Purchase this article with an account.
P. D. Calvert, E. N. Pugh, Jr., J. B. Ames, C. Gherghisan; High Resolution 3D Map of a Recoverin-EGFP Fusion Protein in Living Xenopus Rods. Invest. Ophthalmol. Vis. Sci. 2007;48(13):604.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
To determine the distribution of the calcium-binding regulatory protein, recoverin, which is predicted to be soluble at low calcium concentrations, in living rod photoreceptors at sub-micron spatial resolution and to compare this distribution to that of the soluble cytoplasmic water-space marker, unfused EGFP.
Transgenic tadpoles that contain a fusion construct consisting of a truncated Xenopus opsin (XOP) promoter, the coding sequence of bovine recoverin followed at the 3’ end with that of EGFP were generated by the REMI method, thus allowing rod photoreceptor-specific expression of tracer levels of recoverin with EGFP fused to its c-terminus. Tadpoles that expressed unfused EGFP under the XOP promoter were generated by natural mating of extant adult transgenic frogs. Retinas from stage 48 or greater transgenic tadpoles were isolated into frog Ringer’s solution, sliced perpendicularly to the retinal surface and placed in a live-cell imaging chamber on the stage of a confocal and multiphoton microscope. The distribution of the fusion protein, or of unfused EGFP, was determined by quantitative 3D scanning at submicron resolution.
The distribution of recoverin-EGFP in light adapted Xenopus rod photoreceptors was similar to that of unfused EGFP, but with subtle differences: The brightest fluorescence was found in the myoid region, between the nucleus and the ellipsoid. The outer segment fluorescence intensity was uniform and lower than that of the myoid. Unlike EGFP, recoverin-EGFP appears to aggregate in the myoid region in some cells in a pattern reminiscent of membranous ER and Golgi organelles, which could indicate that the myoid calcium concentration is high. Preliminary results suggest that recoverin-EGFP levels are lower relative to that of unfused EGFP in the synaptic region.
The distribution of recoverin-EGFP in light adapted rods is largely as expected for a soluble protein, but deviations from expectations suggest that calcium may not be uniformly low in the light adapted rod.
This PDF is available to Subscribers Only