Super-resolution mapping in rod photoreceptors identifies rhodopsin trafficking through the inner segment plasma membrane as an essential subcellular pathway
Fig 5
Endogenous IS mouse Rho is located near the cis-Golgi and at the plasma membrane.
(A) SIM z-projection images of a single rod example from an IS-enriched, WT retina section immunolabeled with Rho-C-1D4 (magenta) and centrin antibody (green). Rho-C-1D4 is localized along the boundaries of the IS and CC (yellow arrows). The OS is indicated. (B) SIM z-projection image of a WT full retina section immunolabeled with GM130 (magenta), STX3 (cyan), and centrin-2 (yellow) antibodies. Asterisks indicate strong GM130 puncta staining in the proximal IS/myoid region corresponding to the cis-Golgi in each rod IS. (C) SIM z-projection image of a Rho-GFP IS-enriched retina section immunolabeled with NbGFP-A647 (cyan), along with GM130 (magenta), STX3 (yellow), and centrin (yellow) antibodies. The white arrow indicates a magnified portion of the IS layer, in which an asterisk indicates a GM130 punctum located directly near a Rho-GFP punctum in the myoid of a rod. (D, E) SIM images of WT IS-enriched retinas immunolabeled with either (D) Rho-C-1D4 antibody (magenta) or (E) Rho-N-4D2 antibody (magenta); both are colabeled with STX3 (cyan) and centrin-2 (yellow) antibodies. Partial endogenous Rho colocalization with STX3 at the IS plasma membrane is indicated with white arrowheads, and row average intensity plots are from the magnified portion of the IS corresponding to the region in single rod example images marked with a dashed line. Fluorescence intensities are normalized on both plots for clarity. Throughout the figure, magnified regions are indicated with either a dashed box or a dashed white arrow. Some magnified images are rotated so that the OS end of the rod is at the top of the image. Scale bar values match adjacent panels when not labeled. Numerical values corresponding to all graphical data are provided in Table B in S1 Data. CC, connecting cilium; IS, inner segment; NbGFP-A647, GFP nanobody Alexa 647 conjugate; OS, outer segment; Rho, rhodopsin; SIM, structured illumination microscopy; STX3, syntaxin 3; WT, wild-type.