Figure 12

Amino acid starvation and re-stimulation does not affect the direct interaction between EGFP-mTOR and DsRed-Rheb in perinuclear regions. (A-C) LEFT PANELS: Confocal 488 nm excitation images of EGFP-mTOR (co-expressed with DsRed-Rheb, after 48 h of transfection) and 543 nm excitation images of DsRed-Rheb, 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 and appear not to contain Rheb. A-C) MIDDLE PANELS, Lifetime images of the co-transfected cells (colour coding for the lifetimes is shown in the RIGHT PANELS). (A-C) RIGHT PANELS The lifetime distributions taken for the cell area (within red line) as shown in the MIDDLE PANELS. A, revealing both quenched and unquenched EGFP reflecting the mixture of perinuclear mTOR/Rheb and punctate mTOR), as reflected in the lifetime image; B) the same cells after 10 min of amino acid re-stimulation; C) after 60 minutes of amino acid re-stimulation when the punctate structures had disappeared. At all time-points the area marked by the white circle, mainly ER/Golgi, shows a quenched lifetime i.e. Rheb interacting with mTOR. Bar 8 μm.