title: mTOR direct interactions with Rheb-GTPase and raptor: sub-cellular localization using fluorescence lifetime imaging.
abstract: BACKGROUND
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors: Central Laser Facility, STFC, Rutherford Appleton Laboratory, Research Complex at Harwell, Didcot, Oxon OX110QX, UK.
authors: Rahul B
authors: RB
authors: Yadav
authors: Pierre
authors: P
authors: Burgos
authors: Anthony W
authors: AW
authors: Parker
authors: Valentina
authors: V
authors: Iadevaia
authors: Christopher G
authors: CG
authors: Proud
authors: Rodger A
authors: RA
authors: Allen
authors: James P
authors: JP
authors: O'Connell
authors: Ananya
authors: A
authors: Jeshtadi
authors: Christopher D
authors: CD
authors: Stubbs
authors: Stanley W
authors: SW
authors: Botchway
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D002467:Cell Nucleus / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D003593:Cytoplasm / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D020559:Monomeric GTP-Binding Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D009479:Neuropeptides / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D000076205:Ras Homolog Enriched in Brain Protein; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D000076223:Regulatory-Associated Protein of mTOR; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D059008:Time-Lapse Imaging; D000097573:Red Fluorescent Protein
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D020559:Monomeric GTP-Binding Proteins; D009479:Neuropeptides; D000076205:Ras Homolog Enriched in Brain Protein; D011993:Recombinant Fusion Proteins; D000076223:Regulatory-Associated Protein of mTOR; D020123:Sirolimus; D058570:TOR Serine-Threonine Kinases; D000097573:Red Fluorescent Protein; C490211:RHEB protein, human; C463659:RPTOR protein, human