Bud6 NPF activity is required in vivo for proper actin cable assembly by Bnr1. (A and B) 10-fold serial dilutions of yeast strains grown at 25 and 37°C on YEPD plates. (C) F-actin staining of the indicated strains. Cells were grown to log phase at 25°C in YEPD, fixed, and stained with Alexa Fluor 488–phalloidin. The image containing the bni1Δbud6-35 strain is a composite of three individual images. Bar, 5 µm. (D and E) Quantification of actin cable phenotypes for strains in C. For each strain, >200 budded cells were scored (approximately equal numbers of cells pooled from two independent experiments) and placed into categories: (a) Numerous and robust cables in mother; cables sometimes visible in bud. (b) Fewer or thinner cables in mother; cables visible only occasionally in the bud. (c) Highly depolarized patches and/or highly disorganized cables in the mother (e.g., cable meshworks or cables running perpendicular rather than parallel to the mother–bud axis).