Amino acid starvation and re-stimulation effect on the direct interaction between EGFP-mTOR and DsRed-raptor. (A-C) LEFT PANELS: Confocal 488 nm excitation images of EGFP-mTOR (co-expressed with DsRed-raptor, after 48 h of transfection) and 543 nm excitation images of DsRed-raptor, for HEK293 cells serum-starved overnight and then amino acid starved in D-PBS for 1 h (A) following which the amino acids were added back (B, C). Punctate structures appear after amino acid removal and disappear after adding back the amino acid. (A-C) MIDDLE PANELS, Lifetime images of the co-transfected cells. The colour coding for the lifetime is shown in the right panel which is a lifetime distribution of the area of EGFP-mTOR and DsRed-raptor co-expressing cells (area marked by red line in the middle panel) (A-C) RIGHT PANELS The lifetime distributions taken for the cell area (within red line) as shown in the MIDDLE PANELS. A, revealing quenched EGFP reflecting a direct interaction of mTOR/raptor both in the punctate structures and other regions. Note that the lifetime of EGFP mTOR is slightly reduced, to about 2350 ps, possibly indicating that in the punctate structures there may still be some mTOR-raptor interaction but 'looser' under amino acid starvation conditions (compare Figure 11). (A cells with only EGFP-mTOR expression is shown for comparison, white circle). After amino acid re-stimulation (B, C) the punctate structures disappear and the quenched lifetime for the EGFP returns to <2300 PS.
Yadav et al. BMC Cell Biology 2013 14:3 doi:10.1186/1471-2121-14-3