Spatiotemporal Live-Cell Analysis of Photoreceptor Outer Segment Membrane Ingestion by the Retinal Pigment Epithelium Reveals Actin-Regulated Scission

Abstract

The photoreceptor outer segment (OS) is the phototransductive organelle in the vertebrate retina. OS tips are regularly ingested and degraded by the adjacent retinal pigment epithelium (RPE), offsetting the addition of new disk membrane at the base of the OS. This catabolic role of the RPE is essential for photoreceptor health, with defects in ingestion or degradation underlying different forms of retinal degeneration and blindness. Although proteins required for OS tip ingestion have been identified, spatiotemporal analysis of the ingestion process in live RPE cells is lacking; hence, the literature reflects no common understanding of the cellular mechanisms that affect ingestion. We imaged live RPE cells from mice (both sexes) to elucidate the ingestion events in real time. Our imaging revealed roles for f-actin dynamics and specific dynamic localizations of two BAR (Bin-Amphiphysin-Rvs) proteins, FBP17 and AMPH1-BAR, in shaping the RPE apical membrane as it surrounds the OS tip. Completion of ingestion was observed to occur by scission of the OS tip from the remainder of the OS, with a transient concentration of f-actin forming around the site of imminent scission. Actin dynamics were also required for regulating the size of the ingested OS tip, and the time course of the overall ingestion process. The size of the ingested tip is consistent with the term “phagocytosis.” However, phagocytosis usually refers to engulfment of an entire particle or cell, whereas our observations of OS tip scission indicate a process that is more specifically described as “trogocytosis,” in which one cell “nibbles” another cell.SIGNIFICANCE STATEMENTThe ingestion of the photoreceptor outer segment (OS) tips by the retinal pigment epithelium (RPE) is a dynamic cellular process that has fascinated scientists for 60 years. Yet its molecular mechanisms had not been addressed in living cells. We developed a live-cell imaging approach to investigate OS tip ingestion, and focused on the dynamic participation of actin filaments and membrane-shaping BAR proteins. We observed scission of OS tips for the first time, and were able to monitor local changes in protein concentration preceding, during, and following scission. Our approach revealed that actin filaments were concentrated at the site of OS scission and were required for regulating the size of the ingested OS tip and the time course of the ingestion process.