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Open Access Highly Accessed Research article

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

Rahul B Yadav1, Pierre Burgos1, Anthony W Parker1, Valentina Iadevaia2, Christopher G Proud2, Rodger A Allen3, James P O'Connell3, Ananya Jeshtadi4, Christopher D Stubbs1* and Stanley W Botchway1*

Author Affiliations

1 Central Laser Facility, STFC, Rutherford Appleton Laboratory, Research Complex at Harwell, Didcot, Oxon OX110QX, UK

2 School of Biological Sciences, University of Southampton, Southampton, SO17 1BJ, UK

3 UCB-Pharma, 208 Bath Road, Slough, SL1 3WE, UK

4 School of Life Sciences, Headington Campus, Oxford Brookes University, Oxford, OX3 0BP, UK

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BMC Cell Biology 2013, 14:3  doi:10.1186/1471-2121-14-3

Published: 12 January 2013

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.

Keywords:
FLIM; FRET; mTOR; GFP; Raptor; Rheb