Najafi M et al. (2012), Impact of signaling microcompartment geometry o...

XB-ART-45772

J Gen Physiol 2012 Sep 01;1403:249-66. doi: 10.1085/jgp.201210818.

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Impact of signaling microcompartment geometry on GPCR dynamics in live retinal photoreceptors.

Najafi M , Haeri M , Knox BE , Schiesser WE , Calvert PD .

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G protein-coupled receptor (GPCR) cascades rely on membrane protein diffusion for signaling and are generally found in spatially constrained subcellular microcompartments. How the geometry of these microcompartments impacts cascade activities, however, is not understood, primarily because of the inability of current live cell-imaging technologies to resolve these small structures. Here, we examine the dynamics of the GPCR rhodopsin within discrete signaling microcompartments of live photoreceptors using a novel high resolution approach. Rhodopsin fused to green fluorescent protein variants, either enhanced green fluorescent protein (EGFP) or the photoactivatable PAGFP (Rho-E/PAGFP), was expressed transgenically in Xenopus laevis rod photoreceptors, and the geometries of light signaling microcompartments formed by lamellar disc membranes and their incisure clefts were resolved by confocal imaging. Multiphoton fluorescence relaxation after photoconversion experiments were then performed with a Ti-sapphire laser focused to the diffraction limit, which produced small sub-cubic micrometer volumes of photoconverted molecules within the discrete microcompartments. A model of molecular diffusion was developed that allows the geometry of the particular compartment being examined to be specified. This was used to interpret the experimental results. Using this unique approach, we showed that rhodopsin mobility across the disc surface was highly heterogeneous. The overall relaxation of Rho-PAGFP fluorescence photoactivated within a microcompartment was biphasic, with a fast phase lasting several seconds and a slow phase of variable duration that required up to several minutes to reach equilibrium. Local Rho-EGFP diffusion within defined compartments was monotonic, however, with an effective lateral diffusion coefficient D(lat) = 0.130 ± 0.012 µm(2)s(-1). Comparison of rhodopsin-PAGFP relaxation time courses with model predictions revealed that microcompartment geometry alone may explain both fast local rhodopsin diffusion and its slow equilibration across the greater disc membrane. Our approach has for the first time allowed direct examination of GPCR dynamics within a live cell signaling microcompartment and a quantitative assessment of the impact of compartment geometry on GPCR activity.

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Species referenced: Xenopus laevis
Genes referenced: dlat gprc6a grap2 lat rho rho.2 sfpq tbx2

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	Figure 1. 2-D membrane diffusion model grid. White ellipse represents the disc perimeter, and red lines indicate incisure positions traced onto the grid from the image of a rod outer segment on which an mFRAP experiment was performed. The Gaussian intensity profile represents the initial distribution of photoconverted molecules, and the flux at the disc perimeter and incisure grid points are set to zero. Diffusivities at all other grid points within the ellipse may be set arbitrarily; for this study, they were assumed to be uniform and equal to the empirically derived Dlat of Rho-EGFP (see Fig. 6).
	Figure 2. Transgenic expression of Rho-EGFP. (A) Constructs used to generate transgenic frogs. The color-coded regions above correspond to the colors of the amino acids below (PAGFP is shown). E/PAGFP was placed on the C terminus of Xenopus rod opsin with a single amino acid linker (H). The eight C-terminal amino acids of bovine rhodopsin were added to the C terminus of the GFP sequences. (B) Images of dissociated rod outer segments showing variable expression patterns of Rho-GFP. (Left) Trans-illumination images acquired with 700-nm wavelength illumination. (Right) Epifluorescence images acquired with 488-nm excitation. (C) Fluorescence of the outer segments shown in B averaged radially and plotted against axial distance. The top panel in C shows the fluorescence distribution of the dimmer cell in the top panels of B.
	Figure 3. Rho-PAGFP diffusion in outer segments of intact rods is inhomogeneous in the radial direction and absent on the axial direction. (A; left) Confocal image of a Rho-PAGFPâexpressing rod taken at a single z plane before photoactivation. (Right) Confocal image of the same rod after several multiphoton photoactivation exposures at different positions along the outer segment. Each fluorescent line across the outer segment represents a single multiphoton photoactivation pulse of 0.1-ms duration and 20-mW average power from the Tiâsappire laser tuned to 820 nm. The times indicate the total time after activation for the indicated fluorescence line. Variation in fluorescence intensity was caused by the variable expression of Rho-PAGFP as shown in Fig. 2. (B) Time course of Rho-PAGFP relaxation after multiphoton photoactivation at the radial center of the outer segment. Note the asymmetrical radial diffusion. (C; right) Time course of fluorescence changes monitored by averaging the fluorescence signals in the indicated regions of interest (left), normalized to the preactivation fluorescence in that region.
	Figure 4. End-on imaging of GFP-expressing rod outer segments allows incisure patterns to be visualized. (A) Images of retinal slices arranged with outer segments perpendicular to the chamber floor. (B) Averages of 10â20 488-nm confocal scans at single z planes 10â20 Âµm from the outer segment tip showing the incisure patterns in EGFP and Rho-EGFPâ or Rho-PAGFPâexpressing rods. (C) EM images showing the incisure patterns in frog rods (dark lines in the enface transmission electron micrograph and parallel lines indicated by the yellow arrows in the freeze fracture image). The size of the psf at the first sigma of its Gaussian intensity profile is shown approximately to scale (red ellipse). Enface EM reprinted with permission from Experimental Eye Research (Tsukamoto, 1987). Bar, â¼5 Âµm. Freeze fracture image reprinted with permission from Journal of Comparative Neurology (Corless et al., 1987).
	Figure 5. Rho-PAGFP equilibration across disc faces after photoactivation within discrete lobules is biphasic. (A) mFRAPa of Rho-PAGFP in two cells with different lobule geometries; cell 1 with wide and cell 2 with narrow connections to the larger disc. (Top left) Averaged fluorescence images before photoactivation showing incisure patterns. (Top right) Images showing ellipses and traced incisures used for modeling in Fig. 9. (Bottom) Time course images after photoactivation. (B) Time courses of fluorescence relaxation from the photoactivation sites, normalized to the projected fluorescence immediately after photoactivation, F0. Red circles indicate F/F0 recorded from the images in A. (C) The amplitudes of the fast exponential decay phases obtained from fitting data from 11 experiments (Table 2) plotted against lobule arc length show positive correlation, indicating that the transition between fast and slow phases occurs when diffusing Rho-PAGFP contacts the incisure boundary. (D) Plot of the time constants of the slow exponential decays from 11 experiments versus the width of the conduit connecting the lobule to the larger disc. The time constants fall with increasing conduit width, up to â¼0.75 Âµm, after which other factors appear to limit the equilibration rate.
	Figure 6. Recovery of Rho-EGFP within discrete disc lobules after multiphoton photobleaching is monotonic and rapid. (A) Averaged end-on fluorescence images (top panels) are shown, and the traced disc periphery and incisure patterns with mFRAPb site are indicated by a red dot (bottom panels). (B) mFRAPb recovery curves. The recovery curves were fitted with a cylinder diffusion model (red line) described previously (Calvert et al., 2010). Green line is the data model difference. (C) Dlat plotted as a function of expression level and lobule area. The Pearson correlation coefficients (R) indicated poor correlation for both comparisons.
	Figure 7. Spatially resolved microdensitometry of Xenopus rods. (A) EMCCD images of a rod illuminated with 520 Â± 5 nm, linearly polarized light with electric field vector perpendicular to the long axis of the rod, before and after complete bleach. Red dashed boxes indicate regions shown rotated 90Â° in B. (B) Absorbance images (top panels) and average absorbance along the axial dimensions (bottom panels) of the cell in A at the indicated wavelengths before complete bleach. The electric field vectors were perpendicular to the rod axis in all images, except the 520-nm images where e vector orientation is indicated (â¥ indicates perpendicular and \| \| indicates parallel to the rod axis). (C) Average absorbance values from the central portion of the rods in B, plotted as a function of illumination wavelength. The absorbance with electric vector parallel to the outer segment axis is lower as expected from rhodopsin dichroism (Liebman, 1962). Solid line is a pigment template derived by Govardovskii et al. (2000). (D) Demonstration of the spatial resolution of rhodopsin absorbance variation along the axial dimension of outer segments. (Top) Image of a dark-adapted rod outer segment at 520 nm after scanning 4-Âµm-wide regions spaced 5 Âµm apart with the Tiâsapphire laser tuned to 920 nm (5 mW average power). Scans in each region were repeated the indicated number of times. (Bottom) Axial absorbance profiles before (gray trace) and after (red trace) local Tiâsapphire laser bleaches.
	Figure 8. Axial rhodopsin absorbance is invariant despite large variation in Rho-EGFP levels. (A) A 520-nm transmission image (top) and a 488-nm epifluorescence image (bottom) of an outer segment from a Rho-EGFPâexpressing rod. (B) A 520-nm axial absorbance profile. (C) Axial fluorescence profile.
	Figure 9. A disc membrane diffusion model shows that microcompartment geometries are sufficient to explain heterogeneous Rho-PAGFP relaxation. (A) Model time course images based on the disc geometries of cells 1 and 2 of Fig. 5, with and without incisures. (B) Comparison of model prediction of the Rho-PAGFP relaxation time course with the observed relaxation replotted from Fig. 5.

References [+] :

Arikawa, Localization of peripherin/rds in the disk membranes of cone and rod photoreceptors: relationship to disk membrane morphogenesis and retinal degeneration. 1992, Pubmed