Fig. 2: Cortical actin suppresses signaling network activation.
From: Complementary cytoskeletal feedback loops control signal transduction excitability and cell polarity
a Scanning confocal imaging of polymerizing actin (LimEΔCoil-RFP) and PIP3 (PHCRAC-YFP) in wild type (AX3) Dictyostelium cells before and after treatment with the Arp2/3 inhibitor CK666 (CK). t = 00:00 indicates CK666 addition. b Average (line) and SEM (shaded area) of the total normalized intensity and the variance of the peripheral intensity of LimE (magenta) and PHCRAC (green) patches before and after CK666 addition (dashed line, t = 0). n = 27 cells. c TIRF imaging of activated Ras (RBD-mCherry) in electrofused (“giant”) AX3 cells before and after CK666 and latrunculin addition. Cells are incubated in caffeine to raise basal activity levels. t = 00:00 indicates the addition of CK666 or, in parentheses, latrunculin. d Average and SEM of average RBD membrane intensity in giant AX3 cells before and after CK666 addition (left, dashed line, t = 0) or in CK666 before and after latrunculin addition (right, dashed line, t = 0). n = 22 cells. e Individual traces from d. t = 00:00 indicates CK666 addition; dashed lines correspond to drug addition. f Spinning disk confocal imaging of PIP3 (RFP-PHAKT) in human neutrophil-like (dHL60) cells before and after CK666 and latrunculin addition. t = 00:00 indicates the addition of CK666 or, in parentheses, latrunculin. g Average and SEM of the normalized PHAKT membrane-to-cytosol ratio before and after CK666 (left, n = 51 cells) and subsequently latrunculin (right, n = 65 cells) addition in dHL60 cells. h Individual (dots) and average (lines) PHAKT membrane-to-cytosol ratio normalized to the average level before CK666 (left, n = 51 cells) or latrunculin (right, n = 65 cells) addition. ***=P < 0.005, 2-sided Wilcoxon signed-rank test. i Spinning disk confocal imaging of Ras activation levels (RBD-EGFP) in developed AX3 Dictyostelium cells treated with CK666 (CK), latrunculin (Lat), or buffer (Buff) before and 6 seconds after the addition of 0.1 nM, 0.5 nM, and 10 nM cAMP. j Normalized RBD membrane-to-cytosol ratio before and after cAMP addition (dashed lines) in cells treated with CK666 (purple), latrunculin (yellow) or buffer (cyan). n = 31, 40, and 22 cells. k Individual and average maximum values of normalized RBD membrane-to-cytosol ratio in the 42 seconds after addition of cAMP. ***=p < 0.005, 2-sided Mann-Whitney U test. Time=min:sec; scale bars = 5 µm. Source data are provided as a Source Data file.
