Parameters regulating bleb-driven movement. (A) Blebbing increases as cells prepare for multicellular development. Bleb frequency was measured after different times of starvation (with cyclic-AMP pulsing) in cells randomly moving under buffer, or after addition of 1 µM cyclic-AMP; results are the mean of three separate experiments, in each of which 40–80 cells were analyzed at each time-point. (B) Blebbing increases as the concentration of agarose in the overlay is increased. Blebs given as percentage of total projections (blebs + pseudopods). (C) Decreasing cell height with increasing agarose concentration in the overlay, as determined from confocal images (see Video 5 for reconstructions of cells at different agarose concentrations and for the movement of fluorescent beads in the agarose as cells pass). (D) Dependence of Young’s modulus on the agarose concentration. Agarose elasticity modulus was measured by indentation with a spherical tip at 0.06 mm/s; average of three replicates for each concentration. Ax2 cells expressing the F-actin reporter ABD-GFP, to help bleb identification, were used throughout.