Figure 6.
Asymmetrical subcellular distributions of PHCrac-GFP and inactive G-protein heterotrimers in a nonpolarized cell exposed to a steady cAMP gradient. (A) PHCrac-GFP distribution in a cell exposed to a steady cAMP gradient visualized as the red Alexa 594 fluorescence signals. (B) Quantitative measurement of the cAMP gradient and intracellular distribution of PHCrac-GFP along the line starting from the position of the dispensing micropipette and through the central part of the cell in A. The gray line reflects the relative concentration of cAMP. Assuming the maximal intensity equals 1 μM cAMP, we estimated that the concentration is ∼320 nM at the front side and 240 nM at the back side of the cell. The black line plots the PHCrac-GFP distribution from the front to the back side of the cell. These results are typical of six experiments. (C) Membrane-associated PHCrac-GFP at the front and back regions of nonpolarized cells in response to the difference in cAMP concentration (cAMP). cAMP concentration (cAMP) and membrane associated PHCrac-GFP in the front and back regions of nonpolarized cells were directly measured from six independent experiments. (D) G and PH cell images in a steady gradient. The selected front and back regions for FRET measurement of the distribution of inactive heterotrimeric G-proteins are shown. The entire area of the G cell was illuminated to photobleach YFP, and FRET was monitored as increased CFP emission in the selected front and back regions after photobleaching. FRET efficiency was calculated as [IntensityCFP(post) - IntensityCFP(pre)]/IntensityCFP(post). (E) FRET efficiency that reflects the proportion of inactive G-proteins in the front and back regions of cells in steady gradients with similar steepness (∼20%) but different cAMP concentrations. Means and SEs of FRET efficiency show the proportion of inactive G-proteins in the back (▪) is higher than that in the front (□) in response to both low (1 μM cAMP in the micropipette, n = 16 and p < 0.002) and high (3 μM in the micropipette, n = 21 and p < 0.002) cAMP concentration.