Protrusions made by cells impaired in PI3-kinase signaling. (A) Blebbing is impaired in mutant cells chemotaxing under 0.7% agarose. Analysis with QuimP10 shows that the mutants are deficient in forming high-speed projections, which we equate to blebs (cells analyzed: Ax2 = 30; PI3K1–5⁻ = 19; CRAC⁻/PhdA⁻ = 19). Projections made by the wild type were further classified by eye as pseudopods or blebs (green and red) or various hybrids and unidentified (other marks). (B) Chemotactic orientation of low-speed projections (<1 µm/s, equated to pseudopods) of cells moving under 0.7% agarose. It is apparent that pseudopods are projected less accurately by the mutants (projections analyzed: Ax2 = 374; PI3K1–5⁻ = 257; CRAC⁻/PhdA⁻ = 416). (C) Actin polymerization after acute stimulation of cells with cyclic-AMP. The PhdA and CRAC mutants show a robust fast response (peak before 10 s), similar to the wild type. (D) Formation of F-actin microspikes during turning toward a micropipette releasing cyclic-AMP (red asterisks). Microspikes form in the same time-scale as the first peak of actin polymerization in C and are made by the PhdA/CRAC double mutant as well as the wild type. In A and B, projections by cells moving under 0.7% agarose were identified automatically using modified QuimP10 software and their maximum speed, total displacement, and orientation determined.