Figure 6.

FM4–64 staining of endocytic membranes in wild-type and arf mutant cells. (A–D) Wild-type (SEY6210) and arf1Δ (6210 arf1Δ) cells were incubated in YPD containing 30 μM FM4–64 on ice for 45 min and then warmed to 25°C to initiate endocytosis as described previously (Vida and Emr, 1995). Cells were collected and applied to concanavalin A-treated slides 10 min after the temperature shift, and the images were acquired ∼10 min after cells were applied to the slides. (E–H) The arf1–3 ts mutant (C156–1B) was grown at 23°C and then half of the culture was shifted to 37°C and incubated for 1 h. Cells were harvested and resuspended at ∼2 × 10⁸ cells/ml in YPD media prewarmed to 23°C or 37°C and incubated for 10 min. FM4–64 was added to 30 μM and incubated for 7 min, then the cells were harvested and resuspended in fresh prewarmed media and the incubation was continued. Images of the cells were acquired ∼15–20 min after addition of fresh media.