Dynamic Visualization of mTORC1 Activity in Living Cells

Area of Science:

• Cellular Biology
• Molecular Signaling
• Biochemistry

Background:

• The mechanistic target of rapamycin complex 1 (mTORC1) is a central regulator of cell growth and metabolism, responding to diverse environmental cues.
• Understanding the spatiotemporal dynamics of mTORC1 activity is crucial for deciphering its role in cellular processes, but direct measurement has been challenging.

Purpose of the Study:

• To develop and validate a genetically encoded reporter for real-time monitoring of mTORC1 activity.
• To investigate the spatiotemporal regulation of mTORC1 in response to different signaling pathways.
• To elucidate the relationship between calcium signaling and mTORC1 activation.

Main Methods:

• Development of a genetically encoded Förster Resonance Energy Transfer (FRET)-based reporter for mTORC1 activity (TORCAR).
• Co-imaging of TORCAR activity with calcium dynamics in live cells.
• Subcellular targeting of TORCAR to map mTORC1 activity in distinct cellular compartments.
• Stimulation with growth factors and leucine surrogates to observe signal-specific activity patterns.

Main Results:

• TORCAR successfully reports mTORC1 activity through FRET changes upon phosphorylation.
• Growth factor-induced calcium transients were found to contribute to mTORC1 activation.
• Dynamic activity mapping revealed mTORC1 activity in the cytosol, lysosome, nucleus, and plasma membrane.
• Different stimuli, such as growth factors and leucine surrogates, elicited distinct spatiotemporal patterns of mTORC1 activity.

Conclusions:

• mTORC1 activity is dynamically regulated in both space and time within the cell.
• The TORCAR reporter provides a powerful tool for dissecting mTORC1 spatiotemporal signaling.
• Signal-specific regulation of mTORC1 activity at distinct subcellular locations highlights the complexity of cellular growth control.