Abstract
Purpose :
In rod photoreceptor neurons, discs in the outer segment (OS) are densely packed with the light-sensing protein rhodopsin (Rho). 10% of rod OS discs are replaced daily, and thus constant synthesis and trafficking of new Rho to the OS is critical for maintaining rod homeostasis and normal vision. Our goal was to elucidate the molecular mechanisms of Rho trafficking in the inner segment (IS) region of mouse rods. We applied optimized mouse retina labeling techniques and super-resolution fluorescence microscopy to perform a quantitative spatial analysis of Rho and other trafficking regulator proteins in the rod IS.
Methods :
Dissected mouse retinas were processed to exclude the Rho-dominant OS layer while maintaining the IS and connecting cilium (CC). Rho-GFP knockin mice and GFP-specific nanobody conjugates were used to label Rho for stochastic optical reconstruction microscopy (STORM). Using STORM molecular coordinate data, we performed spatial distance measurements and colocalization analyses. We also performed immunoprecipitation experiments in IS-enriched retinas.
Results :
Our localization studies revealed Rho located along the IS plasma membrane throughout the entire distal IS and contiguous with the CC membrane. A variable cytoplasmic fraction of Rho was also observed near the cis-Golgi region of the IS. STORM localization of Rho at the IS membrane was quantified and validated alongside STX3, an IS plasma membrane protein. Rab11a and the post-Golgi protein alpha-mannosidase III were also localized along the IS plasma membrane. Co-immunoprecipitation assays of IS-enriched retinas confirmed positive Rho interactions with Rab11a, STX3, SNAP25, and dynein-1.
Conclusions :
Using optimized mouse retina preparation techniques and super-resolution single-molecule localization assays, we quantitatively mapped the location of Rho protein in the IS with nanoscale accuracy. We discovered populations of Rho protein localized along the IS plasma membrane, at the IS-CC boundary, and in proximal IS. We also localized other trafficking regulator proteins at the IS plasma membrane, suggesting this is a bioactive site for Rho trafficking events. These data serve as a foundation for identifying new subcellular trafficking mechanisms in rod photoreceptors.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.