Tracking the Activity of mTORC1 in Living Cells Using Genetically Encoded FRET-based Biosensor TORCAR

Area of Science:

• Cellular Biology
• Molecular Signaling
• Biochemistry

Background:

• Mechanistic target of rapamycin complex 1 (mTORC1) is a key regulator of cell growth and proliferation, integrating signals like nutrients and growth factors.
• The precise molecular mechanisms controlling mTORC1 activity remain incompletely understood.
• Spatial compartmentalization of signaling pathways is increasingly recognized for ensuring signal specificity and efficiency.

Purpose of the Study:

• To investigate the spatial regulation of mTORC1 activity in live cells.
• To develop and validate a novel molecular tool for monitoring mTORC1 signaling dynamics.
• To characterize how subcellular localization influences mTORC1 signal transduction.

Main Methods:

• Development of a genetically encoded fluorescence resonance energy transfer (FRET)-based reporter, TORCAR, which acts as a surrogate substrate for mTORC1.
• Utilizing TORCAR to measure mTORC1 phosphorylation-dependent FRET changes in real-time within live cells.
• Employing genetic targeting to direct TORCAR to specific subcellular compartments for localized activity assessment.

Main Results:

• TORCAR successfully reports mTORC1 activity through measurable FRET signal changes upon phosphorylation.
• The tool enables visualization and quantification of mTORC1 signaling at distinct subcellular locations.
• Demonstrated the feasibility of studying spatially compartmentalized mTORC1 signaling using this reporter system.

Conclusions:

• The TORCAR reporter is a valuable tool for dissecting the spatial control of mTORC1 signaling in live cells.
• Understanding mTORC1 compartmentalization is essential for comprehending its role in coordinating cell growth.
• This approach provides new insights into the spatiotemporal regulation of a fundamental cellular pathway.