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A chemorepellent inhibits local Ras activation to inhibit pseudopod formation to bias cell movement away from the chemorepellent

The ability of cells to sense chemical gradients is essential during development, morphogenesis, and immune responses. Although much is known about chemoattraction, chemorepulsion remains poorly understood. Proliferating Dictyostelium cells secrete a chemorepellent protein called AprA. AprA prevents pseudopod formation at the region of the cell closest to the source of AprA, causing the random movement of cells to be biased away from the AprA. Activation of Ras proteins in a localized sector of a cell cortex helps to induce pseudopod formation, and Ras proteins are needed for AprA chemorepulsion. Here we show that AprA locally inhibits Ras cortical activation through the G protein–coupled receptor GrlH, the G protein subunits Gβ and Gα8, Ras protein RasG, protein kinase B, the p21-activated kinase PakD, and the extracellular signal–regulated kinase Erk1. Diffusion calculations and experiments indicate that in a colony of cells, high extracellular concentrations of AprA in the center can globally inhibit Ras activation, while a gradient of AprA that naturally forms at the edge of the colony allows cells to activate Ras at sectors of the cell other than the sector of the cell closest to the center of the colony, effectively inducing both repulsion from the colony and cell differentiation. Together, these results suggest that a pathway that inhibits local Ras activation can mediate chemorepulsion.

Supplemental Figure 2. rAprA does not inhibit Ras cortical activation in grlH¯ cells, and does not alter total Ras levels within 60 minutes. (A) grlH¯ cells expressing Raf1-RBD-GFP (green) were incubated in 0 (control) or 300 ng/ ml rAprA for 0 or 30 minutes then imaged. Image pairs are fluorescence on top and combined fluorescence and DIC below. Bars are 10 µm. Images are representative of cells from 3 independent experiments. (B and C) Whole cell lysates of wild type Ax2 cells incubated with or without 300 ng/ ml rAprA for the indicated times were run on SDS-polyacrylamide gels. Gels were stained with Coomassie (B), or Western blots of the gels were stained with anti-Ras antibodies (C). Images are representative of 3 independent experiments. (D) Ax2 cells were incubated in the indicated concentrations of rAprA for 6 hours. Whole cell lysates were run on duplicate SDS-polyacrylamide gels. One gel was stained with Coomassie and a western blot of the other gel was stained with anti-Ras antibodies. For quantitation of the immunoblot, bands were first normalized to their respective lane on the Coomassie gel and then normalized to the 6-hour control (no rAprA).
Values are mean ± SEM, n=3. *** indicates p < 0.001 compared to control (2-way ANOVA, Holm-Šídák's test). aprA¯ cells were incubated as in panel A, and total cell lysates or Raf1-RBD affinity bead pull-down samples were run on SDS-polyacrylamide gels. Gels were stained with Coomassie, or western blots of the gels were stained with anti-Ras antibody. Graph shows the levels of GTP-bound Ras normalized to the 0-minute control. Values are mean ± SEM from 3 independent experiments. ** indicates p < 0.01, *** p < 0.001 compared to the 0-minute control (Unpaired t-tests, Welch's correction).
Supplemental Figure 5. High cell density decreases Ras levels in cells at the center of colonies. The indicated numbers of wildtype Ax2 cells were spotted onto glass wells and incubated in HL5 for 6 hours. The cells were then fixed and stained with anti-Ras antibodies. Bars are 0.5 mm. Stitched images are representative of 3 independent experiments.
Supplemental Figure 6. AprA inhibits Ras activation at the cortical region in some mutants. Live cells of the indicated cell types expressing Raf1-RBD-GFP (green) were imaged at 0 and 30 minutes of exposure to a uniform concentration of 300 ng/ ml of rAprA. Image shows combined fluorescence and DIC. Bar is 10 µm. Images are representative of 3 independent experiments. Figure 7. Erk2 is required for rAprA induced chemorepulsion. The indicated strains were imaged for 40 minutes after 20 minutes of exposure in growth medium (control) or in a rAprA gradient in growth medium in Insall chambers. A positive forward migration index (FMI) indicates chemorepulsion from the AprA and a negative FMI indicates chemoattraction. Values are mean ± SEM of the averages of 4 independent experiments with at least 45 randomly chosen cells examined in each experiment. On the left side of the graph, *** indicates p < 0.001, **** p < 0.0001 compared with control for that genotype (Unpaired t-tests, Welch's correction). On the right side of the graph, * indicates p < 0.05 compared to Ax2 (Unpaired t-tests, Welch's correction).

Supplemental
Supplemental Figure 8. rAprA redistributes active Ras to the region of the cell away from a source of rAprA in some mutants. Images are of live cells expressing Raf1-RBD-GFP (green) in a rAprA or buffer gradient. * indicates the direction from the cell where rAprA was added. Image shows combined fluorescence and DIC. Bar is 10 µm. Images are representative of 3 independent experiments.