Supplemental Figures

This article contains the following supporting material:

• Supplemental Figure 1.pdf - Effects of cytochalasin A treatment and the sepA1 mutation on TpmA-GFP localization at hyphal tips. (A-C) tpmA::gfp (ACP115) hyphae were grown in YGV at 28°C for 12 h. Hyphae were then exposed to; (A) DMSO (as a solvent control), or (B) 2 μg/ml cytochalasin A. After 20 min, hyphae were imaged by epifluorescence microscopy. Note that cytochalasin A treatment causes depolarization of the hyphal tip. An additional sample was also returned to drug-free media for 30 min to monitor recovery of TpmA-GFP localization at hyphal tips (C). (D) sepA1 tpmA::gfp (ASH630tpmA) hyphae were grown in YGV at 28°C for 12 h, then shifted to 42°C for 60 min. At this point, most hyphae display the dichotomous branching phenotype that is characteristic of sepA mutations. Hyphae were imaged by epifluorescence microscopy. Arrows, TpmA-GFP localization at hyphal tips. Bar, 3 μm.
  
  
• Supplemental Figure 2.pdf - Patterns of filipin staining. (A,B) Hyphae were grown in YGV at 28°C for 12 h. Hyphae were stained with a reduced amount of filipin (12.5 μg/ml) for 5 min without fixation (A) or after 20 min fixation in 3.7% formaldehyde (B). A concentrated patch of filipin staining is observed at the hyphal tip in both samples, though the background is considerably higher in the fixed sample. (C) ASH630 (sepA1) hyphae were incubated in YGV at 42°C for 12h and stained with filipin (25 μg/ml). Staining is observed at the hyphal tip. (D-H) ASH630 (sepA1) and ACP65 (sepA1 mesA1) spores were germinated in YGV at 42°C for 8 h and stained with filipin (25 μg/ml). A patch of filipin staining is observed at the polarization site of sepA1 spores (D,E). By contrast, no patch is observed in mesA1 sepA1 double mutants (F-H). Bar, 3 μm.
  

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