bnr1Δ and bni1-FH2#1 bnr1Δ cells were grown 7 days in YPDA medium at 25°C. Cells were incubated with Con-A-FITC for 1 h, then washed with “old” YPDA and shifted to 37°C for 30 min as described in material and methods. Cells were then re-fed with pre-warmed YPDA medium and grown at 37°C. (A) Percentage of budded cells in bnr1Δ and bni1-FH2#1 bnr1Δ cultures before and after exit from quiescence at 37°C, N>200 for each time point, 2 experiments – error bars show SD. (B) Actin cytoskeleton organization in bnr1Δ and bni1-FH2#1 bnr1Δ cells before and after exit from quiescence at 37°C. Red: Actin Bodies; green: depolarized actin patches and cables; blue: polarized actin patches and cables; yellow: no detectable actin cable and depolarized actin patches (N>200 for each time point, 2 experiments – error bars show SD). (C) Images of typical bnr1Δ and bni1-FH2#1 bnr1Δ cells. Arrows indicate bud scars, arrowheads indicate birth scars; CW: Calcofluor White. Bar 2 µm.

Wild type cells were grown 7 days in YPDA medium at 30°C. Cells were incubated with Con-A-FITC for 1 h and then washed with “old” YPDA. Lat-A (200 µM final concentration) or DMSO were added and cells were further incubated at 30°C for 30 min. Cells were then re-fed with YPDA medium (with or without 1 M sorbitol) containing either 200 µM Lat-A or DMSO and grown at 30°C as described in material and methods. (A) Percentage of new budded cells after exit from quiescence at 30°C, N>200 for each time point, 2 experiments – error bars show SD. (B) Actin cytoskeleton organization in unbudded cells before exit from quiescence or in new budded cells after exit from quiescence at 30°C in YPDA medium containing either DMSO, DMSO and 1 M sorbitol, 200 µM Lat-A or 200 µM Lat-A and 1 M sorbitol. Red: Actin Bodies; green: depolarized actin cytoskeleton; blue: polarized actin cytoskeleton; grey: no detectable F-actin containing structures (N>200 for each time point, 2 experiments – error bars show SD). (C) Images of typical treated and untreated cells. The upper left panel display a typical wild type cell pre-incubated 30 min with Lat-A before re-feeding and the lower right panel, examples of Lat-A treated wild type cells 6 h after re-feeding with a collapsed new bud. Arrows indicate bud scar, arrowhead indicate birth scars; CW: Calcofluor White. Bar 2 µm. Of note, for AlexaFluor Phalloidin images, the maximum intensity is about 20 times lower for Lat-A treated cells than for untreated cells and was therefore greatly enhanced in the figure to document the absence of F-actin structure.

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ARP2 and arp2-1 cells were grown 7 days in YPDA medium at 25°C. Cells were incubated with Con-A-FITC for 1 h, then washed with “old” YPDA and shifted to 37°C for 1 h as described in material and methods. Cells were then re-fed with pre-warmed YPDA medium and grown at 37°C. (A) Percentage of budded cells in ARP2 and arp2-1 cultures before and after exit from quiescence at 37°C, N>200 for each time point, 2 experiments – error bars show SD. (B) Actin cytoskeleton organization in ARP2 and arp2-1 cells before and after exit from quiescence at 37°C. Red: Actin Bodies; green: depolarized actin patches and cables; blue: polarized actin patches and cables; purple: abnormal actin cables and depolarized actin patches (N>200 for each time point, 2 experiments – error bars show SD). (C) Image of typical ARP2 and arp2-1 cells. Arrows indicate abnormally big actin cable-like structures. Bar 2 µm.

(A) sec4-8, sec6-4 and isogenic wild type cells were grown 7 days in YPDA medium at 25°C. Cells were incubated with Con-A-FITC then washed with “old” YPDA and cells were shift to 37°C for 1 h. Cells were then re-fed with pre-warmed YPDA medium and grown either at 25°C or at 37°C as described in material and methods. The percentage of budded cells in wild type (blue), sec4-8 (purple) and sec6-4 (yellow) is indicated before and after exit from quiescence at 25°C or 37°C, N>200 for each time point, 2 experiments – error bars show SD. (B) Image of typical WT, sec6-4 and sec4-8 cells, 4 h after exiting quiescence at 25°C (left) and 37°C (right). (C) Localization of Sec8p-GFP before and after exit from quiescence at 30°C in the absence or in the presence of 200 µM Lat-A. Arrowheads point at Sec8p-GFP dots at the cell periphery. Bar 2 µm.