Actin is required for polarisome focusing. (A) myo2-66 cells carrying an empty plasmid or expressing MYO2 or myo2RD from the genomic URA3 locus were spotted in 10-fold serial dilutions on SD-Ura− plates containing 70 µM methionine and incubated for 3 d at either 25°C (upper panel) or 30°C (lower panel). (B)myo2-66 cells carrying an empty plasmid or expressing MYO2 or myo2RD from the genomic URA3 locus and coexpressing Spa2-mCherry were analyzed by fluorescence microscopy at either 27°C (left panel) or 31°C (right panel). Insets show the DIC image of the same cell in reduced magnification. (C) Intensity profiles of the Spa2-mCherry signals of the bud circumference of the cells in B at 27°C. (D) The polarisome distribution in the bud of the cells from B was classified as focused (gray), cortically spread (spread, blue), or diffuse (black; 27°C: nempty = 226, nMYO2 = 232, nmyo2RD = 225; 31°C: nMYO2 = 223, nmyo2RD = 224). (E) Left: Fluorescence microscopy of WT, bni1Δ, or bni1Δ1240–end cells expressing Spa2-GFP. Right: Intensity profiles of Spa2-GFP of the bud circumference of the cells from the left panel. (F) The polarisome distribution of the cells from E were classified as either focused (gray) or cortically spread (spread, blue; nWT = 234, nbni1Δ = 234, nbni1Δ1240–end = 227). (G) The actin cytoskeleton of WT (upper panel) or myo2RD cells (lower panel) was stained with Alexa Fluor 488–phalloidin in the absence (left) or presence (right) of CK-666. All scale bars: 5 µm.