Constitutive targeting of mTORC1 to FAs uncouples it from regulation by lysosomal positioning. (A) HeLa cells expressing FLAG-RPTOR or FLAG-RPTOR^FAT grown in full-nutrient medium were immunostained for FLAG and the FA protein paxillin. Representative images and fluorescence intensity line profile plots corresponding to lines exemplified by arrows are shown. (B) Scrambled (Scr) or ARL8B siRNA–transfected HeLa cells expressing FLAG-RPTOR or FLAG-RPTOR^FAT grown in full-nutrient medium were FCS starved for 18 h (−FCS) or FCS starved and then recovered in FCS-containing medium for 10 min (10 min FCS) and immunostained for p-S6 and FLAG. (C) The IntDens of peripheral or intracellular signal of p-S6 was quantified. (D) Cells treated as in B were subjected to immunoblot analysis using antibodies as shown. Error bars represent SEM; n = 3 independent experiments (for IntDens, n ≥ 10 cells were quantified per experiment). **, P < 0.01; two-sided Student’s t test. Scale bars, 20 µm (insets, 10 µm). Nuclei were visualized with DAPI. (E) Diagram illustrating the proposed role of FAs in the activation of mTORC1 in response to growth factor–promoting stimuli. aa trans., amino acid transporter; GFR, growth factor receptor. See Discussion for further details.