Photoreceptor discs form through peripherin-dependent suppression of ciliary ectosome release

Abstract

The primary cilium is a highly conserved organelle housing specialized molecules responsible for receiving and processing extracellular signals. A recently discovered property shared across many cilia is the ability to release small vesicles called ectosomes, which are used for exchanging protein and genetic material among cells. In this study, we report a novel role for ciliary ectosomes in building the elaborate photoreceptor outer segment filled with hundreds of tightly packed "disc" membranes. We demonstrate that the photoreceptor cilium has an innate ability to release massive amounts of ectosomes. However, this process is suppressed by the disc-specific protein peripherin, which enables retained ectosomes to be morphed into discs. This new function of peripherin is performed independently from its well-established role in maintaining the high curvature of disc edges, and each function is fulfilled by a separate part of peripherin's molecule. Our findings explain how the outer segment structure evolved from the primary cilium to provide photoreceptor cells with vast membrane surfaces for efficient light capture.

Figure 1.

Ultrastructural analysis of subretinal vesicles in rds^−/− mice. (A) EM images of retinal cross sections from rds^−/− (left) and WT (right) mice at P21. (B) Gaussian histogram of Feret diameters for extracellular vesicles in rds^−/− retinas (n = 250 pooled from two animals). Means ± SD are shown. (C) An example of extracellular vesicle budding from the ciliary membrane of an rds^−/− rod. (D) Immunogold labeling of rhodopsin (4D2 mAb) in the distal cilium and extracellular vesicles of a rod photoreceptor in rds^−/− and WT retina. Insets show magnified areas marked by red and blue rectangles. Bars: (A and D, main images) 500 nm; (C) 200 nm; (D, insets) 100 nm.

Figure 2.

Subretinal vesicles in rds^−/− mice are ciliary ectosomes. (A and B) Immunostaining of rhodopsin (1D4) with the outer segment proteins R9AP (A) and Rom-1 (B) in cross sections of WT and rds^−/− retinas. (C) Immunostaining of Na⁺K⁺-ATPase and R9AP in cross sections of WT and rds^−/− retinas. (D) Rhodopsin immunostaining with N-terminal (4D2) and C-terminal (1D4) antibodies in rds^−/− retinal cross sections with and without Triton X-100 treatment. Below is a cartoon representation of extracellular vesicles and antibodies under each condition. In all panels, mice are at P14 and nuclei are stained with Hoechst (blue). Bars, 10 µm.

Figure 3.

Developmental stages of the photoreceptor cilium in WT and rds^−/− mice. EM images showing the progression of rod outer segment morphogenesis at the indicated postnatal ages for WT and rds^−/− retinas. At P10, red arrows highlight the first discs that form at the end of the cilium in WT rods, as opposed to ectosomes that form in rds^−/− rods. Schematic representations of each developmental stage are shown above their corresponding images. Bars, 200 nm.

Figure 4.

Ectosome occurrence in 10-d-old WT and rds^+/− mice. (A) EM image of a WT rod with ectosomes next to the distal ciliary end. (B) Immunogold labeling of rhodopsin (4D2 mAb) in these ectosomes. (C) EM image of an rds^+/− retina with three ectosome clusters next to developing cilia. Red arrows point to ectosomes. Bars: (A and B) 200 nm; (C) 500 nm. (D) The prevalence of cilia with adjacent ectosomes in WT and rds^+/− retinas. Between 350 and 450 cilia were analyzed in three retinas of each genotype. Data are averaged among three retinas and shown as SEM.

Figure 5.

Transfection of FLAG-tagged recombinant peripherin constructs into rods of rds^−/− mice. (A–C) FLAG immunostaining in cross sections of retinas transfected with peripherin (A), Per-CT (B), and Per-TS constructs (C). A cartoon representation of each construct is shown on the left side of each corresponding panel with peripherin sequences shown in yellow, rhodopsin sequences shown in red, and the FLAG tag shown in green. Nuclei are stained with Hoechst (blue). (D–F) EM images of cilia from rods transfected by peripherin (D), Per-CT (E), and Per-TS (F) constructs. Yellow asterisks mark cilia. (G) FLAG immunostaining of retinal wholemounts transfected by each construct after RPE detachment. Bars: (A–C and G) 10 µm; (D–F) 500 nm. (H) Mean volumes of FLAG-positive objects for each construct in retinal cross sections and wholemounts. The data are averaged from at least six individual retinas of each type and are shown as SEM. *, P = 0.009; n.s., not significant. Student’s t test.

Figure 6.

Transfection of FLAG-tagged rhodopsin into rods of rds^−/− mice. (A) FLAG immunostaining in a retinal cross section. (B) FLAG immunostaining in a detached retinal wholemount. (C) FLAG immunostaining in a wholemount retina transfected with FLAG-tagged peripherin for comparison. A cartoon representation of each construct is shown above its corresponding panel. Bars, 10 µm.

Figure 7.

The tetraspanin core of peripherin is involved in forming highly curved membrane shapes. (A–C) AD293 cells were transfected with either full-length FLAG-tagged peripherin (A), Per-TS (B), or Per-CT (C). Left panels show representative EM images illustrating the accumulation of intracellular tubular membranes in cells transfected with peripherin or Per-TS (representative examples of tubular membranes are marked with yellow arrows) but not in cells transfected with Per-CT. Right panels show immunostaining of each construct probed with anti-FLAG antibodies (green) in relation to markers of the endoplasmic reticulum (calnexin), Golgi (GM130), and plasma membrane (PM; Na⁺K⁺-ATPase). All markers are shown in magenta. Nuclei are stained with Hoechst (blue). Bars: (left) 200 nm; (right) 10 µm.

Figure 8.

Schematic illustration of photoreceptor cilium development with and without peripherin. Shaded red and green areas are topologically equivalent and represent cytoplasmic space in photoreceptor outer and inner segments, respectively. Extracellular space and disc lumens are white.