Figure 1 - Cdc15p affects the organization of a sterol-rich membrane domain during cytokinesis.

(a) Mutant S. pombe cells affected in different aspects of cytokinesis were stained with 5 g ml^-1 of filipin, a sterol dye, and imaged live within 5 min of filipin addition. For temperature-sensitive mutants, exponential-phase cells were grown in YE5S medium at 25 °C and shifted to the restrictive temperature, 35.5 °C, for 3.5 h. In Lat-A cells, 100 M Latrunculin-A was added to cells for 10 min. (b) 3D-reconstructed images of filipin staining in a wild-type cell and a cdc15-287 cell. Two images with different viewing angles are shown for each cell. (c) Filipin staining in cdc15-null cells (cdc15::ura4⁺ germinated spore; see Supplementary Information, Methods). (d) Localization of acylated-GFP in wild-type and cdc15-287 cells. Cells carrying REP3X–Ac–GFP were grown in the absence of thiamine at 25 °C for 14 h and then shifted to 35.5 °C for 3.5 h. Arrowheads indicate the spiral pattern of Ac–GFP. (e) Filipin staining of cdc8-382 cdc15-287 and cdc12-299 cdc15-287 double-mutant cells incubated at 35.5 °C for 3.5 h. (f) Induction of membrane domains in G2-phase-arrested cells by cdc15⁺ overexpression. cdc25-22 cells with REP1–cdc15 were grown in EMM plus thiamine media, washed, and resuspended in EMM plus thiamine (uninduced) or EMM minus thiamine (induced) media, grown for 15 h at 25 °C, shifted to 36 °C for 3 h, and then stained with filipin before imaging. The arrest of cells in interphase was confirmed by DAPI staining (data not shown). Arrowheads show abnormal membrane domains on the sides of cells. (g) Actin-independent induction of medial membrane domains by cdc15+ overexpression in G2-phase-arrested cells. Cells were prepared as in f, but 100 M Lat A or control DMSO (0.5% final concentration) was added to cells 2 h after raising the temperature to 36 °C, before the formation of abnormal membrane domains. Scale bars represent 10 m.