IGF, University of Montpellier, CNRS, INSERM, Montpellier, France.
University Hospital Basel, Department of Biomedecine, Basel, Switzerland.
BMC Biol. 2020 Jul 3;18(1):81. doi: 10.1186/s12915-020-00790-8.
mTOR signaling is an essential nutrient and energetic sensing pathway. Here we describe AIMTOR, a sensitive genetically encoded BRET (Bioluminescent Resonance Energy Transfer) biosensor to study mTOR activity in living cells.
As a proof of principle, we show in both cell lines and primary cell cultures that AIMTOR BRET intensities are modified by mTOR activity changes induced by specific inhibitors and activators of mTORC1 including amino acids and insulin. We further engineered several versions of AIMTOR enabling subcellular-specific assessment of mTOR activities. We then used AIMTOR to decipher mTOR signaling in physio-pathological conditions. First, we show that mTORC1 activity increases during muscle cell differentiation and in response to leucine stimulation in different subcellular compartments such as the cytosol and at the surface of the lysosome, the nucleus, and near the mitochondria. Second, in hippocampal neurons, we found that the enhancement of neuronal activity increases mTOR signaling. AIMTOR further reveals mTOR-signaling dysfunctions in neurons from mouse models of autism spectrum disorder.
Altogether, our results demonstrate that AIMTOR is a sensitive and specific tool to investigate mTOR-signaling dynamics in living cells and phenotype mTORopathies.
mTOR 信号是一种重要的营养和能量感应途径。在这里，我们描述了 AIMTOR，这是一种灵敏的遗传编码 BRET（生物发光共振能量转移）生物传感器，可用于研究活细胞中的 mTOR 活性。
作为原理验证，我们在细胞系和原代细胞培养物中均表明，AIMTOR BRET 强度可通过 mTORC1 的特定抑制剂和激活剂（包括氨基酸和胰岛素）诱导的 mTOR 活性变化而改变。我们进一步设计了几种版本的 AIMTOR，能够对 mTOR 活性进行亚细胞特异性评估。然后，我们使用 AIMTOR 来破译生理病理条件下的 mTOR 信号。首先，我们表明，在肌肉细胞分化过程中以及在不同亚细胞区室（如细胞质、溶酶体表面、核和靠近线粒体）中受到亮氨酸刺激时，mTORC1 活性增加。其次，在海马神经元中，我们发现神经元活性的增强会增加 mTOR 信号。AIMTOR 进一步揭示了自闭症谱系障碍小鼠模型中神经元的 mTOR 信号传导功能障碍。
总之，我们的研究结果表明，AIMTOR 是一种灵敏且特异性的工具，可用于研究活细胞中的 mTOR 信号转导动力学，并表型 mTOR 病。
