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.

Keywords: disc renewal; protocadherin; rhodopsin; rod photoreceptors.

Fig. 1.

Rhodopsin is transported to the newly forming discs via the connecting cilium plasma membrane. Retinal sections were labeled with two rhodopsin antibodies: 1D4 (10 nm gold) against the cytoplasmic and RET-P1 (15 nm gold) against the extracellular/intradiscal epitope. (A) Rhodopsin diagram showing the 1D4 and RET-P1 antibody epitope sites. (B) Note accumulation of rhodopsin-bearing vesicles (white arrowheads) in the IS at the base of the connecting cilium (CC) and rhodopsin confined to the plasma membrane within the CC and the discs within OS. (C) Cross-sections of the CC showing rhodopsin staining restricted to the plasma membrane. Black arrowheads indicate small vesicles with sticklike projections within the CC. (D) Percentage of rhodopsin labeling present on the ciliary plasma membrane. (Scale bar, 100 nm.)

Fig. 2.

Developing discs are evaginations of the rod photoreceptor plasma membrane. (A) Electron micrograph of a mouse rod photoreceptor IS and OS. (B) Slice from an electron tomogram generated from the corresponding photoreceptor A. (C and D) Model generated from the tomography data shows evaginations of the rod photoreceptor plasma membrane forming immature discs that are extracellularly exposed at the perimeter of the OS. Yellow represents the IS plasma membrane, green the developing discs, purple the mature discs, and red the junctions observed between the IS and OS plasma membrane. (E) Schematic diagram showing developing disk as evaginations of the rod photoreceptor plasma membrane with an exposed leading edge. (Scale bar, 500 nm.)

Fig. 3.

Multiple junctions are present between the IS and the leading edge of developing discs. (A) A slice from a tomogram highlighting a region shown at a higher magnification in B. (B) Montage of slices from a tomogram showing junctions between the IS and the leading edge of a developing disk (white arrows). (C) Model generated from the tomography data, showing the spatial positioning of the junctions. Yellow corresponds to the IS, red represents junctions, and green the developing disk. (Scale bars, A, 100 nm; B and C, 25 nm.)

Fig. S1.

Immediate fixation of whole eyes optimally preserves the IS:OS interface. Whole eyes were immediately fixed after removal, or the retina was dissected from the eyecup (isolated retina) and a tissue punch taken from the isolated retina and either chemically fixed or high-pressure frozen. Dissection of the retina and tissue punching resulted in damage to OSs, including loss of close apposition of OSs (black asterisks) and disorganized discs (white asterisks), and the periciliary ridge was often no longer visible. The interface between the inner and OS was particularly sensitive to manipulation of the tissue, resulting in vesicle-like structures at the base of the OS indicated by the arrows in the right-hand panels. (Scale bars, right-hand panels, 2 μm; left-hand panels, 200 nm.)

Fig. 4.

Discrete discs are formed by the fusion of adjacent OS plasma membrane evaginations. (A and B) Two different slices from the same tomogram of a mouse rod photoreceptor. (C) The predicted position of the tomogram slices in A and B. The boxed regions in A show membrane that is open to the extracellular environment and fused in B. (D and E Montage of the tomogram slices corresponding to the boxed regions in A and B; the transition from open to closed membrane and the point of fusion between the adjacent plasma membrane to form a new disk is apparent. (F and G) Models generated from D and E showing the point of plasma membrane fusion in 3D. (Scale bars, A and B, 100 nm; D and E, 50 nm; F and G, 20 nm.)

Fig. 5.

As discs mature, a change in the membrane spacing occurs. (A) Slice from subtomographic average of developing “open” discs and (B) mature “closed” discs. Note that the open discs appear electron dense because of the small luminal space. (C) Measurements of the disk membrane spacing in developing open and (D) mature closed discs. (E) A slice from the tomographic data showing the apparent difference in disk spacing between developing and mature discs. (F) A corresponding schematic diagram highlighting the change in disk spacing as the discs mature. (Scale bars, A and B, 5 nm; E, 100 nm.)

Fig. 6.

Immunogold labeling of proteins involved in rod photoreceptor disk development. (A–C) Photoreceptor-specific cadherin (PCDH21) labels the junctions between the developing discs and the IS. (B and C) Slices from a tomogram where junctions are visible between the IS and OS and are highlighted in yellow (C), emanating from PCDH21 gold labeling shown in red. (D) Peripherin 2 is present on the rims of all discs but is excluded from the tips of the developing disk (white arrows). (E) Syntaxin 3 is present on the plasma membrane of the IS and is likely to be involved in membrane recruitment, but is absent from the OS. (F) Munc-18 labeling is predominantly in the IS. (Scale bars, 200 nm.)

Fig. S2.

Fibers emanate from the site of PCDH21 immuno-gold labeling. Tomographic slices of cryo-immuno-gold labeling of PCDH21 show junctions (yellow in right-hand panels) visible between the IS and OS lining up with PCDH21 gold labeling (red in right-hand panels). A and B are from a 70-nm section, whereas C and D are from a 200-nm section borohydride treated before labeling. (Scale, 200 nm.)

Fig. 7.

Model of maturation of rod photoreceptor discs. (A–D) Schematic showing the rhodopsin-containing plasma membrane of the connecting cilium that is linked to the side of the periciliary ridge by the USH protein network. The evaginating rhodopsin-containing membrane zippers up with neighboring evaginations proximally, leaving the leading edge free to interact with the top of the periciliary ridge via PCDH21-containing junctions. Discs at the base of the evaginations are anchored to the axoneme. The leading edges of neighboring evaginations fuse to form closed discs, accompanied by disassembly of the PCDH21-containing junctions and followed by change in membrane spacing to generate discs with a greater depth. (E and F) 3D models of the base of the OS showing cadherin junctions between developing discs and the periciliary ridge of the IS.