Rod disc renewal occurs by evagination of the ciliary plasma membrane that makes cadherin-based contacts with the inner segment

Abstract

The outer segments of vertebrate rod photoreceptors are renewed every 10 d. Outer segment components are transported from the site of synthesis in the inner segment through the connecting cilium, followed by assembly of the highly ordered discs. Two models of assembly of discrete discs involving either successive fusion events between intracellular rhodopsin-bearing vesicles or the evagination of the plasma membrane followed by fusion of adjacent evaginations have been proposed. Here we use immuno-electron microscopy and electron tomography to show that rhodopsin is transported from the inner to the outer segment via the ciliary plasma membrane, subsequently forming successive evaginations that "zipper" up proximally, but at their leading edges are free to make junctions containing the protocadherin, PCDH21, with the inner segment plasma membrane. Given the physical dimensions of the evaginations, coupled with likely instability of the membrane cortex at the distal end of the connecting cilium, we propose that the evagination occurs via a process akin to blebbing and is not driven by actin polymerization. Disassembly of these junctions is accompanied by fusion of the leading edges of successive evaginations to form discrete discs. This fusion is topologically different to that mediated by the membrane fusion proteins, SNAREs, as initial fusion is between exoplasmic leaflets, and is accompanied by gain of the tetraspanin rim protein, peripherin.rod photoreceptors | disc renewal | rhodopsin | protocadherin R od photoreceptors are specialized neurons of the vertebrate retina responsible for vision in dim light. The cylindrical outer segment (OS) contains ∼1,000 stacked discontinuous membranous discs packed with the light-sensitive pigment rhodopsin. This contrasts with the cone OS, which consists of a series of evaginations of the plasma membrane that remain exposed to the extracellular space (1). OS constituents are synthesized in the inner segment (IS) and then transported to the OS via a nonmotile cilium (connecting cilium) that resembles the transition zone of the primary cilium. Because of the high metabolic demand of phototransduction, OS discs undergo daily renewal, whereby the tip (distal end) is shed and phagocytosed by the retinal pigment epithelium while new discs are formed at the base (proximal end) of the OS (2, 3). How these discs are generated and rhodopsin incorporated remains controversial. The evagination model proposes that the ciliary plasma membrane evaginates to form discs that are initially exposed to the extracellular space and then pinch off to form fully closed discs (4). In contrast, the fusion model proposes that discs originate from rhodopsincontaining vesicles, which undergo homotypic fusion to form new discs (5, 6). Conventional electron microscopic (EM) analyses of chemically fixed specimens have clearly shown discs open to the extracellular space at the base of the OS, supporting the evagination model (4,7,8). Furthermore, studies in amphibians have demonstrated that membrane imper...

Mfsd2a Is a Transporter for the Essential ω-3 Fatty Acid Docosahexaenoic Acid (DHA) in Eye and Is Important for Photoreceptor Cell Development

“…This remarkable rate of membrane turnover and RPE phagocytosis is balanced by daily disc regeneration, but an understanding of the cellular process of regeneration of new photoreceptor discs has been poorly understood. Recently, new electron microscopy methodologies have revealed that OS discs are formed through a process of membrane evagination and indicate a highly regulated and ordered process at play in building photoreceptor discs (11)(12)(13). Rhodopsin is not only a photoreceptor but is a required structural component for disc membrane formation.…”

“…The early postnatal reduction in OS length without photoreceptor cell death was a striking feature of Mfsd2a KO retinas. The OS contains photoreceptor disc membranes that form through membrane biogenesis in the IS (11)(12)(13), occurring on a daily basis to balance photoreceptor disc turnover at the distal OS via phagocytosis by the RPE (10). It can be imagined that this high level of membrane biogenesis to build OS discs would apply unique metabolic demands on the retina.…”

Molecular basis for photoreceptor outer segment architecture

“…The Steinberg et al two-step model is well supported by studies showing that disk differentiation by rim formation (assayed by the presence of peripherin-2/rds) is always accompanied by the appearance of an enclosing plasma membrane in both rods and cones (Arikawa et al, 1992; Burgoyne et al, 2015). The addition of new plasma membrane at specific growth points (Fig.…”

“…Models of disk formation by endosomal expansion (vs. plasma membrane evagination) have enjoyed limited but continued support (Chuang et al, 2007; Obata and Usukura, 1992), including a recent revival that argued for a similar “vesicular targeting” model (Sung and Chuang, 2010). A trio of recent publications have thoroughly examined the question of disk morphogenesis in numerous vertebrate species (discussed below), that together provide a compelling body of evidence that strongly upholds the plasma membrane evagination model for morphogenesis of vertebrate rod photoreceptors (Burgoyne et al, 2015; Ding et al, 2015; Pugh, 2015; Volland et al, 2015). Current thinking suggests that disk membrane morphogenesis in cones follows essentially similar principles (Mustafi et al, 2009).…”

New light on photoreceptor renewal

“…1). This topological inversion occurs as a result of fusion of adjacent newly formed disc membranes to form discrete discs, a fusion event visualised in tomographic series in Burgoyne et al 1 The continuity of the forming discs with the plasma membrane, together with their topology, is not consistent with the vesiculation model, which would predict that the nascent discs would be cytoplasmic vesicles, rather than exposed to the extracellular space. Consistently we did not find rhodopsin-bearing cytoplasmic vesicles in either the base of the OS or in the connecting cilium.…”