Rho3p is required for the activation of full-length Bni1p. (a) Visualization of actin (Act1p) and tropomyosin (Tpm1p) by immunofluorescence microscopy in the indicated cells. (b) Quantitation of the accumulation of cable-like filaments in wild-type or rho deletion cells overexpressing COOH-terminally myc-tagged full-length Bni1p. Cells were double labeled for Act1p and the myc epitope, or Tpm1p and myc, and the myc-positive cells were evaluated for a visible increase over wild-type controls of Act1p (gray) or Tpm1p (white) in the bud. (c) Examples of actin and tropomyosin localization in cells quantitated in b. Arrows indicate accumulation of cable-like filaments in the bud in rho2Δ, rho4Δ, and rho5Δ cells and in rho3Δ yeast containing plasmid-born RHO3. Myc staining indicates expression of Bni1p. (d) Western blot of Bni1p–myc and actin in wild-type and rho3Δ cells. (e) Actin and myc epitope, or tropomyosin and myc epitope, localization in rho deletion cells overexpressing COOH-terminally myc-tagged Bni1pΔRBD. Arrows indicate accumulation of cable-like filaments in the buds of rho2Δ, rho3Δ, rho4Δ, and rho5Δ cells. (f) Quantitation of cells showing a visible increase of actin (Act1p) and tropomyosin (Tpm1p) in cells overexpressing Bni1pΔRBD–myc.

Cdc42p function is required for actin cable organization but not assembly in unbudded cells only. (a) Wild-type and conditional cdc42-1 mutant cells were subjected to the indicated temperature shifts and prepared for immunofluorescence using Tpm1p antibodies. Unbudded cdc42-1 cells (u) lacking polarized actin cables and budded cdc42-1 cells (b) retaining polarized actin cables at 35°C are shown. (b) Quantitation of polarized cables in wild type (filled diamonds) and conditional mutant cdc42-1 (open circles) after shifting to 35°C. (c) Unbudded, small-budded, medium-budded, and large-budded cells from asynchronous cultures of wild type and six conditional cdc42 mutants were scored for the presence of polarized actin cables before (dark gray) and after (light gray) a 1-h shift to 35°C.

Rho1p function is required for formin activation at 37°C. (a) Localization of actin (Act1p) and tropomyosin (Tpm1p) in rho1-2 and wild-type cells before and after a shift to 37°C. (b) Quantitation of the presence of actin cables in cells stained for actin (gray) or tropomyosin (white). (c) Double label actin and myc epitope, and tropomyosin and myc epitope, localization at 18°C or 37°C in rho1-2 or wild-type cells overexpressing full-length Bni1–myc. Arrows indicate enhanced cable-like filament accumulation in the bud. (d) Quantitation of cells from c, scored for an increase over wild-type controls of cable-like actin (gray) and tropomyosin (white) in the bud.

Rho1p signals through Pkc1p to activate the formins. (a) Localization of actin (Act1p) and tropomyosin (Tpm1p) at 18°C or after a 15-min shift to 37°C in rho1-2 cells expressing constitutively activated Pkc1p* from its own promoter. (b) Montages showing actin and myc, or tropomyosin and myc, localization at 18°C or 37°C in rho1-2 cells coexpressing Pkc1p* and full-length Bni1p–myc. Arrows indicate the accumulation of cable-like filaments in the bud. Myc staining indicates expression level of full-length Bni1p. (c) Quantitation of cells from b, scored for a visible increase over wild-type controls of cable-like actin (gray) and tropomyosin (white) in the bud. (d) pkc1Δ BCK1-20 and control PKC1 BCK1-20 cells were stained for actin and tropomyosin at 18°C and after a 15-min shift to 37°C. (e) Cells from d were quantitated for the presence of visible actin cables based on actin (gray) and tropomyosin (white) stain. (f) Growth of serial dilutions of bnr1Δ, rho3Δ, bnr1Δrho3Δ, and bnr1Δrho3Δ [pkc1*] cells on rich media after 2 d. (g) rho3Δbnr1Δ cells bearing Pkc1p* were prepared for immunofluorescence with Act1p and Tpm1p antibodies.

Schematic representation of Bni1p- and Bnr1p-derived constructs. (a) Full-length Bni1p, showing the RBD, FH1 and FH2, and DAD domains. (b) Truncated Bni1p lacking the RBD (Bni1pΔRBD), which is constitutively active in vivo (Evangelista et al., 2002). (c) Bni1pDADCOOH construct containing the Bni1p DAD and COOH-terminal sequences. (d) Full-length Bnr1p, showing the RBD, FH1 and FH2, and DAD domains. (e) Truncated Bnr1p lacking the RBD (Bnr1pΔRBD).

The essential function of Rho3p and Rho4p is in formin activation. (a) Growth of serial dilutions of strains bnr1Δ, rho3Δ, rho3Δbnr1Δ, and rho3Δbnr1Δ [Bni1ΔRBD] at 26°C for 2 d. (b) Localization of actin (Act1p) and tropomyosin (Tpm1p) in control rho3Δbnr1Δ cells and cells expressing Bni1pΔRBD. Actin bar in rho3Δ bnr1Δ cells is indicated. (c) Bni1pΔRBD, Bnr1pΔRBD, and Bni1pDADCOOH can rescue the lethality of rho3Δrho4Δ cells. rho3Δrho4Δ [pRS316-RHO3, pRS315], rho3Δrho4Δ [pRS316-RHO3, pRS315-Bni1ΔRBD], rho3Δrho4Δ [pRS316-RHO3, pRS315-Bnr1ΔRBD], and rho3Δrho4Δ [pRS316-RHO3, pRS315-Bni1DADCOOH] cells were grown on 5-FOA–containing medium to select for loss of the pRS316-RHO3 plasmid. (d) Actin cables, visualized by staining for actin (Act1p) and tropomyosin (Tpm1p), are present in bni1Δ, rho4Δ, bni1Δrho4Δ, bni1Δrho3Δ, and rho3Δrho4Δ [Bni1DADCOOH] cells.

Overexpression of Cdc42p can rescue the synthetic lethality of rho3Δrho4Δ cells. (a) Growth of rho3Δrho4Δ [pRS316-RHO3, pRS425] and rho3Δrho4Δ [pRS316-RHO3, pRS425-CDC42] cells on 5-FOA–containing medium to select for loss of the pRS316-RHO3 plasmid. (b) Normal actin cables are present in rho3Δrho4Δ [pRS425-CDC42] cells.

Bni1pΔRBD induces actin cable assembly in rho1-2 cells at 37°C. (a) rho1-2 or rho1-2 [Bni1ΔRBD] cells grown at 18°C or after shifting to 37°C for 15 min were stained for actin or tropomyosin. (b) The percentage of small- and medium-budded cells with visible actin cables was determined.