Cdc42p regulation of the yeast formin Bni1p mediated by the effector Gic2p

Abstract

Actin filaments are dynamically reorganized to accommodate ever-changing cellular needs for intracellular transport, morphogenesis, and migration. Formins, a major family of actin nucleators, are believed to function as direct effectors of Rho GTPases, such as the polarity regulator Cdc42p. However, the presence of extensive redundancy has made it difficult to assess the in vivo significance of the low-affinity Rho GTPase-formin interaction and specifically whether Cdc42p polarizes the actin cytoskeleton via direct formin binding. Here we exploit a synthetically rewired budding yeast strain to eliminate the redundancy, making regulation of the formin Bni1p by Cdc42p essential for viability. Surprisingly, we find that direct Cdc42p-Bni1p interaction is dispensable for Bni1p regulation. Alternative paths linking Cdc42p and Bni1p via "polarisome" components Spa2p and Bud6p are also collectively dispensable. We identify a novel regulatory input to Bni1p acting through the Cdc42p effector, Gic2p. This pathway is sufficient to localize Bni1p to the sites of Cdc42p action and promotes a polarized actin organization in both rewired and wild-type contexts. We suggest that an indirect mechanism linking Rho GTPases and formins via Rho effectors may provide finer spatiotemporal control for the formin-nucleated actin cytoskeleton.

FIGURE 1:

Timing of protein polarization in wild-type and rewired cells. Wild-type (A–D) (DLY11909, 13890, 13344, 14014) and rewired (F–I) (DLY13072, 13884, 13945, 14026) cells were imaged and scored for the first appearance (arrows) of the indicated proteins at the incipient bud site. Inverted images are shown. Time intervals between polarization of GFP-tagged proteins and the septin Cdc3p-mCherry were quantified in mother and daughter cells (right; D, daughters; M, mothers). (E, J) Summary of the mean ± SEM timing in mother cells. Scale bar, 5 μm.

FIGURE 2:

BNI1 is essential in rewired cells. (A) Synthetic lethality of bni1 BEM1-SNC2. The spore viability of cells with the indicated genotypes was deduced from tetrad dissection of heterozygous diploids (DLY12648 and 12778). (B–E) Rewired cells carrying a temperature-sensitive Bni1p (DLY12226, compared with the control DLY8601) progress through the cell cycle but are defective in polarization and budding. G1-phase cells were enriched by centrifugal elutriation at 4°C and released into 37°C media to resume growth. Samples were fixed and scored for percentage budded (n > 100) (B) and percentage cells with S or G2 DNA content (n = 10,000) (C). (D) DIC images of cells released into 37°C media for 160 min. Most cells did not bud, and those that did formed very wide necks and did not complete cytokinesis. (E) Inverted time-lapse images of G1-phase cells, which were released and filmed at 37°C. The control BEM1-GFP-SNC2 cell (top) started to polarize and bud, whereas a similar-sized BEM1-GFP-SNC2 bni1-12 cell (bottom) grew big and round without establishing polarity. Scale bar, 5 μm.

FIGURE 3:

Bni1p regulation is essential in rewired cells. (A) A catalytic fragment of Bni1p (FH1–FH2) can provide formin function in wild-type but not rewired cells. Full-length Bni1p or the FH1–FH2 fragment was tested for the ability to rescue temperature-sensitive formin function in rewired (bni1-12 BEM1-SNC2; DLY12226, 14024, 12620) and otherwise wild-type (bni1-116 bnr1; DLY7924, 14025, 12627) strains at 37°C. Serial dilutions of cell cultures (containing ∼10⁴, 10³, 10², and 10¹ cells) were spotted on plates with rich media and incubated at the indicated temperature for 2.5 d. (B) FH1–FH2 does not rescue the synthetic lethality of bni1 BEM1-SNC2. Spore viability of cells with the indicated genotypes was deduced from tetrad dissection of heterozygous diploids (DLY12848, 14272). (C) FH1–FH2 is not dominantly deleterious in rewired cells. DIC images of exponentially growing rewired cells expressing (right; DLY14811) or not expressing (left; DLY13095) FH1–FH2 in addition to full-length Bni1p. Images were assembled from multiple fields (denoted with black boxes). Scale bar, 5 μm. (D) Immunoblotting shows expression of the full-length Bni1p (DLY14024) and the FH1-FH2 fragment (DLY12620, 12848, 14272) in the strains used in A and B. Cdc11p (septin) serves as loading control.

FIGURE 4:

The GTPase-binding domain is not necessary for Bni1p regulation. (A) The GBD is not required for Bni1p regulation in rewired cells. DIC images of rewired cells expressing full-length (left; DLY13095) Bni1p or a version lacking the GBD (right; DLY14223). Scale bar, 5 μm. (B) Left, schematic of Bni1p indicating GBD, SBD, and BBD domains that interact with GTP-Cdc42p, Spa2p, and Bud6p, respectively. CC, coiled coil; DAD, Diaphanous autoregulatory domain; DD, dimerization domain; DID, Diaphanous inhibitory domain; FH1, formin homology 1; FH2, formin homology 2. Right, spore viability of cells with the indicated genotypes was deduced from tetrad dissection of heterozygous diploids (DLY14194, 14195, 12959, 12960).

FIGURE 5:

Localization of Bni1p to polarity sites does not require known regulatory domains. (A) The catalytic fragment (FH1–FH2; DLY13426) localizes diffusely in the nucleus and cytoplasm, but addition of N-terminal Bni1p sequences suffices to target the fragment to the bud tip and mother–bud neck, whether or not the GBD and SBD regions are included (DLY13916, 13433, 13431). Representative images of the indicated GFP-tagged constructs are shown. (B) To assess actin-independent localization, cells were treated with 200 μM Lat A at 24°C for 2.5 h (DLY13916, 13433, 13431, 13426). Bem1p-Tomato marks the polarization sites, and the indicated Bni1p constructs (except for the catalytic fragment alone) colocalize with Bem1p. Additional spots of GFP-Bni1p staining remain at old mother–bud necks, presumably reflecting problems with cytokinesis in the absence of F-actin. Scale bar, 5 μm.

FIGURE 6:

The Cdc42p effector Gic2p may regulate Bni1p. (A, B) Gic2p binds to a region of Bni1p distinct from the GBD, SBD, or BBD. (A) Gic2p-Myc was coimmunoprecipitated with overexpressed GFP-Bni1p full-length (FL; DLY14515) and Bni1p^3Δ (DLY14516) but not with FH1–FH2 (DLY14517). The stronger binding to full-length Bni1p is consistent with previous findings that Gic2p can also bind to Bud6p/Spa2p. Negative controls: DLY 14513, 13857. (B) The uncharacterized N-terminal Bni1p regions upstream of the GBD (ND1), between the GBD and SBD (ND2), and between the SBD and FH1 (ND3) were fused to the catalytic fragment and expressed as GFP-tagged proteins from the GAL1 promoter (DLY14885, 14886, 14887). The ND2 construct (but not the others) coimmunoprecipitated Gic2p-Myc. (C) Gic1p and Gic2p become critical in rewired cells and when the Bni1p GBD, SBD, and BBD are deleted. The gic1Δ gic2Δ growth defect (DLY14169) at 37°C is greatly exacerbated when combined with BEM1-GFP-SNC2 (DLY14170) or with BNI1^3Δ (DLY14601). Serial dilutions of the indicated strains were spotted on yeast extract/peptone/dextrose media and grown for 2.5 d at the indicated temperature. (D, E) DIC images of exponentially growing cells of indicated genotypes at 24°C, showing strong synthetic polarity defects even at permissive temperature. Scale bar, 5 μm.

FIGURE 7:

The Gic2p N-terminus can replace the Bni1p regulatory regions. (A) The Gic2p N-terminal Cdc42p-binding domain is sufficient for Bni1p regulation in rewired cells (DLY12621, 12626). Function of the indicated constructs was tested as in Figure 3A. (B) 3HA-tagged Bni1p and the GIC2^N-FH1-FH2 construct are expressed at comparable levels. Blot probed with anti-HA and anti-Cdc11p (loading control). (C) GIC2^N-FH1FH2 can function as the sole formin in rewired cells. Spore viability of cells with the indicated genotypes was deduced from tetrad dissection of heterozygous diploids (DLY 12848, 12594, 12744). (D) DIC images of rewired cells expressing full-length Bni1p (left; DLY12888) or Gic2^N-FH1-FH2 (middle, right; DLY12892, 12887) in place of endogenous Bni1p (left, middle) or both Bni1p and Bnr1p (right). (E) GFP-Gic2^N-FH1-FH2 (DLY13430) localizes to the bud tip (top) and to the polarization site (marked with Bem1p-tdTomato) in the absence of F-actin (bottom). (F) F-actin distribution in cells containing the indicated construct as the sole formin (DLY13703, 13446, 13444, 13452). (G) GFP-Sec4p (vesicle marker) localization in the same strains as in F. (H) GFP-Sec4p localization categories (cartoon) were scored in small- and medium-budded cells of the strains in F and G. Scale bar, 5 μm.

FIGURE 8:

Schematic of the Bni1p regulatory network. Cdc42p interacts directly with Bni1p (1), but this interaction is not necessary for Bni1p regulation. Two other “polarisome” proteins (Spa2p and Bud6p) are localized to sites of Cdc42p action (2) and interact with Bni1p (3), but even in combination these pathways (1–3) are dispensable for Bni1p regulation. We identified a novel path (4) from Cdc42p to Bni1p via the Cdc42p effector Gic2p, which was previously found to interact with Spa2p and Bud6p (5). Genetic interactions suggest that paths 1, 2–3, and 4 act in parallel, and biochemical findings suggest that paths 4 and 5 operate in parallel. Solid lines indicate interactions shown by in vitro biochemical assays. Dashed lines indicate interactions identified with immunoprecipitation and/or cofractionation. Dotted lines with question marks indicate interactions suggested by colocalization.