Discs of mammalian rod photoreceptors form through the membrane evagination mechanism

Abstract

Photoreceptor discs are membrane organelles harboring components of the visual signal transduction pathway. The mechanism by which discs form remains enigmatic and is the subject of a major controversy. Classical studies suggest that discs are formed as serial plasma membrane evaginations, whereas a recent alternative postulates that discs, at least in mammalian rods, are formed through intracellular vesicular fusion. We evaluated these models in mouse rods using methods that distinguish between the intracellular vesicular structures and plasma membrane folds independently of their appearance in electron micrographs. The first differentiated membranes exposed to the extracellular space from intracellular membranes; the second interrogated the orientation of protein molecules in new discs. Both approaches revealed that new discs are plasma membrane evaginations. We further demonstrated that vesiculation and plasma membrane enclosure at the site of new disc formation are artifacts of tissue fixation. These data indicate that all vertebrate photoreceptors use the evolutionary conserved membrane evagination mechanism to build their discs.

Figure 1.

New discs at the rod outer segment base are accessible to tannic acid. (A) EM image of a mouse retina section at the interface between rod outer and inner segments stained with tannic acid–uranyl acetate. Arrowheads point at densely stained, tightly packed new discs at the outer segment base; note that mature discs are less densely stained and slightly swollen. (B) Permeabilization of the plasma membrane with 0.5% saponin results in equal density staining of new and mature discs by tannic acid–uranyl acetate. BB, basal body; CC, connecting cilium; IS, inner segment; Mt, mitochondria; OS, outer segment. Bar, 1 µm.

Figure 2.

New discs form as plasma membrane evaginations. (A and D) Representative magnified images of the rod outer segment base. (B and E) Traces of the plasma membrane folds from the images in panels A and D. Open discs are black, mature discs are orange, and discs that are enclosed in this section plane but still highly accessible to tannic acid are magenta. (C) Magnified view of the area boxed in A. Arrowheads point to triple-layered membranes of new discs, and arrows point to mature discs that do not display this pattern. Bars: (A–E) 200 nm; (C) 50 nm. IS, inner segment.

Figure 3.

Different shape and peripherin content at opposing edges of new discs. (A) Edges of new discs at the axonemal side of the outer segment base. (B) Edges of new discs at the side opposite to the axoneme. (C) and (D) Examples of two rod outer segments immunogold-labeled for peripherin. (E) and (F) High magnification images of two rod outer segment bases immunogold-labeled for peripherin. (G) The mean number of gold particles per disc measured in four regions illustrated in the cartoon to the right. Particles within 100 nm from the discs edges were counted. The data are averaged from 15 rods; error bars represent SEM. The P value for the difference between c and d is 7.2 × 10⁻⁶ and between a and c is 0.04 from a Student’s t test. Arrowheads point to the hairpin-shaped disc edges; arrows point to paperclip-like disc edges. Bars: (A and B) 50 nm; (C–F) 200 nm. Ax, axoneme; IS, inner segment.

Figure 4.

Rhodopsin topology in the membranes of newly formed discs. (A and B) Low- and high-magnification images of rhodopsin immunogold labeling by the 1D4 antibody recognizing the C terminus of rhodopsin. (C and D) Low- and high-magnification images of rhodopsin immunogold labeling by the 4D2 antibody recognizing the N terminus of rhodopsin. (E) Relative abundance of gold particles in the extracellular space adjacent to the membranes of newly formed discs. The number of particles produced by rhodopsin immunogold labeling by each antibody was counted within a 30-nm region of the extracellular space between membranes of new discs and the inner-segment plasma membrane. This region is depicted by light gray in the cartoon on the right and particles used in the count are marked by arrowheads in B and D. These counts were normalized by the total number of gold particles counted in the region of newly formed open discs of the same cell (marked by dark gray in the cartoon to the right from the graph). The number of analyzed cells labeled by each antibody was 13; error bars represent SEM; P = 0.0009. (F) Cartoon representation of rhodopsin orientation in the membrane of newly formed discs. Bars: (A and C) 200 nm; (B and D) 50 nm. IS, inner segment.

Figure 5.

Perturbations of rod outer segment structure caused by variations in tissue preparation procedure. (A–C) Representative EM images of rod outer segment bases fixed according to Chuang et al. (2007) and treated with tannic acid–uranyl acetate. (D–F) Representative EM images of rod outer segment bases obtained after introducing an additional heparin saline perfusion step before paraformaldehyde/glutaraldehyde (PFA/GA) fixation. (G and H) Representative EM image of a rod outer segment base obtained from eyecups immersed in PFA/GA (G) or GA (H) fixative. (I) Quantification of the fraction of rods containing vesicular structures at the outer segment base. The number of cells analyzed for each condition was 138 (saline PFA/acrolein), 343 (saline PFA/GA), 57 (PFA/GA), and 31 (GA). The data are averaged from cell counts in three mice treated under each condition; error bars represent SEM. Arrowheads point to the membrane fusion points. Bar, 200 nm. See also Fig. S1.