Extended Data Figure 4: Spatiotemporal organization of different mutated charge sensors and uniform membrane controls.
(A) Representative live cell images of Dictyostelium cells co-expressing GFP-R(+8)-Pre and PHCrac-mCherry under chemotactic gradient stimulation. Solid magenta arrowhead indicates the direction of micropipette (filled with 1 μM cAMP) for gradient stimulation. Dashed magenta arrowhead indicates the introduction of needle (t=0s) which is manifested by the transient global response in PHCrac channel. Cells were pre-treated with Latrunculin A. (B-D) Live-cell time-lapse images and line scan intensity profiles of Dictyostelium cells expressing PHcrac-mCherry, along with GFP-R(+7)-Pre (B) or GFP-R(+4)-Pre (C) or GFP-R(+2)-Pre (D), during ventral wave propagation, displaying decreasing extent of back-state preference of the surface charge sensors. The first time points were showed in Figure 2J (in grayscale colormap). (E and F) The 360° membrane kymographs of cells shown in Figure 2K, indicating R(+7)-Pre consistently moves away from PIP3-rich protrusions (E), whereas R(+2)-Pre is uniform over the cortex (F). (G) Live-cell images, line scan intensity profiles, and representative line-kymographs of ventral waves in Dictyostelium cells co-expressing PHcrac-mCherry and membrane marker cAR1-GFP, demonstrating that cAR1 does not distribute to front- or back- state regions and it is consistently uniform over the membrane. (H) Live-cell time-lapse images of migrating Dictyostelium cells co-expressing PHcrac-mCherry and cAR1-GFP showing cAR1 is symmetric over the membrane. Black arrows: PIP3- rich protrusions where cAR1 was present as well. (I) Live-cell images, linescan intensity profiles, and representative line-kymographs of ventral waves in RAW 264.7 cells co-expressing PHAKT-mCherry and membrane marker, LYN-GFP, showing consistent uniform profile of LYN over the membrane and no depletion in front-state area. (J) Live-cell time-lapse images of migrating Dictyostelium cells co-expressing PHcrac-mCherry and GFP-Palm/Pre, showing a symmetric profile of Palm/Pre over the membrane. Black arrows: Protrusions/front-states. In (G-J), the “Fire invert” LUT of Fiji/ImageJ was used so that it can clearly show any small inhomogeneity.