The goal of this project is to understand how the actin cytoskeleton of the photoreceptor inner segment contributes to the morphogenesis, maintenance and survival of photoreceptors, and hence to vision. The photoreceptor inner segment cytoskeleton is comprised of parallel bundles of actin filaments terminating in fingerlike calycal processes that surround the base of the outer segment. This research tests the hypothesis that these bundles are critical to photoreceptor morphogenesis, to inner and outer segment alignment, and to alignment of the inner segment with the pupil. It also examines the hypothesis that these bundles provide tracks for myosin-dependent transport of cargo between the perinuclear myoid region and the outer segment. Several proteins localize specifically to these structures including two actin cross-linkers fascin and fimbrin and Myo3A, a class III myosin. Patients with a mutation in fascin suffer from retinitis pimentosa, and Myo3A over-expression also causes abnormalities in the photoreceptor actin cytoskeleton. While the nature of the cellular defect and how it contributes to retinal dysfunction has not been studied yet for the fascin mutation, it illustrates the importance of the inner segment actin cytoskeleton in the retina. This research tests the hypothesis that the cross-linking and regulated nucleation of actin filament bundles orchestrates their proper assembly and maintenance, and thus is required for maintenance of healthy photoreceptors. This study will initially focus on fascin, fimbrin and Myo3A. In order to study the actin filament bundles of photoreceptors, two animal model systems will be employed, fish and Xenopus, each of which is uniquely suited to answer certain questions. The morphogenesis of the inner segment actin filament bundles and calycal processes during development will be studied using trout retinas, since they possess a proliferative zone containing all stages of photoreceptor development. We will also identify other protein components of the photoreceptor inne/outer segment cytoskeleton using fish (Bass and green sunfish), because homogeneous preparations of photoreceptor inner outer segments can be used, and the size of the fish makes biochemistry tractible. These studies will provide new information about the constituents, assembly and functions of the inner segment filament bundles and calycal processes and will thereby increase or understanding of mechanisms fundamental to photoreceptor morphogenesis, function and survival.
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