Sahan Ayse Z, Metha Sohum, Zhang Jin
Department of Pharmacology, University of California, San Diego, CA, USA.
Biomedical Sciences Graduate Program, University of California, San Diego, CA, USA.
Methods Mol Biol. 2025;2882:139-162. doi: 10.1007/978-1-0716-4284-9_7.
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.
雷帕霉素机制靶点复合物1（mTORC1）是一种营养感应复合物，它整合来自多个途径的输入信号以促进细胞生长和增殖。mTORC1定位于许多细胞区室，包括细胞核、溶酶体和质膜。然而，关于mTORC1的空间调节以及mTORC1在这些位置的特定功能知之甚少。为了解决这些问题，我们之前开发了一种基于荧光共振能量转移（FRET）的mTORC1活性报告基因（TORCAR），以可视化活细胞内mTORC1活性的动态变化。在这里，我们描述了一个详细的方案，用于使用亚细胞靶向的TORCAR构建体，通过活细胞荧光显微镜研究亚细胞mTORC1活性。
