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.