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

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.