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

作者：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

来源：BMC Cell Biology, 2013, 14(1): 3.

DOI：10.1186/1471-2121-14-3

• FLIM
• FRET
• mTOR
• GFP
• Raptor
• Rheb

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. %26lt;br%26gt;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 similar to 2400 ps was reduced by energy transfer to similar 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. %26lt;br%26gt;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.

• 出版日期2013-1-12