The actin cytoskeleton is critical for maintaining PI(4,5)P₂ gradient in filamentous cells. (A) F-actin is not required for an asymmetric PI(4,5)P₂ distribution in budding cells. Cells (wt) expressing the PI(4,5)P₂ reporter were treated with 200 μM LatA for 15 min. Average signal concentration over the long axis of small budded cells as described in Fig. 4 A (n_i.ex. = 3; n = 25 cells) with SD in gray. (B) F-actin is critical for PI(4,5)P₂ gradient in hyphae. Sum projections (10 z-sections) of representative cells, as described in Fig. 4 B. Cells as in A were treated with FCS for 30 min and then incubated in the presence or absence LatA as in A (n_i.ex. = 5). (C) Quantification of PI(4,5)P₂ gradients in cells responding to FCS. Signal concentration over the long axis of each the cell as described in Fig. 4 C. Cells were treated as described in B. Averages (n = 48 cells) shown with SD in gray. (D) Depolymerization of the actin cytoskeleton does not affect PI(4,5)P₂ reporter expression levels. Immunoblot showing the levels of the reporter (α-GFP) and loading control (α-Cdc11) in the strains and conditions used in C.