Figure 7.
RacC acts downstream of GxcM to regulate cortical actin assembly. (A) Yeast two-hybrid assay showing the interaction between Fbp17 and the GBD domain of WASP with the constitutively active (CA) forms of the indicated 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) Co-IP of GFP, GFP-RacC, or GFP-Rac1A with GxcM-RFP in the presence or absence of EDTA. IP was performed with RFP-trap and samples were probed with GFP or RFP antibody. (C and D) Time-lapse imaging of racC– cells migrating under agarose along a folic acid gradient. (C)racC− cells expressing GxcM-GFP and LimEΔcoil-RFP. (D)racC− cells expressing GxcM-GFP and RFP-ArpC4. Angle-series plots on the right show fluorescent intensity distribution of the indicated proteins along the perimeter of the cell, with 0° and +180°/−180° corresponding to the migrating front and rear, respectively. Solid lines represent the mean and shades represent mean ± SD. n, number of cells analyzed. Scale bars, 5 μm. Source data are available for this figure: SourceData F7.

RacC acts downstream of GxcM to regulate cortical actin assembly. (A) Yeast two-hybrid assay showing the interaction between Fbp17 and the GBD domain of WASP with the constitutively active (CA) forms of the indicated 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) Co-IP of GFP, GFP-RacC, or GFP-Rac1A with GxcM-RFP in the presence or absence of EDTA. IP was performed with RFP-trap and samples were probed with GFP or RFP antibody. (C and D) Time-lapse imaging of racC– cells migrating under agarose along a folic acid gradient. (C)racC− cells expressing GxcM-GFP and LimEΔcoil-RFP. (D)racC− cells expressing GxcM-GFP and RFP-ArpC4. Angle-series plots on the right show fluorescent intensity distribution of the indicated proteins along the perimeter of the cell, with 0° and +180°/−180° corresponding to the migrating front and rear, respectively. Solid lines represent the mean and shades represent mean ± SD. n, number of cells analyzed. Scale bars, 5 μm. Source data are available for this figure: SourceData F7.

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