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J. Sammons, S. D. McAlear, A. Marsh, A. K. Gross; Monitoring Rhodopsin Trafficking in Live Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1895.
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We have expressed rhodopsin fused to photoactivatable GFP with the addition of rhodopsin’s C-terminal 8 amino acids (rho-paGFP-1D4), and studied its localization, biochemical and spectral properties in order to use as a tool in trafficking studies in cultured cells and in transgenic Xenopus laevis.
A synthetic gene encoding rho-paGFP-1D4 was transfected into COS cells or into IMCD cells, a polarized ciliated mouse cell line derived from the inner medullary collecting duct. The fusion protein was purified from COS cells, reconstituted with 11-cis retinal and solubilized. UV/Visible spectra were taken in the dark and after exposure to light to monitor for proper folding of the fusion protein. The localization of the expressed protein in both transiently transfected COS and IMCD cells was monitored using immunocytochemistry and by photoactivation of the fusion protein. Stable IMCD cell lines expressing low amounts of rho-paGFP-1D4 were prepared to monitor ciliary localization of the fusion protein and trafficking in cultured polarized cells. Transgenic Xenopus laevis expressing rho-paGFP-1D4 were prepared to examine the localization and trafficking of the photoactivated protein in rod cells using 2-photon fluorescence confocal microscopy.
Rho-paGFP-1D4 forms a rhodopsin-like pigment with 11-cis retinal, and undergoes appropriate spectral changes after exposure to visible light. Additionally, the fusion protein traffics to the cilium in stably transfected polarized IMCD cells and to the rod outer segments of transgenic X. laevis. The paGFP moiety of the fusion protein is able to be photoactivated in individual rods from transgenic X. laevis.
These data suggest that rho-paGFP-1D4 has similar folding and photoisomerization properties to those of wild type rhodopsin, and is properly localized in polarized cells. Thus it is functionally similar to wild type rhodopsin and can be used to measure trafficking in cultured cells or transgenic retinas.
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