Diffusion and light-dependent compartmentalization of transducin

Abstract

Diffusion and light-dependent compartmentalization of transducin are essential for phototransduction and light adaptation of rod photoreceptors. Here, transgenic Xenopus laevis models were designed to probe the roles of transducin/rhodopsin interactions and lipid modifications in transducin compartmentalization, membrane mobility, and light-induced translocation. Localization and diffusion of EGFP-fused rod transducin-α subunit (Gα(t1)), mutant Gα(t1) that is predicted to be N-acylated and S-palmitoylated (Gα(t1)A3C), and mutant Gα(t1) uncoupled from light-activated rhodopsin (Gα(t1)-Ctα(s)), were examined by EGFP-fluorescence imaging and fluorescence recovery after photobleaching (FRAP). Similar to Gα(t1), Gα(t1)A3C and Gα(t1)-Ctα(s) were correctly targeted to the rod outer segments in the dark, however the light-dependent translocation of both mutants was markedly impaired. Our analysis revealed a moderate acceleration of the lateral diffusion for the activated Gα(t1) consistent with the diffusion of the separated Gα(t1)GTP and Gβ(1)γ(1) on the membrane surface. Unexpectedly, the kinetics of longitudinal diffusion were comparable for Gα(t1)GTP with a single lipid anchor and heterotrimeric Gα(t1)β(1)γ(1) or Gα(t1)-Ctα(s)β(1)γ(1) with two lipid modifications. This contrasted the lack of the longitudinal diffusion of the Gα(t1)A3C mutant apparently caused by its stable two lipid attachment to the membrane and suggests the existence of a mechanism that facilitates axial diffusion of Gα(t1)β(1)γ(1).

Fig. 1. Expression of EGFP-Gα_t1 and mutants in retinas of transgenic X. laevis

(A). Map of the transgene. L – linker. The Sal I site was used to linearize the transgene plasmid for transgenesis. (B) A piece of retina of a transgenic tadpole expressing EGFP- Gα_t1. EGFP-fluorescence/DIC overlay. Bar – 20 μm. (C) Samples of retinal extracts from tadpoles expressing EGFP-fused Gα_t1 (1), Gα_t1A3C (2), and Gα_t1-Ctα_s (3) (equivalent to 2 retinas at about stage 50 X. laevis tadpole with an average fluorescence intensity in the eye) were immunoblotted with rabbit anti-GFP antibodies (Invitrogen) (left) or with anti-Gα_t1 antibodies K-20 (Santa Cruz Biotech) (right). Arrow indicates frog Gα_t1.

Fig. 2. Localization of G α_t1, Gα_t1A3C, and Gα_t1-Ctα_s in dark- and light-adapted rods

(A) EGFP-fluorescence in living rods expressing EGFP-fusion proteins of Gα_t, Gα_t1A3C, and Gα_t-Gα_sCt. Transgenic tadpoles were dark adapted overnight (D). After dark adaptation, some tadpoles were exposed to light (800 lux/40 min) in a Petri dish (L). Bar -10 μm. (B) Mean fluorescence intensities in the OS (red line) and IS (yellow line) of light-adapted rods were quantified using Z-stack projection images and ImageJ. The ratio of the intensities (I_OS/I_IS) (mean±SE) was determined for control Gα_t1 rods (2 tadpoles, 8 rods) and Gα_t1A3C rods (2 tadpoles, 8 rods) (p=0.003).

Fig. 3. Light-dependent translocation of total transducin in EGFP-Gα_t1A3C rods

Localization of total Gα_t1 (red, anti-Gα_t1 K-20 immunofluorescence) and EGFP-Gα_t1A3C (green, EGFP-fluorescence) in the dark (D) and after light exposure (800 lux, 40 min) (L). OS – outer segment, IS – inner segment. Bar -5 μm.

Fig. 4. Membrane association of Gα_t1A3C

Soluble GTPγS-extracted (S) and membrane (M) fractions were obtained from control EGFP-Gα_t1 and EGFP-^Gα_t1A3C tadpoles as described in Materials and Methods. The fractions were analyzed by Western blotting with anti-Gα_t1 antibodies K-20 (Santa Cruz Biotech) (right). Open and filled arrows indicate EGFP-fused and frog Gα_t1, respectively.

Fig. 5. Representative measurement of lateral diffusion of EGFP-Gα_t1β₁γ₁

(A). Pre- and post-bleach images of a transgenic ROS at different time points. The retina was pre-incubated in Ringer’s buffer containing 10 mM hydroxylamine under ambient light conditions to monitor diffusion of Gα_t1β₁γ₁. 150 images (256×256 pixels) were collected at 196 msec/frame with no intervals between the scans. (B) Intensity integrated along y-axis for each x-axis pixel of the selected box region from the first post-bleach image. The plot is used to determine the width of a bleach region with a Gaussian profile and the depth of bleach according to the protocol of Wang et al. (2008). (C) Intensities of the bleached region during the experimental time course corrected for background and fading (black) and the fitting curve to a one-dimensional diffusion equation 3 in Wang et al. (2008).

Fig. 6. Representative measurement of longitudinal diffusion of EGFP-Gα_t1β₁γ₁

(A). Pre- and post-bleach images of a transgenic ROS at different time points. The retina was pre-incubated in Ringer’s buffer containing 10 mM hydroxylamine to monitor diffusion of Gα_t1β₁γ₁. 150 images were collected with a 2-sec interval between the scans. (B) Intensities of the bleach region during the experimental time course corrected for background and fading (black), and the fitting curve to one-dimensional diffusion equation 3 in Wang et al. (2008).