PROJECT SUMMARY Human vision begins in the retina where light capture stimulates an electrical signal within the outer segment organelle of photoreceptor neurons. The outer segment is a modified primary cilium built with hundreds of flattened membranous discs that maximize the light-absorbing surface. Phototoxicity damages the disc membranes requiring that the outer segment be continuously renewed through the addition of new discs at the base. Outer segment length is maintained by phagocytosis of old discs from the distal tip by the adjacent retinal pigment epithelial cells. The unique architecture of the outer segment compartment is critical to photoreceptor health and mutations that disrupt disc stacks underly many forms of human retinal degeneration. The purpose of this proposal is to investigate components that contribute to forming and maintaining discs in order to better understand the pathogenesis of inherited retinal degeneration. Rod photoreceptors express three proteins containing the glutamic acid rich protein (GARP) domain: the β1-subunit of the cyclic nucleotide-gated channel (CNG), GARP1, and GARP2. In mice, genetic ablation of all three GARP proteins results in extraneous membrane outgrowth from the outer segment base, a hallmark of defects in disc synthesis. I have new preliminary data indicating that the key disc morphogenesis protein, protocadherin 21 (PCDH21), is dysregulated in this model. These data led to my hypothesis that GARP proteins help to recruit PCHD21 to the leading edge of the budding nascent discs and the loss of GARP proteins results in reduced rates of disc growth that contributes to retinal degeneration. I have further identified that the CNGβ1 subunit is necessary to stabilize PCDH21 at the outer segment base, making it the primary focus of Aim 1. I will confirm that loss of PCDH21 from the outer segment base drives defects in disc morphogenesis and test whether CNGβ1 is sufficient to regulate PCDH21 and disc formation. Recent insights into the molecular mechanisms underlying disc formation have revealed the urgent need to quantitatively measure rates of disc synthesis, yet the tools to do so lack sensitivity and versatility. As a solution, I developed an inducible mouse model that expresses a photoconvertible, outer segment targeted, transmembrane reporter that I will use to quantify new disc addition. In Aim 2, I will characterize this novel mouse model followed by a quantitative comparison of rates of disc growth between wildtype, GARPKO, and CNGβ1-KO mouse rods to understand how dysregulated disc formation contributes to retinal degeneration. Completion of these aims will provide mechanistic insight into the role GARP proteins play in disc formation and outer segment growth kinetics as well as provide a quantitative tool for future investigation of disc renewal in health and disease.