Blebbing and movement under an agarose overlay are regulated by PI3-kinase and its downstream effectors, CRAC and PhdA. (A) Blebbing in response to cyclic-AMP is severely impaired when PI3-kinase activity is inhibited, either in a mutant lacking five PI3-kinases (PI3K1–5⁻) or by adding the PI3-kinase inhibitor, LY294002, to Ax2 cells. Blebbing in mutants of downstream PIP3-binding proteins is unimpaired in PKB/PKBR1 double-null cells, but significantly impaired in CRAC⁻ and PhdA⁻ cells, where it can be substantially rescued by re-expression of the corresponding GFP fusion protein. A double CRAC/PhdA-null mutant blebs very poorly. (B) Movement speed is reduced under 0.7% agarose overlays in PI3-kinase, CRAC, and PhdA mutants. (C) Wild-type and PI3-kinase quintuple knock-out cells under 2% agarose. (D) PKB/PKBR1 double-mutant cell produces a bleb (dot) in a re-orientation experiment performed as in Fig. 6, and providing evidence that blebbing is unimpaired in this mutant. Cyclic-AMP shock assay: cells were stimulated with 1 µM cyclic-AMP, and blebs counted manually. Under agarose migration speed: the speed of cells moving under 0.7% agarose toward a well containing 4 µM cyclic-AMP was measured. Bar, 10 µm.