FIGURE 9.

Priming amino acids and Leu are required to localize mTOR to lysosomes. Starved HeLa cells were preincubated in the absence (−) or in the presence (+) of 5 mm Asn or Ser for 30 min followed by incubation for 20 min in the absence or in the presence of 5 mm Leu. Alternatively, the cells were supplemented for 30 min with complete amino acids at concentrations present in growth media (AAs). A, representative images of cells treated in the absence of amino acids or in the presence of Asn, Leu, or Asn followed by Leu or AAs. The cells were fixed, stained for LAMP2 and mTOR, and imaged as described under “Experimental Procedures.” The images are maximum intensity projections of 16 z slices. Shown are LAMP2 (red), mTOR (green), merge (LAMP2 and mTOR), and the co-localization (Coloc) channel showing voxels where LAMP2 and mTOR co-localization was indicated by Mander's coefficient (MC). Not shown are images of cells treated with Ser (similar to cells treated with Asn) and Ser followed by Leu (similar to Asn followed by Leu). B, quantification of mTOR and LAMP2 co-localization using thresholded Mander's coefficients, i.e. the number of voxels with above-threshold fluorescence in both channels (mTOR and LAMP2) divided by the number of voxels with above-threshold fluorescence for mTOR. The data shown are averages of two independent experiments (24–30 cells each) ± S.D.