Outer Segment tip ingestion by the RPE

Project Summary An important function of the retinal pigment epithelium (RPE) is to ingest and degrade outer segment (OS) disk membranes from the photoreceptor cells. This catabolic role in the turnover of the phototransductive disk membranes is essential for the viability of the photoreceptor cells, with defects in the underlying processes leading to retinal pathogenesis. The proposed research focuses on the first event: the ingestion of the OS tips. We have developed high-speed live-cell imaging of this dynamic process by primary RPE cells. 3D analyses show that following formation of an f-actin cup around the OS tip, transient foci of actin filaments form at the site of scission of the OS, indicating that OS tip ingestion occurs by trogocytosis, involving dynamic polymerization of actin. The first two specific aims will combine studies on primary RPE cells in culture and in vivo analyses to investigate molecular mechanisms associated with the actin polymerization in this ingestion. The third aim addresses the daily timing of OS tip ingestion by performing experiments in which this event can be quantified by isolating it from the effects of phagosome degradation on phagosome number. The proposed experiments challenge current assumptions about how the OS tips are ingested by the RPE, and the daily timing of this cellular process. They are expected to generate novel findings that will establish a more accurate fundamental understanding of mechanisms underlying OS tip ingestion.