Fig. 7

Cell protrusions are sites of high R-RAS2 activity and Akt phosphorylation. a, Confocal microscopy sections at the plane of contact with the coverslips of CBM-MBC21 control and knockdown cells plated on coverlips coated with poly-L-lysine plus hyaluronic acid (HA). After incubation, cells were fixed and stained with anti-phospho-Akt (S473) and phalloidin to show their co-localization at sites rich in F-actin. The nucleus of the cells is stained with DAPI (blue). A detail of inset a is shown to the right to illustrate strong co-localization at cell protrusions. Co-localization of F-actin and phospho-Akt was measured by analysis of all pixels in 10–19 cell protrusions per condition and calculating the Pearson’s correlation coefficient. The violin plot shows all data points, the median and the 75% and 25% percentiles. Statistical significance was assessed by carrying out Mann-Whitney tests. b, Confocal microscopy sections at the plane of contact with the coverslips of CBM-MBC21 control and knockdown cells plated on coverlips coated with poly-lysine plus hyaluronic acid (HA) or poly-L-lysine alone. After incubation, cells were fixed and stained with anti-phospho-Akt (S473) and with anti-Hag (for R-RRAS2) to show their co-localization at cell protrusions. The nucleus of the cells is stained with DAPI (blue). A detail of inset b is shown to the right to illustrate strong co-localization at cell protrusions. Co-localization of R-RAS2 and phospho-Akt was measured by analysis of all pixels in 7–16 cell protrusions per condition and calculating the Pearson’s correlation coefficient. The violin plot shows all data points, the median and the 75% and 25% percentiles. Statistical significance was assessed by carrying out Mann-Whitney tests. c, Schematic of the R-RAS2 biosensor designed to measure its active form (GTP-bound) in living cells. Human R-RAS2 was appended a FRET donor (cerulean) and a FRET acceptor (venus) to its N-terminal end. Cerulean is preceded at its N-terminus by the Ras-binding domain (RBD) of the catalytic subunit of PI3Kδ (p110δ). The exchange of GDP by GTP in R-RAS2 allows the RBD to bind the effector loops of R-RAS2 and brings cerulean into closer proximity to venus, allowing an increase in FRET efficiency. d, Confocal microscopy sections at the plane of contact with the coverslips of CBM-MBC21 control cells plated transfected with the R-RAS2 biosensor and plated on coverslips coated with poly-L-lysine plus hyaluronic acid (HA). The example shows one cell with protruding cell extensions and the fluorescence of the biosensor in the cerulean, venus and FRET channels. The colored micrograph shows the FRET ratio resulting of dividing the fluorescence intensity in the FRET channel by this of the cerulean channel. The colored bar to the right shows the scale of FRET ratios. The FRET ratio was calculated for three types of cell sites: plasma membrane at cell protrusions (1), non-protruding plasma membrane (2) and cytoplasm (3). e, Violin plot showing all data points, the median and the 75% and 25% percentiles of the FRET ratios calculated in 109–150 cell sites of the three types in a total of seven cells. Statistical significance was assessed using a one-way ANOVA Tukey’s multiple comparison test