understand how cytoskeletal motor proteins power motile processes critical to the differentiation and function of the vertebrate photoreceptor. Since photoreceptor survival is jeopardized if the function of specific motors is compromised by mutation or aging, a better understanding of motor protein function in normal photoreceptors will provide insight into mechanisms of photoreceptor degeneration and vision loss. In this application we examine the roles of the cytoskeletal motors kinesin II and myosin VIIA in photoreceptor morphogenesis and outer segment turnover, investigate the contribution of ciliary axoneme-dependent transport to outer segment formation and maintenance, and further characterize a new myosin we have discovered, myosin III, which is a vertebrate homologue of Drosophila ninaC. Our strategy for functional analysis of these motors entails assessing the effects of compromising their function in chick photoreceptors in vitro and in vivo using two complementary strategies: (a) transient knockout of motor protein expression using antisense technology; and (b) retroviral-mediated overexpression of dominant negative tail constructs to inactivate endogenous motor proteins and saturate cargo binding sites. Kinesin II is concentrated at the basal body and axoneme of vertebrate photoreceptors. This motor has been shown to be required for morphogenesis and maintenance of motile and sensory flagella, where it mediates intraflagellar transport (IFT), the movement of rafts of multipeptide particles between the axoneme and plasma membrane. We propose to use degenerate primer PCR to seek vertebrate homologues of IFT particle peptides and investigate the possible roles of kinesin II and axoneme-dependent transport in outer segment morphogenesis and maintenance using the chick photoreceptor assay system. Mutations in the human myosin VIIA gene produce Usher's Syndrome type 1B, a form of retinitis pigmentosa accompanied by deafness. Myosin VIIA has been shown to be concentrated at the photoreceptor connecting cilium but its function is not known. We propose to test the hypothesis that disk morphogenesis is powered by the interaction of myosin VIIA with actin filaments associated with the axoneme at the distal connecting cilium. We have recently found that vertebrate retinas selectively express a homologue of the Drosophila myosin (ninaC). Since flies with null mutations for ninaC exhibit altered phototransduction and light-induced photoreceptor degeneration, we are interested in determining the properties and functions of the vertebrate myosin III. To this end we will use heterologous expression for biochemical characterization and in vitro motility assays, and functional analysis in the chick photoreceptor system.