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JJ Peterson, BM Tam, OL Moritz, DS Papermaster, PA Hargrave, WC Smith; Time Course of Arrestin-GFP Migration in Xenopus Photoreceptors in Response to Light and Dark . Invest. Ophthalmol. Vis. Sci. 2002;43(13):1395.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:To study the time scale of arrestin migration during light and dark adaptation. Methods: F1 progeny from transgenic Xenopus laevis expressing a Xenopus arrestin-GFP fusion protein in rod photoreceptors were fixed at different intervals during light and dark adaptation. For the light-adaptation series, tadpoles were dark adapted overnight, and fixed (3.7% formaldehyde/87% methanol) following exposure to light for 15, 30, 60, and 120 minutes. For the dark-adaptation series, tadpoles were dark adapted overnight, light adapted for 60 minutes, and then dark adapted for 15, 30, 60, 120, and 270 minutes and fixed. Frozen retinal sections were analyzed using fluorescence confocal microscopy. Results: In fully dark-adapted retinas, arrestin-GFP was retained almost exclusively in the rod inner segment (RIS). The fusion protein was not apparent in the outer segment except in the ciliary stalk. After 15, 30 and 60 minutes of light exposure, arrestin-GFP migrated to the proximal third, half, and distal portions of the rod outer segments (ROS), respectively. No further increase in the ratio of ROS to RIS GFP fluorescence was observed after 60 minutes. However, at no point in light adaption was the RIS totally devoid of arrestin-GFP. Intriguingly, although the migration front was easily observed by intrinsic fluorescence, neither anti-arrestin nor anti-GFP antibodies effectively labeled the fusion protein in the ROS although both antibodies were able to detect the fusion protein in the RIS. After only 15 minutes of dark adaptation, arrestin-GFP localization changes dramatically from being homogeneously distributed throughout the ROS to being highly concentrated within the RIS and ciliary stalk. Conclusion: We have clearly demonstrated that arrestin-GFP migrates from the RIS to the ROS within 60 minutes of light onset. The intrinsic fluorescence of arrestin-GFP is easily detected throughout the photoreceptor, whereas antibodies to either protein component of the fusion did not detect the fusion protein when located in the ROS for reasons that are still unclear. During dark adaptation, the majority of the arrestin-GFP relocalized to the RIS within 30 minutes.
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