Figure 6: LEGI-BEN for folic acid in Dictyostelium and fMLP response in human neutrophils.
From: Evolutionarily conserved coupling of adaptive and excitable networks mediates eukaryotic chemotaxis
(a–c) Ras activation in Dictyostelium cells stimulated with folic acid. (a) Responses (n=32 cells each) to 2 s (red) and long (orange) stimuli. The response to a 2s cAMP stimulus (n=10 cells, blue) is plotted for comparison. (b) Responses (n=15 cells, red) to the second 2s stimulus applied after variable intervals (T) following the first 2 s stimulus. The response to cAMP stimuli (n=30 cells, blue) is plotted for comparison. (c) Responses (n=20, 19, 18 cells, respectively; 3 experiments) to a 2 s folic acid stimuli applied 50 s after first stimuli of 10, 60 and 120 s. (d) Ras activation in Dictyostelium cells stimulated with 5 μM folic acid at 0 s followed by 0.3 μM cAMP at12 s (red, n=15 cells; 4 experiments) or 45 s (blue, n=13 cells; 5 experiments). (e) Ras activation in Dictyostelium cells responding to folic acid with or without prior adaptation to cAMP. Non-adapted cells were stimulated by 2 s folic acid (blue). The same cells were then treated with 200 μM cAMP for 3 min and stimulated with a 2s folic acid pulse (red; n=12 cells; 2 experiments). For a–e, responses were defined as the peak cytosolic RBD-GFP decrease normalized to the pre-stimulus cytosolic levels (a,d,e) or to the first response (b,c). (f–h) PH-GFP responses in human HL60 neutrophils responding to fMLP. (f) Responses (n=4 cells each) to 10 s (blue) and 120 s (red) stimulation. (g) Responses (n=5 cells each) to a second 10s stimulation administered after various intervals (T) following first stimuli of 10 s (blue) or 120 s (red). (h) Responses (n=5 cells each) to a second 10 s stimulation administered 150 s after removal of the first stimulation of various durations (T). In all experiments, HL-60 cells were immobilized with the JLY cocktail (see Methods)60. The responses were defined as peak changes in the (membrane+cytosol)/(cytosol) of PH grey values. For g and h, normalization was to the responses to the first stimuli. Error bars: s.d. for a,b and s.e.m. for c–h.
