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Fig. 9. Sterol lipid distribution was disrupted in OSH mutants. (A) The pattern of filipin fluorescence observed in wild-type cells (Osh⁺; SEY6210) was disrupted in osh OSH1 OSH3 (JRY6306) and osh OSH3 OSH6 (JRY6312) mutant cells. Bright fluorescence at the plasma membrane was observed in 98% of wild-type cells (54 out of 55 cells counted), 98% of osh OSH1 OSH3 cells (54/55), and in 66% of osh OSH3 OSH6 cells (19/34). Bright internal fluorescence was observed in 25% of wild-type cells (11/44), 36% of osh OSH1 OSH3 cells (19/53), and in 76% of osh OSH3 OSH6 cells (26/34). (B) osh P^MET3-OSH2 (JRY6326) cells were cultured in the presence and absence of methionine and treated with filipin. In each series, left panels show cell morphology by DIC, and the right panels show the corresponding filipin fluorescence. Cells grown in the absence of methionine (-Met) displayed normal cell morphology and the pattern of filipin staining was comparable to that seen in wild-type cells. In the presence of methionine (+Met), Osh depleted osh P^MET3-OSH2 cells were larger in size, bud morphology and septation were defective, and the intensity of intracellular filipin fluorescence increased. (C) Filipin fluorescence was examined in wild-type cells (SEY6210), and in the temperature-sensitive mutants osh osh4-1 (CBY926), and osh osh4-2 (CBY928). At 23°C, more intracellular filipin fluorescence was observed in the OSH mutants compared with wild-type cells, and after 60 minutes at 37°C the intensity of intracellular fluorescence increased in mutant cells. In both (B) and (C), images of filipin fluorescence do not represent equal exposures; exposures were longer for wild-type cells.

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