Characterization of RacC. (A) Yeast two-hybrid assay showing interactions between Fbp17 and the GBD domain of WASP with the CA forms of 19 Rac proteins. Yeast was transformed with the indicated constructs and selected for the presence of prey and bait plasmids by growth on double-dropout (DD) agar plate lacking leucine and tryptophan. Interactions were assayed by growth on quadruple-dropout (QD) agar plate additionally lacking histidine and adenine. AD, Gal4-activation domain; BD, Gal4-binding domain. (B) Top: Design of the racC knockout construct. Bottom: Targeted clones were confirmed by PCR. (C) The indicated cells were plated clonally with bacteria (K. aerogenes) on standard medium agar for 5 d. Scale bar, 5 mm. (D–F) Time-lapse imaging of randomly migrating WT cells. (D) Cells expressing GFP-RacCG15V and LimEΔcoil-RFP. (E) Cells expressing GFP-RacCQ64L and LimEΔcoil-RFP. (F) Cells expressing GFP-RacCQ64L and RFP-ArpC4. Scale bars, 5 μm. (G) Left: Localization of GFP-myosin II in WT and racC− cells during random migration. Right: Quantification of the relative cortical localization of GFP-myosin II. Data were from three independent experiments; the scatter plot shows data points with mean ± SEM; n, number of cells analyzed. Scale bars, 5 μm.
