Fig. 6. AC is required for formation of oriented actin-filament bundles and cell polarization. Cell polarization was initiated in chick heart fibroblasts by washing out Lat A. Cells were then incubated live for 15, 25, 30 or 60 minutes after the washout, fixed and stained with Alexa-Fluor-380-conjugated or FITC-conjugated phalloidin. (A) Control cells expressing GFP only. (B) Cells expressing inactive pseudo-phosphorylated AC only (XAC1 E3 mutant). (C) Cells expressing LIMK1 EE508 only to block AC function. (D) Rescue: cells co-expressing LIMK1 EE508 and active, non-phosphorylatable AC (XAC1 A3 mutant). Cells expressing GFP only were identified by GFP expression (inset in A); LIMK-EE508-expressing cells by GFP expression off a separate promoter (insets in C and right insets in D); and XAC1-E3- and XAC1-A3-expressing cells by anti-XAC-antibody staining (insets in B and left insets in D), which has minimal cross-reactivity with endogenous AC (Shaw et al., 2004). Note the lack of formation of oriented actin-filament bundles in cells in which AC function is blocked (B,C). (E) Quantification of the formation of oriented actin-filament bundles in the cell population. (F) Quantification of cell polarization in the cell population. Graphs are expressed as mean ± s.e.m. of triplicate experiments (n=600 cells per condition). Bar, 10 µm.