JCS036269 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Colocalisation of yeast formins and Bud6p. Representative images showing formins fused to GFP (in green) and CFP-Bud6p (in red). Images are 2D projections of five-plane z-stacks. (a-d) Cells expressing CFP-Bud6p and Bni1p-GFP. Both fusions exhibited colocalisation at the bud (a). As the bud grew, Bud6p accumulated at the bud neck (b,c, arrowheads). Bni1p only reached the bud neck shortly before cytokinesis (d). (e-h) Cells expressing CFP-Bud6p and Bnr1p-GFP. Bud6p labelled the incipient bud in the absence of Bnr1p (e, right cell, arrowhead). After bud emergence, Bud6p labelled the bud tip while Bnr1p was present at the bud neck (e, top left cell, f). As the bud grew, cells began to accumulate Bud6p at the bud neck (e, centre cell; g, arrowheads). Bnr1p-GFP did not label cells at cytokinesis (compare g with h). Bar, 2 µm.

• Supplemental Figure S2 -

  Fig. S2. Actin organisation in bni1^CTΔ bud6Δ, bnr1Δ bud6Δ and bnr1Δ bni1^ts cells. (A,B) Actin organisation following a 30-minute shift of a bnr1Δ bni1-FH2#1^ts strain to 34°C. In agreement with Sagot et al. (Sagot et al., 2002), inactivation of Bni1-FH2#1p led to the bulk loss of actin cables and accumulation of cells with depolarised patches. (A) Representative images of fixed wild-type (S288C), bnr1Δ (PY3505) and bnr1Δ bni1-FH2#1 (PY3744) cells (Sagot et al., 2002) stained by rhodamine-phalloidin prior to (upper panel) or following (lower panel) a 30-minute shift from 25°C to 34°C, for the indicated strains. Bar, 2 µm. (B) Quantitation of actin organisation was carried out as in Fig. 2. (C) A bud6Δ bni1^CTΔ exhibited an intermediate phenotype relative to the single mutants (suggestive of a partial bypass due to Bni1^CTΔp, yet pointing to Bud6p contribution via Bnr1p) contrary to the strong synergism shown by bud6Δ and bni1Δ in Fig. 2E). (D) Modest synergism between bnr1Δ and bud6Δ (underscoring Bud6p functions via Bni1p). Accordingly, bud6Δ could not worsen actin organisation in a bnr1Δ bni1^CTΔ double mutant (Fig. 2F). Quantitation of actin organisation for the indicated strains was carried out as in Fig. 2.

• Supplemental Figure S3 -

  Fig. S3. Bni1^CTΔp does not colocalise precisely with Bud6p. Representative images for localisation of Bni1p-GFP or Bni1^CTΔp-GFP (in green) and Bud6p-CFP (in red) are shown. Images are 2D projections of five-plane z-stacks. Cells expressing Bud6p-CFP and Bni1p-GFP exhibited colocalisation at the bud cortex consistent with Buttery et al. (Buttery et al., 2007). (A). Bni1^CTΔp-GFP and Bud6p-CFP exhibited some colocalisation and also individually labelled foci (B). Bar, 2 µm.

• Supplemental Figure S4 -

  Fig. S4. Localisation of full-length Bni1p but not Bni1^CTΔp is dependent on Bud6p. (A) Percentage of cells labelled by either full-length Bni1p-GFP or truncated Bni1^CTΔp-GFP in the indicated strains. (B) Mean fluorescence intensity of Bni1p-GFP or Bni1^CTΔp-GFP in the bud of 20 cells of the indicated strains. For Bni1p-GFP a bud6Δ mutation significantly reduced the intensity of cortical labelling. Moreover, synergism between bud6Δ and spa2Δ was observed. By contrast, only spa2Δ affected Bni1^CTΔp localisation. Formins were expressed at endogenous levels and images obtained without fixation. Under these conditions, the yield of labelled cells was much higher than reported by previous studies (Fujiwara et al., 1998; Sagot et al., 2002), offering a dynamic range to determine the genetic interaction between spa2Δ and bud6Δ. In addition, an effect of bud6Δ on localisation of full-length Bni1p could be observed by contrast with previous studies using overexpression (e.g. Sagot et al., 2002). (C) Representative images of the strains analysed are 2D projections of five-plane z-stacks. Bar, 2 µm.

• Supplemental Figure S5 -

  Fig. S5. No formin is localised to the division site and incipient bud in bni1Δ cells, yet this mutant exhibited Bud6p-dependent microtubule-cortex interactions. (A) Representative images for localisation of Bnr1p-GFP (in green) and CFP-Bud6p (in red) in wild type (a-d) and a bni1Δ mutant (e-f) during the interval from mitotic exit to bud emergence. Images are 2D projections of five-plane z-stacks. In agreement with Buttery et al. (Buttery et al., 2007), Bnr1p-GFP did not decorate the division site and the presumptive bud site in wild-type cells (a,b). Bnr1p-GFP behaved similarly in bni1Δ cells (e,f). Following bud emergence, Bnr1p-GFP began to accumulate at the bud neck in both cases (c,d,g,h). (B) Modes of MT-cortex interaction (as described in Fig. 3C) from mitotic exit until bud emergence to cover the same cell cycle interval illustrated in A.

• Supplemental Figure S6 -

  Fig. S6. Orientation of Kar9p-decorated microtubules in formin mutants. (A) Localisation by cortical compartment of Kar9p in association with MT plus ends was quantitated from digital images of cells containing short spindles. Over 100 MTs were scored. Single bnr1Δ, bni1Δ or bni1^CTΔ exhibited a modest compromise in orientation of MT plus ends decorated by Kar9p. By contrast, Kar9p polarised localisation was severely compromised in bnr1Δ bni1^CTΔ, consistent with the severe compromise in actin cable organisation (see Sagot et al., 2002). (B-F) Representative images for localisation of Kar9p-GFP (in green) and CFP-Tub1p (in red). (B) Wild-type cells typically oriented Kar9p-decorated MTs towards the bud with their plus ends positioned inside the bud or near the bud neck. (C-F) Images illustrating Kar9p position in formin mutants. Bar, 2 µm.