This proposal addresses one of the most fundamental unsolved problems in vision: the molecular and cellular mechanism responsible for building and maintaining the light-sensitive organelle of vertebrate photoreceptor cells, the outer segment. The outer segment is a ciliary structure filled with a stack of disc membranes, which provide vast surfaces for light capture and harbor proteins comprising the phototransduction machinery. Discs are renewed on a daily basis in order to counteract the adverse effects of light exposure, and the fidelity of disc renewal is critical for maintaining photoreceptor health and normal vision. Previous studies established that photoreceptor discs are formed as serial evaginations of the plasma membrane at the outer segment base. Yet, the molecular mechanisms responsible for performing these membrane transformations remain poorly understood. The research strategy outlined in this proposal is built upon the recently uncovered analogy between disc formation in photoreceptor cells and a fundamental property of many other cilia types - the ability to release small extraciliary vesicles, called ectosomes. The photoreceptor cilium also has an innate ability to release massive amounts of ectosomes. However, in normal photoreceptors this process is suppressed by the disc-specific protein, peripherin-2, which retains the budding membranes at the outer segment base, thereby enabling them to be morphed into discs. The formation of both ciliary ectosomes and photoreceptor discs requires the action of the actin cytoskeleton, and recent evidence suggests that ectosome release also relies on the ESCRT protein complex. Therefore, Aim 1 will explore whether a similar interplay between the actin cytoskeleton and ESCRT proteins is responsible for performing the first steps of photoreceptor disc formation.
Aim 2 will explore the mechanism by which peripherin-2 transforms the functional state of the photoreceptor cilium from releasing ectosomes to retaining membranes at the outer segment base and transforming them into discs. Elucidating these mechanisms is critical for advancing our understanding of basic photoreceptor cell biology and pathobiological mechanisms underlying photoreceptor degeneration frequently associated with defects in outer segment morphogenesis.
The studies proposed in this application address the molecular and cellular mechanisms responsible for the formation and maintenance of the light-sensitive compartment of vertebrate photoreceptor cells, the outer segment. Because of adverse effects of daily light exposure, the outer segment material has to be replaced approximately every ten days; this requires an exquisite efficiency and precision in performance of the molecular machinery responsible for supporting the integrity of this cellular compartment. Dysfunction of outer segment morphogenesis causes some of the most severe types of inherited retinal degeneration, which highlights the significance of our proposed studies for developing strategies of disease prevention and future therapeutic intervention.
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