Role of dynamin in photoreceptor disc morphogenesis

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PROJECT SUMMARY/ABSTRACT The photoreceptor outer segment is a modified primary cilium containing constantly renewing stack of disc membranes. Around 10% of the outer segment is replaced every day, with new discs formed at the outer segment base and old discs shed at the distal outer segment end. Disc morphogenesis starts with evagination of the ciliary plasma membrane, is followed by membrane flattening and lateral outgrowth, and concludes with complete or partial disc enclosure (in rods and cones, respectively). Complete disc enclosure involves a unique membrane scission event separating the disc membrane from the surrounding outer segment plasma membrane. The molecular basis for this event remains entirely unknown. During endocytosis in other eukaryotic cells, membrane scission is performed by dynamins, which release membrane vesicles into the cytoplasm. Therefore, Aim 1 of this proposal explores the role of dynamins in the process of rod disc scission and Aim 2 seeks to identify the key of proteins performing this important function. My research strategy will utilize ultrastructural analysis of knockout mice lacking specific dynamin isoforms, various approaches to studying subcellular protein localization, targeted proteomic search for endocytic proteins present in highly purified rod outer segment preparations and absolute protein quantification using isotope-labeled mass spectrometry. The feasibility of these approaches is validated in preliminary experiments. Taken together, the proposed studies will allow us to better understand how disc morphogenesis is performed on the molecular level, which is highly relevant to understanding the basic biology of these fascinating cells and uncovering the pathophysiological mechanisms of inherited blinding diseases in human patients.