Three-dimensional organization of nascent rod outer segment disk membranes

Abstract

The vertebrate photoreceptor cell contains an elaborate cilium that includes a stack of phototransductive membrane disks. The disk membranes are continually renewed, but how new disks are formed remains poorly understood. Here we used electron microscope tomography to obtain 3D visualization of the nascent disks of rod photoreceptors in three mammalian species, to gain insight into the process of disk morphogenesis. We observed that nascent disks are invariably continuous with the ciliary plasma membrane, although, owing to partial enclosure, they can appear to be internal in 2D profiles. Tomographic analyses of the basal-most region of the outer segment show changes in shape of the ciliary plasma membrane indicating an invagination, which is likely a first step in disk formation. The invagination flattens to create the proximal surface of an evaginating lamella, as well as membrane protrusions that extend between adjacent lamellae, thereby initiating a disk rim. Immediately distal to this initiation site, lamellae of increasing diameter are evident, indicating growth outward from the cilium. In agreement with a previous model, our data indicate that mature disks are formed once lamellae reach full diameter, and the growth of a rim encloses the space between adjacent surfaces of two lamellae. This study provides 3D data of nascent and mature rod photoreceptor disk membranes at unprecedented z-axis depth and resolution, and provides a basis for addressing fundamental questions, ranging from protein sorting in the photoreceptor cilium to photoreceptor electrophysiology.

Keywords: EM tomography; cilium; disk morphogenesis; photoreceptor.

Fig. 1.

Fixation of disk membrane morphology. (A and B) Transmission electron micrographs of the basal disks of mouse rod OSs, preserved by transcardiac perfusion with Karnovsky’s fixative and conventional processing (A) or by brief transcardiac perfusion with Karnovsky’s fixative, followed by HPF-FS (B). (C and D) Transmission electron micrographs of the basal disks of monkey (C) and cat (D) rod OSs, preserved by transcardiac perfusion with Karnovsky’s fixative and conventional processing. In A–D, the basal-most disks appear to be open to the extracellular space (and thus are lamellae), as indicated by arrows. (E) A typical elongated, well-preserved mitochondrion from the inner segment (adjacent to the basal OS disks) from a cat rod. (Scale bar: 500 nm.) A–E are at the same magnification. B and C are z-slices from tomograms; other panels are from transmission electron micrographs of 70-nm sections.

Fig. 2.

Continuity of nascent disks with the plasma membrane. (A–D) Monkey. (E–H) Cat. A–C and E–G show different z-plane images from tomograms of the basal disks of rod OSs. Basal disks appear enclosed in one plane of section (red arrow in A and E), but are actually open to the extracellular space in another plane of section (yellow arrows in C and G). D and H show 3D renderings of the membrane structures. The arrows (red, blue, and yellow) indicate corresponding areas in the z-plane images (A–C and E–G) and the respective 3D models (D and H). (Scale bars: 250 nm in A; 100 nm in D.) A–C and E–G are at the same magnification, as are D and H.

Fig. 3.

EM tomography of nascent disk initiation and rim formation in monkey (A–E) and mouse (F–J). (A–C) Different z-plane images from a tomogram of monkey rod OS basal disks showing different profiles of a membrane structure (red, blue, and yellow arrows) that appears as a bilobed invagination in 3D renderings (D and E). E is from Movie S5; plasma membrane is green, mature disks are dark blue. (F–H) Different z-plane images from a tomogram of mouse rod OS basal disks. Arrows indicate the location of rim formation; below the arrows, the nascent disks appear as lamellae, and above them, they appear enclosed by the plasma membrane. (I) 3D rendering of a view from outside the cell. The space above the most mature lamella (red arrow) is partially closed (to the right of the blue arrow); to the left of the blue arrow, this space is still open. (J) Lower-magnification view of the 3D model. The inner segment and a calycal process (light blue) have been modeled separately from other plasma membrane (green). J is a frame of Movie S7. (Scale bars: 250 nm in A; 50 nm in D; 500 nm in E.) A–C and F–H are at the same magnification, as are D and I, and E and J.

Fig. 4.

Number of open disks throughout the daily cycle at the base of monkey and mouse rod OSs. Error bars indicate ± SD. The probability of no significant difference among the different times of day was determined by ANOVA to be < 0.001 for each species.

Fig. 5.

Diagram of the organization of the nascent disks of a rod OS. Horizontal sections at different locations of the vertical section are shown. (A) An invagination that appears to initiate flattening of a protrusion of the ciliary plasma membrane. (B) Lateral expansion of the invagination (arrows in A), leading to the beginning of rim growth (arrows). (C) Lamella growth outward. (D and E) Growth of the rim leading to the enclosure of extracellular space between two adjacent lamellae (D), and thus the formation of a mature disk that is discrete from the plasma membrane (E).