Ropelewski P , Imanishi Y .

P30 EY011373 NEI NIH HHS , R01 EY028884 NEI NIH HHS , R21 EY027292 NEI NIH HHS , T32 EY007157 NEI NIH HHS

             extracellular vesicle

	Visual abstract
	Figure 1- Class I mutant rhodopsin is released as microvesicles before being engulfed by RPE cells. A) Live retina explant was imaged at 9 DPF. Vesicles (arrowheads) were observed in the extracellular space surrounding rods expressing class I mutant rhodopsin, RhoQ344ter-Dend2. B) Cross sections of retinas expressing RhoQ344ter-Dend2 (Dend2, green) labeled with phalloidin-Alexa Fluor 633 (Actin, red) and Hoechst 33342 dye (Nuclei, blue). Microvesicles containing RhoQ344ter-Dend2 were in close contact with the actin filaments of the RPE microvilli (Zoom, arrowheads) and within RPE cytoplasmic space (Merge, indicated by double-headed arrows). Microvesicles were observed both at 9 DPF (top panels) and 21 DPF (bottom panels). Large OS fragments (single asterisk) or cell bodies (double asterisks) also existed within the RPE cell layer (double-headed arrows). C) Cross sections of retinas expressing Rho-Dend2-1D4 (Dend2, green) were labeled with phalloidin-Alexa Fluor 633 (Actin, red) and Hoechst 33342 dye (Nuclei, blue). OS fragments (Zoom, single asterisk) containing Rho-Dend2-1D4 were visible in cytoplasmic space (Merge, double-headed arrows) of the RPE cells. Retinas were imaged either at 9 DPF (top panels) or at 21 DPF (bottom panels). D) Size distribution of green fluorescent structures/vesicles found in the RPE of animals expressing RhoQ344ter-Dend2 (Q344ter, based on 180 structures from n = 4 animals) or Rho-Dend2-1D4 (wild type, based on 180 structures from n = 4 animals) at 21 DPF. Scale bars:  Zoom, 1 μm; other panels, 10 μm.
	Figure 2- NKAα is not released in extracellular vesicles under normal physiological conditions. A) Cross sections of retinas expressing Dend2-NKAα were labeled with phalloidin-Alexa Fluor 633 (Actin, red) and Hoechst 33342 dye (Nuclei, blue). Retinas were 9 DPF (top two rows) or 21 DPF (bottom two rows). For each panel, either Dend2-NKAα fluorescence was imaged directly (Dend2, green) or NKAα (both endogenous and Dend2-NKAα) was visualized by immunofluorescence (+NKAα ab, green). B) Live retina explant was imaged at 9 DPF. NKAα-containing extracellular vesicles were not observed in the vicinity of rod cells expressing Dend2-NKAα. Scale bars, 10 μm.
	Figure 3- IS fragments containing NKAα are phagocytosed by RPE at a late stage of photoreceptor degeneration. A) Cross sections of retinas that express either no transgenic protein (Nontransgenic) or class I mutant rhodopsin fused to Dendra2 (RhoQ344ter-Dend2). Retinas were 9 DPF (top two rows) or 21 DPF (bottom two rows). Sections were labeled with anti-NKAα antibody (NKAα, cyan), phalloidin-Alexa Fluor 633 (Actin, red), and Hoechst 33342 dye (Nuclei, blue). Photoreceptor-derived NKAα was not shed in microvesicles at either 9 or 21 DPF, whereas large vesicular structures (arrowheads) containing NKAα were occasionally seen in contact with RPE microvilli or inside RPE layer at 21 DPF. B) Histogram indicating the size distribution of structures containing NKAα within the RPE layer. Retinas expressing RhoQ344ter-Dend2 were analyzed at 21 DPF. C) A cross section of 21 DPF retina expressing RhoQ344ter-Dend2 and labeled with anti-NKAα (NKAα, cyan), phalloidin-Alexa Fluor 633 (Actin, red) and Hoechst 33342 dye (Nuclei, blue). In this view, a large inner segment fragment containing NKAα (arrowhead) is being engulfed by an RPE cell. Scale bars, 10 μm.
	Figure 4- NKAα proteins are degraded intracellularly in rod photoreceptors. A) Representative images of rod cells from animals expressing Dend2-NKAα and treated with either the vehicle (DMSO, control), 100 nm BA1, 100 nm Bort, a combination of both BA1 and Bort (Combo), or 250 nm Mariz for 24–144 h. Scale bar, 5 μm. B) Estimation plot analysis of Dend2-NKAα concentration. Average fluorescence intensities of Dend2-NKAα were measured in the cytoplasmic region of individual rods (each dot). The error bars represent the mean ± 1.96 SEs (95% confidence interval). The average fluorescence intensities are indicated on the y-axis [arbitrary unit (a.u.)]. *p < 0.001 by Student’s t test (each treatment condition vs control). At least 92 rod cells (n = 92) from four independent animals were subjected to quantification for each condition.
	Figure 1. Class I mutant rhodopsin is released as microvesicles before being engulfed by RPE cells. A, Live retina explant was imaged at 9 DPF. Vesicles (arrowheads) were observed in the extracellular space surrounding rods expressing class I mutant rhodopsin, RhoQ344ter-Dend2. B, Cross sections of retinas expressing RhoQ344ter-Dend2 (Dend2, green) labeled with phalloidin-Alexa Fluor 633 (Actin, red) and Hoechst 33342 dye (Nuclei, blue). Microvesicles containing RhoQ344ter-Dend2 were in close contact with the actin filaments of the RPE microvilli (Zoom, arrowheads) and within RPE cytoplasmic space (Merge, indicated by double-headed arrows). Microvesicles were observed both at 9 DPF (top panels) and 21 DPF (bottom panels). Large OS fragments (single asterisk) or cell bodies (double asterisks) also existed within the RPE cell layer (double-headed arrows). C, Cross sections of retinas expressing Rho-Dend2-1D4 (Dend2, green) were labeled with phalloidin-Alexa Fluor 633 (Actin, red) and Hoechst 33342 dye (Nuclei, blue). OS fragments (Zoom, single asterisk) containing Rho-Dend2-1D4 were visible in cytoplasmic space (Merge, double-headed arrows) of the RPE cells. Retinas were imaged either at 9 DPF (top panels) or at 21 DPF (bottom panels). D, Size distribution of green fluorescent structures/vesicles found in the RPE of animals expressing RhoQ344ter-Dend2 (Q344ter, based on 180 structures from n = 4 animals) or Rho-Dend2-1D4 (wild type, based on 180 structures from n = 4 animals) at 21 DPF. Scale bars:  Zoom, 1 μm; other panels, 10 μm.
	Figure 2. NKAα is not released in extracellular vesicles under normal physiological conditions. A, Cross sections of retinas expressing Dend2-NKAα were labeled with phalloidin-Alexa Fluor 633 (Actin, red) and Hoechst 33342 dye (Nuclei, blue). Retinas were 9 DPF (top two rows) or 21 DPF (bottom two rows). For each panel, either Dend2-NKAα fluorescence was imaged directly (Dend2, green) or NKAα (both endogenous and Dend2-NKAα) was visualized by immunofluorescence (+NKAα ab, green). B, Live retina explant was imaged at 9 DPF. NKAα-containing extracellular vesicles were not observed in the vicinity of rod cells expressing Dend2-NKAα. Scale bars, 10 μm.
	Figure 3. IS fragments containing NKAα are phagocytosed by RPE at a late stage of photoreceptor degeneration. A, Cross sections of retinas that express either no transgenic protein (Nontransgenic) or class I mutant rhodopsin fused to Dendra2 (RhoQ344ter-Dend2). Retinas were 9 DPF (top two rows) or 21 DPF (bottom two rows). Sections were labeled with anti-NKAα antibody (NKAα, cyan), phalloidin-Alexa Fluor 633 (Actin, red), and Hoechst 33342 dye (Nuclei, blue). Photoreceptor-derived NKAα was not shed in microvesicles at either 9 or 21 DPF, whereas large vesicular structures (arrowheads) containing NKAα were occasionally seen in contact with RPE microvilli or inside RPE layer at 21 DPF. B, Histogram indicating the size distribution of structures containing NKAα within the RPE layer. Retinas expressing RhoQ344ter-Dend2 were analyzed at 21 DPF. C, A cross section of 21 DPF retina expressing RhoQ344ter-Dend2 and labeled with anti-NKAα (NKAα, cyan), phalloidin-Alexa Fluor 633 (Actin, red) and Hoechst 33342 dye (Nuclei, blue). In this view, a large inner segment fragment containing NKAα (arrowhead) is being engulfed by an RPE cell. Scale bars, 10 μm.
	Figure 4. NKAα proteins are degraded intracellularly in rod photoreceptors. A, Representative images of rod cells from animals expressing Dend2-NKAα and treated with either the vehicle (DMSO, control), 100 nm BA1, 100 nm Bort, a combination of both BA1 and Bort (Combo), or 250 nm Mariz for 24–144 h. Scale bar, 5 μm. B, Estimation plot analysis of Dend2-NKAα concentration. Average fluorescence intensities of Dend2-NKAα were measured in the cytoplasmic region of individual rods (each dot). The error bars represent the mean ± 1.96 SEs (95% confidence interval). The average fluorescence intensities are indicated on the y-axis [arbitrary unit (a.u.)]. *p < 0.001 by Student’s t test (each treatment condition vs control). At least 92 rod cells (n = 92) from four independent animals were subjected to quantification for each condition.

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