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. 2022 Nov 1;15(12):dmm049871.
doi: 10.1242/dmm.049871. Epub 2022 Nov 24.

Microvesicle release from inner segments of healthy photoreceptors is a conserved phenomenon in mammalian species

Tylor R Lewis  1 , Sebastien Phan  2 , Keun-Young Kim  2 , Isha Jha  2 , Carson M Castillo  1 , Jin-Dong Ding  1 , Benjamin S Sajdak  3   4 , Dana K Merriman  3 , Mark H Ellisman  2 , Vadim Y Arshavsky  1   5
Affiliations

Microvesicle release from inner segments of healthy photoreceptors is a conserved phenomenon in mammalian species

Tylor R Lewis et al. Dis Model Mech. .

Abstract

Many inherited visual diseases arise from mutations that affect the structure and function of photoreceptor cells. In some cases, the pathology is accompanied by a massive release of extracellular vesicles from affected photoreceptors. In this study, we addressed whether vesicular release is an exclusive response to ongoing pathology or a normal homeostatic phenomenon amplified in disease. We analyzed the ultrastructure of normal photoreceptors from both rod- and cone-dominant mammalian species and found that these cells release microvesicles budding from their inner segment compartment. Inner segment-derived microvesicles vary in their content, with some of them containing the visual pigment rhodopsin and others appearing to be interconnected with mitochondria. These data suggest the existence of a fundamental process whereby healthy mammalian photoreceptors release mistrafficked or damaged inner segment material as microvesicles into the interphotoreceptor space. This release may be greatly enhanced under pathological conditions associated with defects in protein targeting and trafficking. This article has an associated First Person interview with the first author of the paper.

Keywords: Microvesicle; Mitochondria; Photoreceptor; Retina; Rhodopsin; Vision.

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Conflict of interest statement

Competing interests The authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
Extracellular vesicles are located next to inner segments of wild-type (WT) and pcd5J mouse photoreceptors. (A) Representative transmission electron microscopy (TEM) images of retinal sections from 22-day-old homozygous pcd5J mice stained with osmium tetroxide. Boxed areas in A are magnified in A′ and A″. (B) A low-magnification TEM image of a WT mouse retinal section stained with osmium tetroxide. Boxed areas are magnified in B′ and B″. (C) An example of a cluster containing several vesicles. (D) An example of two neighboring vesicles with different staining densities. (E) The frequency of vesicles located next to photoreceptor inner segments in WT mice at P30, P60 and P180. The data are expressed as the number of vesicles per inner segment within a region of the 70 nm-thick retinal section containing 200 photoreceptors. Each data point represents an individual mouse; the bars represent mean±s.e.m. One-way ANOVA test revealed no statistically significant differences across ages (P=0.7775). In all panels, arrows point to extracellular vesicles. IS, inner segment; OLM, outer limiting membrane; OS, outer segment. Scale bars: 0.5 µm. A total of four pcd5J and nine WT mice were analyzed.
Fig. 2.
Fig. 2.
Microvesicles released from the inner segment plasma membrane. (A) Representative z-sections at the depths of 0, +18, +36 and +54 nm obtained from a 3D electron tomogram of a 250 nm-thick WT mouse retinal section stained with tannic acid/uranyl acetate. Full tomogram is shown in Movie 1. Arrowheads depict a microvesicle with a staining pattern matching that of the cytoplasm in the process of being released. Tomogram pixel size is 1.4 nm; scale bar: 0.2 µm. (B) Representative z-sections at the depths of 0, +18, +36 and +54 nm obtained from a 3D electron tomogram of a 250 nm-thick WT mouse retinal section stained with tannic acid/uranyl acetate. Full tomogram is shown in Movie 2. Arrowheads depict a clear microvesicle in the process of being released. Tomogram pixel size is 1.4 nm; scale bar: 0.2 µm. The data are selected from a total of six tomograms documenting microvesicle release from the plasma membrane obtained from three WT mice.
Fig. 3.
Fig. 3.
Extracellular vesicular structure connected to inner segment mitochondrion through a short membrane tunnel. (A) Representative z-sections at the depths of 0, +18, +36 and +54 nm obtained from a 3D electron tomogram of a 750 nm-thick WT mouse retinal section stained with tannic acid/uranyl acetate. Full tomogram is shown in Movie 3. Arrowheads depict a membrane tunnel connecting the budding vesicle with an inner segment mitochondrion. Tomogram pixel size is 1.5 nm; scale bar: 0.2 µm. (B) The corresponding 3D segmentation in two views: from the top as in A and from the bottom. Full segmentation is shown in Movie 5. IMM, inner mitochondrial membrane (blue); Mito, mitochondrion; OMM, outer mitochondrial membrane (green); PM, plasma membrane (red). The data are selected from a total of 12 tomograms documenting extracellular vesicular structures connected to mitochondrial membranes obtained from three WT mice.
Fig. 4.
Fig. 4.
Extracellular vesicular structure connected to inner segment mitochondrion through a complex membrane tunnel. (A) Representative z-sections at the depths of 0, +14, +28 and +42 nm obtained from a 3D electron tomogram of a 250 nm-thick WT mouse retinal section stained with tannic acid/uranyl acetate. Full tomogram is shown in Movie 4. Arrowheads depict a membrane tunnel connecting the budding vesicle with an inner segment mitochondrion. Tomogram pixel size is 1.4 nm; scale bar: 0.2 µm. (B) The corresponding 3D segmentation in two views: from the top as in A and from the bottom. Full segmentation is shown in Movie 6. IMM, inner mitochondrial membrane (blue); Mito, mitochondrion; OMM, outer mitochondrial membrane (green); PM, plasma membrane (red). The data are selected from a total of 12 tomograms documenting extracellular vesicular structures connected to mitochondrial membranes obtained from three WT mice.
Fig. 5.
Fig. 5.
Examples of microvesicles located next to the inner segments of WT rat and 13-lined ground squirrel (13LGS) photoreceptors. (A-C) Microvesicles with a variety of staining patterns in TEM images of retinal sections from WT rats stained with tannic acid/uranyl acetate. (D,E,F) Low-magnification TEM images of WT 13LGS retinal sections stained with tannic acid/uranyl acetate. Boxed areas are magnified in D′,E′,F′. In all panels, arrows point to microvesicles; arrowhead points to a microvesicle budding off of a cone inner segment. IS, inner segment. Scale bars: 0.2 µm (A-C); 2 µm (D,E,F); 0.5 µm (D′,E′,F′). A total of four rats and four 13LGS were analyzed.
Fig. 6.
Fig. 6.
Microvesicles can be endocytosed by the photoreceptor inner segment. (A) TEM images of WT mouse retinal sections stained with tannic acid/uranyl acetate, which show microvesicles adjacent to endocytic, clathrin-coated pits. (B) TEM images of WT 13LGS retinal sections stained with tannic acid/uranyl acetate, which show microvesicles adjacent to endocytic, clathrin-coated pits. (C) TEM images of WT rat retinal sections stained with tannic acid/uranyl acetate. Endocytic, clathrin-coated pits are observed either with (left) or without (right) an adjacent microvesicle. In all panels, arrows point to microvesicles; arrowheads point to darkly stained clathrin-coated pits. IS, inner segment. Scale bar: 0.5 µm. A total of nine mice, four 13LGS and four rats were analyzed.
Fig. 7.
Fig. 7.
A subset of microvesicles contain rhodopsin. (A,B) TEM images from immunogold labeling of WT mouse retina using the 4D2 anti-rhodopsin antibody. Boxed area is magnified in A′. The majority of gold particles label the outer segment; microvesicles can be either labeled or unlabeled. (C) An example of a microvesicle labeled with the 1D4 anti-rhodopsin antibody. In all panels, arrows point to microvesicles labeled with anti-rhodopsin antibodies; arrowheads point to unlabeled microvesicles. Note that the granular structure of inner segment ribosomes can be particularly well distinguished from the gold particles in higher-magnification images. IS, inner segment; OS, outer segment. Scale bars: 0.5 µm. A total of 37 microvesicles were analyzed, of which 14 were labeled with anti-rhodopsin antibodies.
Fig. 8.
Fig. 8.
Rhodopsin is not required for microvesicle release from rod inner segments. Representative TEM image of a retinal section from a Rho−/− mouse stained with tannic acid/uranyl acetate. Boxed areas in A are magnified in A′ and A″. Arrows point to microvesicles; arrowhead points to a microvesicle in the process of budding from the inner segment. IS, inner segment. OS, outer segment. Scale bars: 0.5 µm. A total of four Rho−/− mice were analyzed.
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