Visualization of Subcellular mTOR Complex 1 Activity with a FRET-Based Sensor (TORCAR).

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• The mechanistic target of rapamycin complex 1 (mTORC1) is a crucial nutrient-sensing complex regulating cell growth and proliferation.
• mTORC1 localizes to various cellular compartments, but its spatial regulation and specific functions at these sites remain poorly understood.

Purpose of the Study:

• To develop and present a detailed protocol for investigating subcellular mTORC1 activities.
• To enable visualization of dynamic changes in mTORC1 activity within live cells at specific subcellular locations.

Main Methods:

• Utilized a Förster resonance energy transfer (FRET)-based mTORC1 activity reporter (TORCAR).
• Developed subcellularly targeted TORCAR constructs for precise localization.
• Employed live-cell fluorescence microscopy for dynamic monitoring.

Main Results:

• Successfully visualized dynamic changes in mTORC1 activity within live cells.
• Demonstrated the utility of targeted TORCAR constructs for studying subcellular mTORC1.
• Established a protocol for investigating spatial mTORC1 regulation.

Conclusions:

• The described protocol provides a powerful tool for dissecting the spatial control and function of mTORC1.
• Live-cell imaging of subcellular mTORC1 activity offers new insights into nutrient-sensing pathways.
• This method facilitates further research into mTORC1's role in cell growth and disease.