Figure 6. Movement of Arrestin from the OS to the IS Requires Dissociation of the Rhodopsin-Arrestin Complex.
(A) Eyecups were prepared from dark-adapted mice in the dark and either kept in DMEM (+ Glucose) or ATP depleted. They were then exposed to light at room temperature for 1 hr, treated with hydroxylamine for 15 min, and fixed. Localization of arrestin was determined by immunofluorescence microscopy.
(B) Eyecups were prepared in the dark and either ATP depleted for 1 hr in the dark or kept in DMEM. They were then illuminated for 1 hr with ambient light at room temperature to cause movement of arrestin to the OS and then transferred to the dark. At the indicated times, eyecups were fixed, and localization of arrestin was determined.
(C) Kinetics of arrestin translocation to the IS in vivo. Mice were exposed to light (600 lux) for 1 hr and then returned to the dark. At the indicated times, animals were sacrificed. Enucleated eyes were placed in fixing solution, and cornea and lens were rapidly removed to allow instantaneous fixing.
(D) Kinetics of rhodopsin dephosphorylation in vivo. The contralateral eyes from the animals used in (B) were homogenized in 0.5 ml of 7 M urea, and the phosphorylation status of rhodopsin in the eyecups was determined by mass spectrometry.