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Subcellular translocation of phototransduction proteins in response to light has previously been detected by immunocytochemistry. This movement is consistent with the hypothesis that migration is part of a basic cellular mechanism regulating photoreceptor sensitivity. In order to monitor the putative migration of arrestin in response to light, we expressed a functional fusion between the signal transduction protein arrestin and green fluorescent protein (GFP) in rod photoreceptors of transgenic Xenopus laevis. In addition to confirming reports that arrestin is translocated, this alternative approach generated unique observations, raising new questions regarding the nature and time scale of migration. Confocal fluorescence microscopy was performed on fixed frozen retinal sections from tadpoles exposed to three different lighting conditions. A consistent pattern of localization emerged in each case. During early light exposure, arrestin-GFP levels diminished in the inner segments (ISs) and simultaneously increased in the outer segments (OSs), initially at the base and eventually at the distal tips as time progressed. Arrestin-GFP reached the distal tips of the photoreceptors by 45-75 min at which time the ratio of arrestin-GFP fluorescence in the OSs compared to the ISs was maximal. When dark-adaptation was initiated after 45 min of light exposure, arrestin-GFP rapidly re-localized to the ISs and axoneme within 30 min. Curiously, prolonged periods of light exposure also resulted in re-localization of arrestin-GFP. Between 150 and 240 min of light adaptation the arrestin-GFP in the ROS gradually declined until the pattern of arrestin-GFP localization was indistinguishable from that of dark-adapted photoreceptors. This distribution pattern was observed over a wide range of lighting intensity (25-2700 lux). Immunocytochemical analysis of arrestin in wild-type Xenopus retinas gave similar results.
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12697419
???displayArticle.link???Exp Eye Res ???displayArticle.grants???[+]
Fig. 1. Bovine arrestin and Ar-GFP binding to rhodopsin. Wild-type bovine arrestin and Ar-GFP fusion were tested for specific binding to bovine and bullfrog
rhodopsin, respectively. (A) Coomassie blue stained SDS–PAGE gel of wild-type bovine arrestin bound to bovine rhodopsin in ROS membranes. Arrow
indicates arrestin (the band migrating below the 45 kDa marker is rhodopsin). (B) Western blot of SDS–PAGE gel of Xenopus Ar-GFP bound to bullfrog
rhodopsin in ROS membranes. Proteins were transferred to PVDF membrane and stained using anti-GFP polyclonal sera. Anti-rabbit alkaline phosphatase
conjugate served as the secondary antibody. Arrow indicates the Ar-GFP fusion protein. Fractions bound to the following forms of rhodopsin were separated by
SDS–PAGE: Non-phosphorylated rhodopsin in the dark (R); phosphorylated rhodopsin in the dark (RP); light-activated, non-phosphorylated rhodopsin (R*);
and light-activated, phosphorylated rhodopsin (R*P). Molecular mass standards are indicated to the left. Bound arrestin and Ar-GFP were quantified by
scanning densitometry and represented in the bar graphs, averaging 4 gels for arrestin (A) and 6 blots for Ar-GFP (B) (^SEM).
Fig. 2. Ar-GFP localization in retinal sections of transgenic tadpoles in response to various lighting intensities following overnight dark-adaptation. Each image
is representative of 5–11 retinas examined. (A) Light adaptation at 2700 lux. Green fluorescent confocal images of transgenic tadpoles expressing Ar-GFP that
were dark-adapted overnight and either fixed at time ¼ 0 (D.ON), or fixed following light-adaptation for t ¼ 15; 30, 45, 60, 90, or 150 min. Lower right-hand two
panels are controls, where a tadpole expressing unfused GFP was dark-adapted overnight or light adapted for 60 min. Arrow indicates the banding pattern (b)
observed in outer segments. Arrowhead indicates dense concentration of Ar-GFP in the area of the axoneme (a) adjoining the interconnecting cilium. Outer
segment (os); Inner segment (is); Nucleus (n); Synapse (s). (B) Light adaptation at 860 lux. Green fluorescent confocal images of transgenic tadpoles expressing
Ar-GFP that were dark-adapted overnight and light adapted for t ¼ 17; 30, 45, 67, 90, or 240 min. (C) Light adaptation at 25–50 lux. Green fluorescent confocal
images of transgenic tadpoles expressing Ar-GFP that were dark-adapted overnight and light adapted for t ¼ 17; 45, 76, 143, or 251 min. (D) Higher
magnification image from the retina of a tadpole light adapted for 45 min at 860 lux. The Ar-GFP fluorescence is distributed in a banded pattern in the OS region.
A, B, C Scale bar: 20 mm. D Scale bar: 10 mm.
Fig. 3. The localization of arrestin in wild-type tadpoles detected immunocytochemically is consistent with that of Ar-GFP in transgenic tadpoles. Sections
from wild-type Xenopus tadpole retinas were labelled using an anti-arrestin monoclonal antibody xAr1-6 (red channel). Immunocytochemically detected
arrestin is localized in the IS and axonemes of dark-adapted retinas (DA (ON)), in a banded, proximal-to-distal gradient in the OS during early light adaptation
LA (450), and in the IS and axonemes after extended light adaptation LA (2400) at 860 lux. Photoreceptors from retinal sections stained with secondary antibody
conjugate alone L. (45/NP) are not labelled. Nuclei are stained with Sytox green (blue channel). Outer segment (os); Inner segment (is); Nucleus (n); Synapse
(s): Axoneme (a); Bands (b). Scale bar: 20 mm.
Fig. 4. Quantitative densitometric assessment of Ar-GFP localization
during light- and dark-adaptation. (A) Assessment of Ar-GFP localization
during light adaptation under three separate lighting conditions (2700 lux—
diamonds; 860 lux—squares; 25–50 lux—circles). Regardless of light
intensity the ratio of OS:IS fluorescence consistently increased during early
light adaptation, and gradually decreased during extended light adaptation.
The OS:IS fluorescence ratio was calculated for each time point using
densitometric analysis of confocal images from a minimum of 5 retinas per
time point. (B) Assessment of migration during dark-adaptation. Ar-GFP
migrates rapidly to the IS during dark-adaptation. Following 45 min of light
adaptation at 2700 lux, tadpoles were fixed following 5, 10, 15, 30 or
45 min in the dark. (See filled triangles at t ¼ 50; 55, 60, 75, or 90 min).
Light adaptation curves from Fig. 4(A) are included for reference. The first
data point (t ¼45 min) represents OS:IS ratios from tadpole retinas light
adapted for 45 min. The OS:IS fluorescence ratio was calculated for each
time point using densitometric analysis of confocal images from 7 to 8
retinas per time point.
Fig. 5. Ar-GFP localization in retinal sections from Ar-GFP tadpoles during
dark-adaptation following 45 min of light adaptation at 2700 lux. Darkadaptation
is characterized by a steady, uniform decrease of Ar-GFP in the
OS. Fluorescence confocal images were collected from tadpoles darkadapted
for 5 min (D. 50), 10 min (D. 100), 15 min (D. 150), 30 min (D.300),
and 45 min (D. 450). Each image is representative of 7–8 retinas examined.
Outer segment (os); Inner segment (is); Axoneme (a); Nucleus (n); Synapse
(s). Scale bar: 20 mm.
