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Alecia K Gross, Vladimir Parpura, Joshua D Sammons; Monitoring Kinetic Changes of Proper and Improper Rhodopsin Transport ex vivo. Invest. Ophthalmol. Vis. Sci. 2014;55(13):6010.
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© ARVO (1962-2015); The Authors (2016-present)
The highly efficient mechanism for transporting rhodopsin to the rod outer segments is still poorly understood. We have used our recently generated rho-paGFP-1D4 knock-in mouse to monitor real-time differences in mutant rhodopsin (Q344ter, devoid of the known rhodopsin sorting motif) and wild-type (WT) rhodopsin transport in live retinal tissue to test the hypothesis that mislocalized mutant rhodopsin traffics at a different rate than does outer segment transported rhodopsin. We have inhibited active transport with NaN3 to delineate active versus passive rhodopsin transport of mutant and WT rhodopsin.
Mice expressing endogenous rhodopsin or knock-in proteins rho-paGFP-1D4 or Q344ter rhodopsin were bred to generate three models of rhodopsin transport; rho-paGFP-1D4/+, rho-paGFP-1D4/rho-paGFP-1D4, and rho-paGFP-1D4/Q344ter. Retinas were removed from freshly euthanized mice and sliced into 300 µm sections. Confocal microscopy was utilized to photoactivate a portion of the rod inner segments of the retina and fluorescence movement monitored over time.
Between the three rhodopsin transport models, a slightly larger portion of the protein moved toward the synapse in the rho-paGFP-1D4/Q344ter model than in the other two models. However, in all three models, more rhodopsin transported toward the rod outer segment (ROS) than the synapse. Inhibition of active transport with NaN3 reduced the amount of rhodopsin moving toward the ROS in all three models, though at different amounts, revealing transport within the rho-paGFP-1D4/Q344ter model is active. Furthermore, transport toward the synapse was also reduced upon NaN3 addition with no difference between models suggesting rhodopsin transport toward the synapse is active and independent of rhodopsin’s C-terminal sequence.
Our results confirm active transport of rhodopsin and Q344ter rhodopsin toward the ROS. Additionally, these data suggest an active mechanism of rhodopsin transport toward the synapse that is independent of the rhodopsin sorting motif. Whether the low amounts of rhodopsin at the synapse have a function, or whether its transport is through mass action remains to be elucidated.
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