Nuclear mTORC1 Live-Cell Sensor nTORSEL Reports Differential Nuclear mTORC1 Activity in Cell Lines
Yifan Wang1, Canrong Li1, Yingyi Ouyang1
1School of Medicine, Shenzhen Campus of Sun Yat-Sen University, Sun Yat-Sen University, Shenzhen 518107, China.
Abstract:
The mammalian or mechanistic target of rapamycin complex 1 (mTORC1) is activated on the surface of lysosomes and phosphorylates substrates at various subcellular locations, including the lysosome, cytosol, and nucleus. However, the signaling and biological functions of nuclear mTORC1 (nmTORC1) are not well understood, primarily due to limited tools for monitoring mTORC1 activity in the nucleus. In this study, we developed a genetically encoded nmTORC1 sensor, termed nTORSEL, based on the phosphorylation of the eukaryotic initiation factor 4E (eIF4E) binding protein 1 (4EBP1) by mTORC1 within the nucleus. nTORSEL, like its predecessor TORSEL, exhibits a fluorescent punctate pattern in the nucleus through multivalent protein-protein interactions between oligomerized 4EBP1 and eIF4E when nmTORC1 activity is low. We validated nTORSEL using biochemical analyses and imaging techniques across representative cell lines with varying levels of nmTORC1 activity. Notably, nTORSEL specifically detects physiological, pharmacological, and genetic inhibition of nmTORC1 in mouse embryonic fibroblast (MEF) cells but not in HEK293T cells. Therefore, nTORSEL is an effective tool for investigating nuclear mTORC1 signaling in cell lines.
Insights
Researchers developed nTORSEL, a novel sensor to monitor nuclear mechanistic target of rapamycin complex 1 (mTORC1) activity. This tool aids in understanding nuclear mTORC1 signaling pathways and their biological functions.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Mammalian/mechanistic target of rapamycin complex 1 (mTORC1) regulates cellular processes.
- mTORC1 activation occurs at lysosomes, phosphorylating substrates in the nucleus, cytosol, and lysosomes.
- Nuclear mTORC1 (nmTORC1) signaling and functions remain poorly understood due to limited monitoring tools.
Purpose of the Study:
- To develop a genetically encoded sensor for monitoring nmTORC1 activity.
- To investigate the signaling and biological functions of nmTORC1.
- To provide a tool for studying nmTORC1 in various cellular contexts.
Main Methods:
- Development of a genetically encoded nmTORC1 sensor (nTORSEL) based on eIF4E binding protein 1 (4EBP1) phosphorylation.
- Utilizing fluorescent microscopy and biochemical analyses for validation.
- Testing nTORSEL in cell lines with varying nmTORC1 activity levels.
Main Results:
- nTORSEL successfully detects nmTORC1 activity by monitoring 4EBP1 phosphorylation in the nucleus.
- The sensor exhibits a punctate nuclear pattern dependent on nmTORC1 activity.
- nTORSEL specifically identified inhibition of nmTORC1 in mouse embryonic fibroblast (MEF) cells.
Conclusions:
- nTORSEL is an effective tool for investigating nmTORC1 signaling.
- The sensor enables specific detection of nmTORC1 activity and inhibition.
- This tool facilitates further research into the roles of nmTORC1 in cellular functions.
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