Figure 3
From: The spatial distribution of GPCR and Gβγ activity across a cell dictates PIP3 dynamics
Gβγ and PIP3 attenuation (A) RAW264.7 cells expressing α2AR-CFP, Akt-PH-Venus and mCh-Gγ3 exhibited simultaneous mCh-Gγ3 translocation and PIP3 production upon α2AR activation (at 1 min). The translocated mCherry-Gγ3 stayed at IMs (white arrows), while PIP3 level reached an equilibrium after attenuation. In addition to 515 nm excitation for YFP, 594 nm excitation was used to capture mCherry. The corresponding plot shows mCh-Gγ3 (red) and PIP3 (green) dynamics in the cytosol of the cells (n = 20). (B) RAW264.7 cells expressing α2AR-CFP, Akt-PH-Venus and mCh-Gγ3-CC mutant exhibited PIP3 production upon α2AR activation. As expected, the images and the plot show that translocation deficient Gγ mutant (Gγ3-CC) remained at the PM despite receptor activation. PIP3 showed no significant attenuation (n = 16). (C) The whisker box plots show the extents of % PIP3 attenuation in RAW264.7 cells with endogenous Gβγ (Control), or Gγ3 WT, or Gγ3-CC mutant. Extent of PIP3 attenuation was quantified using the increase in the mean cytosolic fluorescence due to PIP3 attenuation. The observed % attenuations were significantly different from each other (p < 0.05). Kymographs show PIP3 levels on the plasma membrane. Averages were plotted using cells from n ≥ 3 independent experiments. ‘n’ denotes the number of cells’ data used to plot the average curve. The error bars represent SEM (standard error of mean). The scale bar = 5 µm. IMs internal membranes.
