In situ characterization of the mTORC1 during adipogenesis of human adult stem cells on chip
In situ characterization of the mTORC1 during adipogenesis of human adult stem cells on chip
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芯片上人类成体干细胞脂肪形成过程中 mTORC1 的原位表征
DOI：
10.1073/pnas.1601207113
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发表时间：
2016
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影响因子：
--
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--
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哺乳动物雷帕霉素靶标(MTOR)是一种整合营养、能量和代谢物信号的中央激酶。该激酶形成两个不同的复合体：mTORC1和mTORC2。MTORC1对脂肪组织的再生起着重要但未明确的调节作用。在不断变化的细胞微环境中，mTOR的分析通常受到调控机制的复杂性的阻碍，包括蛋白质相互作用和/或磷酸化。在这里，我们开发了一个微流控大规模集成芯片平台，用于在超过3wk的标准化营养条件下，在128个分离的微室中培养和分化人脂肪干细胞。通过测定HASC培养中的脂质积累率来衡量干细胞分化的进程。在原位蛋白质分析方面，我们建立了一种多重原位邻近连接分析(MPLA)，它可以在单个细胞中选择性地检测其两个复合体中的mTOR，并在同一芯片上实现。结合这项技术，有可能揭示在脂肪形成过程中mTORC1的丰度、磷酸化状态和与溶酶体协调定位的调节。对4个芯片上培养的100多万个细胞的高含量图像分析和原位聚乳酸信号的参数分析表明，mTORC1和溶酶体在时间和空间上是协调的，但在成脂过程中其组成并不是一致的。
Mammalian target of rapamycin (mTOR) is a central kinase integrating nutrient, energy, and metabolite signals. The kinase forms two distinct complexes: mTORC1 and mTORC2. mTORC1 plays an essential but undefined regulatory function for regeneration of adipose tissue. Analysis of mTOR in general is hampered by the complexity of regulatory mechanisms, including protein interactions and/or phosphorylation, in an ever-changing cellular microenvironment. Here, we developed a microfluidic large-scale integration chip platform for culturing and differentiating human adipose-derived stem cells (hASCs) in 128 separated microchambers under standardized nutrient conditions over 3 wk. The progression of the stem cell differentiation was measured by determining the lipid accumulation rates in hASC cultures. For in situ protein analytics, we developed a multiplex in situ proximity ligation assay (mPLA) that can detect mTOR in its two complexes selectively in single cells and implemented it on the same chip. With this combined technology, it was possible to reveal that the mTORC1 is regulated in its abundance, phosphorylation state, and localization in coordination with lysosomes during adipogenesis. High-content image analysis and parameterization of the in situ PLA signals in over 1 million cells cultured on four individual chips showed that mTORC1 and lysosomes are temporally and spatially coordinated but not in its composition during adipogenesis.
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