Abstract
Purpose :
Rhodopsin (Rho) is synthesized in rod photoreceptor inner segments (IS) and its trafficking is critical for rod homeostasis. Mutations that disrupt Rho trafficking can cause retinitis pigmentosa. Mutated Rho is often mislocalized at the endoplasmic reticulum (ER) of rod IS, which led to our goal to accurately map the endogenous ER network in rods as the site of Rho biosynthesis before it is trafficked to the outer segment.
Methods :
We used the nanoscopy methods structured illumination microscopy (SIM) and stochastic optical reconstruction microscopy (STORM), as well as an adeno-associated virus (AAV) to express SNAP-tag fused Sec61b under a rod-specific promoter. The SNAP-Sec61b construct was first validated in heterologous cells using STORM. The AAV construct was subretinally injected in mice to label the ER membranes in rods; labeling was compared to endogenous immunolabeled Sec61b using SIM. Rho mRNA localization was performed using RNAscope fluorescence detection with multiplex immunolabeling and SIM. Ribosomes were immunolabeled in the IS using a S6 antibody.
Results :
SNAP-Sec61b plasmid was validated in cultured cells as a robust dense marker of the ER ultrastructure. Endogenous labeling of Sec61b revealed a punctate pattern throughout individual rods. In comparison, using AAV, Sec61b was located throughout the entire IS, demonstrating that ER fills the rod IS and the cytoplasmic space surrounding the nuclei. Ribosomes with polysome chains studded on rough ER were distributed throughout the IS. Using RNAscope and SIM, we identified Rho mRNA throughout the IS using multiple mRNA probes. Notably, Rho mRNA localized in the distal IS region near the base of the connecting cilium.
Conclusions :
Our results demonstrate that overexpression of an ER fusion marker, combined with self-labeling tags such as the SNAP-tag and SIM can reconstruct the ER network in rod with fluorescence. These results further show that Rho mRNA and ribosomes are distributed throughout the IS. Together, our results demonstrate that Rho biosynthesis is not confined to a particular subregion of the IS but distributed through the cytoplasm. These results provide new context to the organization of Rho biosynthesis and the secretory system and will drive future studies to identify the mechanisms that drive these cellular events.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.