Abstract
Abstract: :
Purpose: Arrestin and transducin translocate in vertebrate photoreceptors in reciprocal directions in response to light. In the dark–adapted eye, arrestin is concentrated in the inner segment (IS) and migrates to the outer segment (OS) in response to light adaptation; transducin is concentrated in the OS in the dark, and moves to the IS in response to light adaptation. The goal of this study was to determine if arrestin and transducin are using the cytoskeleton as a molecular framework on which to move, and if so, which cytoskeletal elements are being utilized. Methods: Wild–type tadpoles of Xenopus laevis and transgenic tadpoles expressing a fusion of green fluorescent protein (GFP) at the C–terminus of arrestin were treated with cytoskeletal poisons to target specific cytoskeletal elements. For microfilaments, tadpoles were treated for 5 hours with 25 µM cytochalasin D (CD). For microtubules, tadpoles were treated for 7–11 hours with 500 µM thiabendazole (TB). Tadpoles were either dark adapted or light adapted after exposure to the reagent, and then fixed and processed for confocal microscopy. Arrestin localization was analyzed using intrinsic GFP fluorescence or anti–arrestin immunocytochemistry; transducin localization was detected using anti–Tα immunocytochemistry. Results: Arrestin translocation was unaffected by treatment of tadpoles with CD, both to the OS in response to light, and back to the IS in response to dark adaptation. In contrast, transducin translocation from the IS to the OS during dark adaptation was significantly delayed by CD. In tadpoles treated with TB, arrestin translocation from the IS to the OS in response to light was not affected, but its return from the OS to the IS in response to dark adaptation was significantly delayed. Similarly, transducin migration from the OS to the IS in response to light adaptation was significantly slowed. Conclusions: Our results indicate that arrestin translocation relies on the microtubular cytoskeleton, particularly in moving arrestin from the OS to the IS. Transducin translocation appears to utilize both cytoskeletal elements, using microtubules to translocate from the OS to the IS, and microfilaments to translocate from the IS to the OS.
Keywords: photoreceptors • cytoskeleton • microscopy: confocal/tunneling