Peripherin-2 and rom1 self-assembly shapes photoreceptor outer segment disk rim membranes

Abstract

The outer segment (OS) is the highly specialized primary cilium that rod and cone photoreceptors use to sense light. Each OS comprises an evenly ordered stack of hundreds of membranous disks that scaffold the first steps of visual phototransduction. Disk structure, OS organization, and photoreceptor viability rely on a transmembrane scaffold of the tetraspanin peripherin-2 (Prph2) and its homologue Rom1 that self-assembles to shape the extreme membrane curvature that characterizes OS disk rims. Mutations in PRPH2 are among the leading causes of human inherited retinal degenerations (IRDs), yet the mechanisms underlying Prph2 and Rom1 self-assembly and membrane curvature scaffolding in both normal and disease states remain poorly understood.Recent structural advances have revealed that the disk rim scaffold comprises three parallel belts of linear polymers of Prph2/Rom1 non-covalent dimers linked together by inter-dimer disulfide bonds. This dissertation describes complementary biochemical approaches for characterizing Prph2/Rom1 polymers in detergent extracts from native rod outer segment (ROS) and transfected HEK293 cell membranes. A reoptimized velocity sedimentation protocol was developed to capture the complete size range and heterogeneity of the extracted Prph2/Rom1 polymers. We developed and validated a BN-PAGE method for resolving individual size species of Prph2/Rom1 polymers and then applied it to characterize Prph2 and Rom1 assembly in WT ROS, Rom1 knockout mice, and transfected HEK293 cells. These approaches establish that native Prph2 assembles primarily as linear chains of disulfide-linked polymers of highly variable length and that Rom1 promotes higher-order Prph2 polymerization. Collectively, these findings provide a mechanistic framework for Prph2/Rom1-mediated disk rim morphogenesis and establish BN-PAGE as a robust, high-throughput platform for biochemical phenotyping of pathogenic PRPH2 variants of uncertain significance. This work advances fundamental understanding of photoreceptor membrane architecture and offers translational insights into the molecular basis of IRDs.