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Updated: May 15, 2026
Spatio-Temporal Manipulation of Small GTPase Activity at Subcellular Level and on Timescale of Seconds in Living Cells
Published on: March 9, 2012
mTOR direct interactions with Rheb-GTPase and raptor: sub-cellular localization using fluorescence lifetime imaging
Rahul B Yadav1, Pierre Burgos, Anthony W Parker
1Central Laser Facility, STFC, Rutherford Appleton Laboratory, Research Complex at Harwell, Didcot, Oxon OX110QX, UK.
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.
Insights
This study confirms direct interactions between Rheb GTPase, mTOR, and raptor in living cells using advanced imaging. It also reveals nuclear localization of mTOR, challenging previous assumptions about Rheb
Area of Science:
- Cellular Biology
- Molecular Signaling
- Biophysics
Background:
- The mammalian target of rapamycin (mTOR) pathway is crucial for cellular regulation and disease.
- Direct interactions between mTOR, Rheb GTPase, and raptor in live cells remain unverified.
- Rheb GTPase localization was previously thought to be exclusively cytoplasmic.
Purpose of the Study:
- To investigate the direct physical interactions between mTOR, Rheb, and raptor in living cells.
- To determine the sub-cellular localization of mTOR, including potential nuclear presence.
- To validate the role of Rheb GTPase as an upstream regulator of mTOR.
Main Methods:
- Utilized green and red fluorescent protein (GFP and DsRed) fusion proteins.
- Employed highly sensitive single photon counting fluorescence lifetime imaging microscopy (FLIM) in live cells.
- Applied fluorescence energy transfer-FLIM (FRET-FLIM) to detect molecular interactions.
Main Results:
- Demonstrated both cytoplasmic and significant nuclear localization of mTOR.
- Provided evidence of direct physical interaction between mTOR and Rheb GTPase.
- Confirmed direct physical interaction between mTOR and raptor.
- Observed changes in mTOR localization upon amino acid withdrawal/re-addition, but not with rapamycin.
Conclusions:
- Established direct physical interactions between mTOR, Rheb, and raptor in living cells using GFP-technology and FRET-FLIM.
- Showcased the utility of FRET-FLIM for studying signaling component interactions in real-time.
- Extended understanding of mTOR pathway regulation and localization dynamics.
