Fig. 4.

Arf6 GEFs facilitate Salmonella invasion via Arf1-ARNO-WAVE. (A) Influence of Arf6 GEF depletion on Salmonella ruffle formation. HeLa cells were transfected with scrambled, EFA6, or BRAG2 siRNA, or with EFA6 or BRAG2 siRNA together, 72 h before infection with WT Salmonella (10 min). Error bars represent ± SEM. (B) Influence of Arf6 GEF depletion on ARNO-driven Salmonella invasion. HeLa cells were transfected with siRNA as in A 72 h before treatment with SecinH3 and infection with Salmonella (10 min) encoding pM975 that expresses GFP inside pathogen-containing vacuoles. Error bars represent ± SEM. (C) Localization of RFP-tagged Arf1 in HeLa cells coexpressing control CFP alone, CFP-tagged WT, or CI EFA6. HeLa cells were stained with Alexa Fluor 594–phalloidin to visualize actin. (Insets) Magnified areas. Arrows indicate intracellular vacuoles. (Scale bar: 8 µm.) (D) Actin assembly on the membrane driven by Arf6 GEFs in the presence of ARNO. Proteins recruited to EFA6 and BRAG2-associated membrane platforms (depicted in cartoons) from extract with (+) or without (−) purified ARNO analyzed by immunoblotting with indicated antibodies (Right). Anti-GST was used to detect GST-tagged EFA6 and BRAG2. (E) Actin assembly by Arf6 GEFs in the presence of CI ARNO (E156D) and the ARNO mutant defective for Arf6-binding (K336A). Proteins recruited to EFA6 and BRAG2-associated membrane platforms (depicted in cartoons) from extract containing indicated ARNO derivatives, analyzed by immunoblotting as in D. (F) Actin assembly via CI EFA6. Proteins recruited to WT and CI EFA6 from extract containing purified WT ARNO (+), analyzed by immunoblotting as in D.