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The mechanistic target of rapamycin complex 1 (mTORC1) is a nutrient-sensing complex that integrates inputs from several pathways to promote cell growth and proliferation. mTORC1 localizes to many cellular compartments, including the nucleus, lysosomes, and plasma membrane. However, little is known about the spatial regulation of mTORC1 and the specific functions of mTORC1 at these locations. To address these questions, we previously developed a Förster resonance energy transfer (FRET)-based mTORC1 activity reporter (TORCAR) to visualize the dynamic changes in mTORC1 activity within live cells. Here, we describe a detailed protocol for using subcellularly targeted TORCAR constructs to investigate subcellular mTORC1 activities via live-cell fluorescence microscopy.

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

  • 实验方案

  • 发表日期：

    2025年

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  • Ayse Z. Sahan,
  • Sohum Metha,
  • Jin Zhang

摘要

The mechanistic target of rapamycin complex 1 (mTORC1) is a nutrient-sensing complex that integrates inputs from several pathways to promote cell growth and proliferation. mTORC1 localizes to many cellular compartments, including the nucleus, lysosomes, and plasma membrane. However, little is known about the spatial regulation of mTORC1 and the specific functions of mTORC1 at these locations. To address these questions, we previously developed a Förster resonance energy transfer (FRET)-based mTORC1 activity reporter (TORCAR) to visualize the dynamic changes in mTORC1 activity within live cells. Here, we describe a detailed protocol for using subcellularly targeted TORCAR constructs to investigate subcellular mTORC1 activities via live-cell fluorescence microscopy.