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Fig. 1. Vps1p is required for normal actin cytoskeleton organization. (A) Actin cytoskeleton in several yeast mutants at different temperatures. Strains (YMC451) vps1
, (YMC452) dnm1, (YMC453) mgm1, (YMC454) vps26 and wild-type W303-1-B (W303) cultured to mid-log phase in YEPD at 24°C were shifted to 30°C or 37°C for 3 hours before being subjected to actin staining (bar, 5 µm). (B) Quantitative illustration of the populations of vps1 and W303 with actin abnormalities. Actin depolarization shown in the left panel was calculated only in budded cells with small- and medium-sized buds, and the actin aggregation shown in the right panel was based on the total cell population. (C) The abnormal budding pattern and chitin deposition in vps1 cells. Strains YMC456 (vps1, diploid) and the wild type YMC455 (W303, diploid) were grown in YEPD to log phase at 24°C. Aliquots of the cultures were then shifted to either 30°C or 37°C for 3 hours before being subjected to Calcofluor staining. The budding patterns and chitin distributions of cells incubated at different temperatures were scored and shown in Table 4 (bar, 5 µm). (D) Prolonged half-life of Ste3p in vps1 cells. YMC451 (vps1) (
) and the wild-type W303-1-B (W303) (
) cells that contain GAL1-STE3-EGFP were induced to express Ste3p-GFP for 90 minutes in 2% of galactose at 25°C, followed by addition of 3% glucose to repress the expression. The cultures were immediately shifted to 30°C or 37°C. The amount of Ste3p-EGFP at indicated times after glucose addition in W303 and vps1 cells were detected by western blotting (lower panels). The levels of Ste3-EGFP were quantified using a densitometer and plotted in upper panels.
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