mTOR direct interactions with Rheb-GTPase and raptor: sub-cellular localization using fluorescence lifetime imaging

• Published in: BMC cell biology Vol. 14; no. 1; p. 3
• Main Authors: Yadav, Rahul B, Burgos, Pierre, Parker, Anthony W, Iadevaia, Valentina, Proud, Christopher G, Allen, Rodger A, O'Connell, James P, Jeshtadi, Ananya, Stubbs, Christopher D, Botchway, Stanley W
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 12.01.2013; BioMed Central; BMC
• Subjects: Adaptor Proteins, Signal Transducing - analysis; Adaptor Proteins, Signal Transducing - genetics; Adaptor Proteins, Signal Transducing - metabolism; Animals; Binding proteins; Cell growth; Cell Nucleus - metabolism; CHO Cells; Cricetinae; Cricetulus; Cytoplasm - metabolism; Endoplasmic Reticulum - metabolism; FLIM; Fluorescence microscopy; Fluorescence Resonance Energy Transfer; FRET; Gene expression; GFP; Golgi Apparatus - metabolism; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; HEK293 Cells; HeLa Cells; Humans; Lasers; Life sciences; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Microscopy; Monomeric GTP-Binding Proteins - analysis; Monomeric GTP-Binding Proteins - genetics; Monomeric GTP-Binding Proteins - metabolism; mTOR; Neuropeptides - analysis; Neuropeptides - genetics; Neuropeptides - metabolism; Phosphorylation; Protein Binding - drug effects; Protein-protein interactions; Proteins; Raptor; Ras Homolog Enriched in Brain Protein; Recombinant Fusion Proteins - analysis; Recombinant Fusion Proteins - biosynthesis; Recombinant Fusion Proteins - genetics; Red Fluorescent Protein; Regulatory-Associated Protein of mTOR; Rheb; Scanners; Sirolimus - pharmacology; Time-Lapse Imaging; TOR Serine-Threonine Kinases - analysis; TOR Serine-Threonine Kinases - genetics; TOR Serine-Threonine Kinases - metabolism; Transfection; United Kingdom
• ISSN: 1471-2121; 1471-2121
• DOI: 10.1186/1471-2121-14-3

The mammalian target of rapamycin (mTOR) signalling pathway has a key role in cellular regulation and several diseases. While it is thought that Rheb GTPase regulates mTOR, acting immediately upstream, while raptor is immediately downstream of mTOR, direct interactions have yet to be verified in living cells, furthermore the localisation of Rheb has been reported to have only a cytoplasmic cellular localization. In this study a cytoplasmic as well as a significant sub-cellular nuclear mTOR localization was shown , utilizing green and red fluorescent protein (GFP and DsRed) fusion and highly sensitive single photon counting fluorescence lifetime imaging microscopy (FLIM) of live cells. The interaction of the mTORC1 components Rheb, mTOR and raptor, tagged with EGFP/DsRed was determined using fluorescence energy transfer-FLIM. The excited-state lifetime of EGFP-mTOR of ~2400 ps was reduced by energy transfer to ~2200 ps in the cytoplasm and to 2000 ps in the nucleus when co-expressed with DsRed-Rheb, similar results being obtained for co-expressed EGFP-mTOR and DsRed-raptor. The localization and distribution of mTOR was modified by amino acid withdrawal and re-addition but not by rapamycin. The results illustrate the power of GFP-technology combined with FRET-FLIM imaging in the study of the interaction of signalling components in living cells, here providing evidence for a direct physical interaction between mTOR and Rheb and between mTOR and raptor in living cells for the first time.