Fig. 6. Actin polymerization and chemotactic response upon cAMP stimulation of aggregation competent cells. (A) F-actin polymerization responses of GDI1^– and complementation mutants. The relative F-actin content was determined by TRITC-phalloidin staining of cells fixed at the indicated time points after stimulation with 1 µM cAMP. Each data point represents the average of at least three independent measurements. For the sake of clarity, error bars are not shown. Standard deviations fell between 4% and 11% of the average values. In GDI1^– cells, the initial response of actin polymerization and depolymerization was reduced, and was restored after re-expression of GDI1. (B) Chemotactic movement toward a micropipette filled with 0.1 mM cAMP. Cells were starved for 6 h, allowed to sit on a glass coverslip and stimulated with a micropipette filled with 0.1 mM cAMP. Images of chemotaxing cells were captured every 30 s for up to 60 min. DIAS software was used to analyze cell movement and generate the migration paths. The asterisks indicate the position of the micropipette tip. GDI1^– cells display an apparently normal chemotactic response. (C) Morphology of aggregation competent wild-type and GDI1^– cells. Cells were prepared as in (B), fixed and stained with mAb Act-1 to visualize actin. GDI1^– cells are well polarized and build cell streams. Scale bar, 10 µm.