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Fig.1. Fluorescence intensity and volumes of CHO cells expressing EGFP. (A) Transmission image of pDP3 cells. (B) Fluorescence intensity of the cells in A, measured with the CLSM; this image is the average of two successive x-y scans taken at the z-level where the apparent cell diameters were maximal. (C) Three-dimensional surface renderings of the cells of panels A and B, from the viewpoint of the objective in the inverted microscope, and presenting for each cell an iso-intensity surface at a level ∼3% of the maximum fluorescence. (D) Mean fluorescence intensity of 485 pDP3 cells (•) and 193 non-expressing control CHO cells (○), plotted as a function of the cell volume. The green filled symbols plot the data of the cells illustrated in panels A-C. The symbols to the right give the mean±s.d. of each population, scaled relative to the mean of the pDP3 cell fluorescence. The dashed lines show the boundaries of 15 bins into which the data were pooled to examine dependencies of the estimated aqueous volume fraction on cell volume and EGFP levels.
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Fig.2. (A) Quantitation of EGFP mass per CHO cell with western blotting and fluorimetry. Immunofluorescence of a gel stained with primary antibody against GFP. Lane 1 (red band) was injected with the lysate of 5×104 pDP3 cells, while lanes 2-7 contained incremented amounts of recombinant EGFP; lane 8 (green bands), molecular size calibration. (B) Fluorescence intensity of the bands of the gel in A as a function of loaded protein: each point corresponds to the intensity of the lane above it. The line is a least squares regression line fitted to the points in the linear range of the blot. The protein mass in the pDP3 lysate band (lane 1) is estimated from the projection of the band intensity (leftmost circle) onto the regression line, yielding for this experiment 0.2 pmols, or 4.0 amols per cell. (C) Fluorimetric analysis of cell lysates. Lysates of 106 pDP3 or non-expressing control cells in 100 μl of non-denaturing buffer were scanned in the CLSM in the same manner as recombinant EGFP (see Materials and Methods). Filled symbols show fluorescence (mean±s.d.) of lysates: •, pDP3 cells (n=12 scans of 8 different lysates); , non-expressing cells (n=7 lysates). The unity slope regression line through the pDP3 lysate data corresponds to 57 nM recombinant EGFP, yielding 5.7 pmols total mass, or 5.7 amols/cell, in good correspondence with the value 5.4±1.1 amols/cell obtained from averaging estimates from regression analysis of the lysates of the 12 experiments.
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Fig.3. Features of rod cells expressing EGFP. (A) Transmission image of a small piece of Xenopus retina in the recording chamber. (B) Three-dimensional rendering of CLSM scans of the piece of retina shown in A. The scan data are displayed using a linear gradient over the lower 50% of the fluorescence range to enhance visibility of weakly fluorescing rods. (The rod identified by the arrow in B is singled out for further analysis in Fig. 4A. The red arrows point to a small area of high fluorescence, a feature examined in Fig. 6.) (C,D) An outline of each rod identified in the 3D rendering was made: rods nearer the chamber floor have been outlined in red, while those further back in the stack have been outlined in yellow. In panel C these outlines have been superimposed on the transmission micrograph, illustrating the correspondence between the two images.
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Fig.4. Quantification of the spatial distribution of EGFP in living Xenopus rods. (A) EGFP distribution in a single rod. The voxels corresponding to the rod identified in Fig. 3B (white arrow) were excised from the stack of 2D CLSM scans, and projected onto a plane parallel to the OS axis: the panel is a false-color representation of the fluorescence distribution in the average of three scans spanning a thickness of 1.5 μm. The black line through the image is the automatically determined `z-spline' path through the cell's core along which a fluorescence intensity profile was computed. (B) Fluorescence intensity profiles of 8 rods from the piece of retina illustrated in Fig. 3, segregated with colors to indicate different levels of fluorescence: red for most intense, green for mid-level and blue for least intense (the profile of the rod of panel A is identified with a thickened red line). Panels A and B are aligned on the abscissa, and the profiles of all rods have been aligned at the junction between the IS and OS (x=0). The colored symbols at the left give the mean (±s.d.) fluorescence of the voxels of the entire IS of each rod having the top 5th percentile of intensities (comprising, on average, 460 voxels per rod). In the OS region the spline goes through the center or `core' of the rod discs, whereas in the IS the spline randomly encounters high and low fluorescence voxels. (C) The profile distribution of each rod in panel B has been normalized by the value of the corresponding colored circle plotted at the left of the graph. The thickened black line plots the average of the normalized intensity profiles of the 15 rods `cut' from the piece of retina illustrated in Fig. 3. The black symbol to the left represents the normalization value (unity), and the error bar gives the average coefficient of variation (s.d./mean) of the top 5th percentile of voxels of the inner segments of the 15 rods. (D) Grand average normalized intensity distributions of rods expressing EGFP. Data such as shown in panel C were pooled from 57 rods whose profiles were extracted from CLSM scans of 11 pieces of retina from 5 Xenopus tadpoles and froglets ranging from 4 weeks to 9 months of age. Error bars are 95% confidence intervals. (The average trace from panel C is shown in light gray for comparison.)
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Fig.5. Quantitative test of the EGFP equilibration hypothesis in Xenopus rod cells. (A) Each point gives the average concentration of EGFP in a rod OS derived from a profile analysis such as illustrated in Fig. 5A,B, plotted as a function of the maximal concentration of EGFP in the rod IS. The gray lines have a slope of G and plot the prediction of the EGFP equilibration hypothesis that the OS should exhibit 50% of the fluorescence intensity of the brightest voxels of the IS. The inset shows the distribution in the lowest corner of the main plot, i.e. the initial 15 μM of the abscissa on an expanded scale. The red circles plot scan data of cells after a complete bleaching exposure; pre-bleach scans of the same cells were unchanged. (B) Display of the brightest 5% of the voxels (green) and the dimmest 5% (red) of the rod identified by the arrow in Fig. 3B; three orientations of the rod are provided. Autofluorescence levels of control rods were comparable with those of CHO cells (Fig. 1D) under our experimental conditions, and thus negligible in comparison with cells having average [EGFP] in the micromolar range.
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Fig.6. Distribution of Arr-EGFP in dark adapted rods. (A) Pseudocolor representation of the distribution of Arr-EGFP in a dark-adapted rod. (B) Profile distributions along splines of the rod of panel A (thickened red trace), along with those of nine other rods from the same piece of retina, scaled by the intensity of the voxels of the inner segment having the top 5% fluorescence intensity. (C) Average fluorescence distributions (red trace) of Arr-EGFP in 15 rods from two animals compared with the average distribution of EGFP (black trace; cf. Fig. 4D); error bars are 95% confidence intervals. (D) Three-D rendering of the rod in A in which the dimmest 5% of the voxels are shown in red, whereas the 5% most intensely fluorescing voxels are shown in green.
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Fig.7. Redistribution of Arr-EGFP fusion protein upon exposure of the retina to light and test of conservation of total protein. (A) Pseudocolor representation of the fluorescence intensity of a Xenopus rod expressing the fusion protein, Arr-EGFP 30 minutes after a 30 second light exposure that bleached all the rhodopsin; the dark adapted profile of the same rod is shown in Fig. 8A. (B) Intensity distributions along the rod in the dark (black) and 30 minutes (green for the IS; red for the OS) after the bleaching exposure. (C) Test of the hypothesis that total Arr-EGFP in the rod is conserved before and after the bleaching exposure. The mass of Arr-EGFP in the OS (red circles) and in the IS (green circles) was determined in the dark, and at 30 and 60 minutes after the bleaching exposure, and the sum (black circles) computed. For 10 rods from four separate pieces of retina, the same analysis was followed, and the data of each rod was normalized by the total Arr-EGFP in the cell in the dark (left axis). The scale on the right gives the Arr-EGFP mass for the average cell of the population; individual rods had up to 30 amols. Error bars are 95% confidence intervals. Conservation of Arr-EGFP is represented by the fact that the total (black circles) remains constant over the ca. 1.5 hour experiment. (D) 3D renderings of the distribution of Arr-EGFP of panel A at 30 minutes after the bleaching exposure: the leftmost images displays the fluorescence in a linear gradient; the middle and rightmost images show the brightest 5% voxels (green), and the dimmest 5% (red), in two orientations of the rod.
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Fig.8. Xenopus rod aqueous spaces revealed by EGFP and confirmed by immunohistochemical labeling of cytochrome c and α-tubulin, and by EM. (A) Confocal image of transverse frozen section of retina with only the EGFP channel activated. Arrows point to bright dots that localize just below the OS, which align with a green line that projects up the OS. (B) Same section as in A, showing immunostaining of cytochrome c (red) and the nucleus with DAPI (blue). The green fluorescence in the same location as the DAPI labeling shows EGFP to be present in the nucleus. Note the presence of three cone nuclei (deep blue) which show no green labeling; these cones give rise to dark regions in the images of A and B. (C) Confocal image of transverse frozen section labeled with antibody against acetylated α-tubulin (red), which identifies the cilium and axonemes of two rods (arrows) and two cones (no arrows). (D) Same image as in C, but with the EGFP channel activated: the bright green dots are seen to localize at the projection of the axoneme to the top of the ellipsoid region. The EGFP channel is relatively less intense than in A and B because some of the protein is lost owing to the use of Triton to enable the antibody access to α-tubulin (Kaplan et al., 1987). When z-stacks of such confocal images are scanned, every single rod shows the features illustrated in these panels. (E-H) Montage of EM images of the ellipsoid and basal disc region of Xenopus rods: in these panels one can see a region devoid of mitochondria immediately surrounding the basal body of the cilium (arrows). Scale bars, 1 μm.
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