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Abhishek Chadha, Stefanie Volland, David S Williams; Routing of opsin to the cilium. Invest. Ophthalmol. Vis. Sci. 2016;57(12):568.
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© ARVO (1962-2015); The Authors (2016-present)
The trafficking and localization of opsin to the outer segment of the photoreceptor cilium is essential for normal photoreceptor function. Opsin could either be trafficked directly to the cilium, or it could be routed via the adjacent membrane. Our goal is to determine the route rod opsin takes.
We used hTERT-RPE1 cells, which, like photoreceptor cells, feature a ciliary pocket. These cells were transfected with fluorescently-tagged rod opsin constructs. This approach was complemented with photoreceptor cells from RHO-GFP mice. The efficiency of delivery of fluorescently-tagged RHO to cilia was performing by fluorescence recovery after photobleaching (FRAP) experiments.
To test whether RHO trafficking is direct or whether it occurs via the plasma membrane, we generated surface-tagged RHO constructs, which we labeled with non-cell-permeable, labeled streptavidin. Multi-color FRAP experiments in RPE1 cells revealed that all the recovery of RHO in the cilium, determined by the rate and amount of recovery, could be accounted for by movement of cell surface RHO. Complementary experiments in both RPE1 and mouse photoreceptor cells demonstrated that crosslinking surface RHO resulted in a reduction in the rate of recovery of ciliary RHO. ImmunoEM studies with mouse photoreceptors demonstrated localization of RHO to the ciliary plasma membrane, rather than intraciliary regions.
Our results indicate that RHO transport to the cilium occurs via the adjacent plasma membrane, and that RHO moves along the ciliary plasma membrane.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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