Mapping of activated Ras relative to filamentous actin during the uptake of budded yeast. Cells were double-labeled with GFP fusions of Ras-binding domains (RBDs) and mRFP-LimEΔ. The RBDs of (a-c) human Raf-1 and (d, e) Dictyostelium NdrC kinase were used. Top panels show brightfield images of phagocytes and yeast (marked by dots or circles), middle panels fluorescence images of filamentous actin labeled with mRFP-LimEΔ, and bottom panels the simultaneously recorded localization of RBDs. (a) A stage of partial uptake showing actin accumulation at the neck of the bud, which is not paralleled by an enrichment of activated Ras (arrowheads). (b) Two stages of complete uptake, showing strong increase in the actin label at the bud, whereas the RBD label at this site remains low. (c) Sequence showing severing of a particle. At the time the outer part of the particle is cleaved off (26 seconds), the site of separation is not discriminated by its Ras activity. (d) Three stages of a complete uptake. The actin is increasing around the neck of the particle when the RBD label is already fading out. (e) Uptake of a particle that is finally released. Within the sequence comprising 1 minute, actin strongly accumulates at the bud neck, whereas RBD shows strongest binding at 13 seconds around the large mother cell. Numbers in (b-e) indicate seconds after the first frame. Bars = 10 μm; the bar in (e) applies also to (b-d).