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.

中文翻译：

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AIMTOR是用于实时成像的BRET生物传感器，可揭示亚细胞mTOR信号传导和功能异常。

mTOR信号传导是必需的营养和能量感应途径。在这里，我们描述了AIMTOR，这是一种敏感的遗传编码BRET（生物发光共振能量转移）生物传感器，用于研究活细胞中的mTOR活性。作为原理上的证明，我们在细胞系和原代细胞培养物中均显示，mTOR活性变化（由氨基酸和胰岛素等mTORC1的特定抑制剂和活化剂诱导）可改变AIMTOR BRET强度。我们进一步设计了AIMTOR的几种版本，可以对mTOR活性进行亚细胞特异性评估。然后，我们使用AIMTOR来解析生理病理条件下的mTOR信号。第一，我们显示，mTORC1活性在肌肉细胞分化过程中增加，并在不同亚细胞区室（如溶质）和溶酶体，细胞核以及线粒体附近响应亮氨酸刺激。第二，在海马神经元中，我们发现神经元活动的增强会增加mTOR信号传导。AIMTOR进一步揭示了自闭症谱系障碍小鼠模型神经元中的mTOR信号功能障碍。总之，我们的结果表明AIMTOR是研究活细胞和表型mTORopathies中mTOR信号动力学的灵敏且特定的工具。AIMTOR进一步揭示了自闭症谱系障碍小鼠模型神经元中的mTOR信号功能障碍。总之，我们的结果表明AIMTOR是研究活细胞和表型mTORopathies中mTOR信号动力学的灵敏且特定的工具。AIMTOR进一步揭示了自闭症谱系障碍小鼠模型神经元中的mTOR信号功能障碍。总之，我们的结果表明AIMTOR是研究活细胞和表型mTORopathies中mTOR信号动力学的灵敏且特定的工具。