Figure 2.

Macropinocytosis is stimulated by expressing activated-Ras in wild-type cells. (a) Expression of RasG or RasS or their activated versions (as GFP fusions) fails to stimulate macropinocytosis in Ax2 cells, where Ras is already somewhat activated due to deletion of the RasGAP NF1. (b,c) The GFP-Ras proteins are targeted to the plasma membrane of wild-type DdB cells as expected. (d) Expression of Ras and especially activated-Ras stimulates macropinocytosis in wild-type DdB cells, which have intact NF1. (e,f) Though the activated GFP-Ras proteins are recruited fairly uniformly to the plasma membrane of Ax2 cells, PIP3 and active-Rac accumulate locally at macropinocytic cups (asterisks). Ras was expressed using pPI432 (GFP-RasG), pPI332 (GFP-RasG G12T), pPI434 (GFP-RasS) or pPI445 (GFP-RasS G12V). Active Rac was visualised using PakB-CRIB-mCherry (pTW050) and PIP3 using PH-PkgE-mCherry (pPI363). DdB cells grown on bacteria were adapted in HL5+10% FCS over-night before fluid uptake assay or imaging.