Fig. 4. Sterol localisation to the middle of the cell requires a functional secretory pathway but not an intact F-actin or microtubule cytoskeleton. (A,C) cdc25-22 cells were synchronised and released to the permissive temperature (24°C). Cells were released into medium containing (A) 100 µM LatA or 1% (v/v) DMSO as solvent control, or (C) 100 µM BFA or 1% (v/v) EtOH as solvent control. Samples were taken 60 minutes after release and stained with filipin. (B,D) Sterol localisation detected by filipin staining in (B) nda3-KM311 cells at 18°C and cells overexpressing mad2⁺ under the nmt1-promotor, and (D) cdc7-24 cells at 36°C. Arrows indicate strong medial staining in A and faint medial staining in C and D. Arowheads indicate additional asymmetric patches of sterol-rich membrane.