Total workflows of the single-molecule imaging analysis in living cells: a tutorial guidance to the measurement of the drug effects on a GPCR
Total workflows of the single-molecule imaging analysis in living cells: a tutorial guidance to the measurement of the drug effects on a GPCR
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DOI：
10.1101/2020.06.08.141192
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发表时间：
2020-06
期刊：
影响因子：
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通讯作者：
Masataka Yanagawa;Y. Sako
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文献类型：
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作者：
Masataka Yanagawa;Y. Sako
单分子成像（SMI）是一种测量细胞膜上膜蛋白动态变化的有效方法。单分子跟踪（SMT）分析提供了有关扩散动力学，低聚物尺寸分布和颗粒密度变化的信息。蛋白质-蛋白质相互作用的亲和力和开/关速率可以从双色SMI分析中估计。然而，它是很难的学员，以确定量化的信息，从SMI电影。本方案指导了详细的工作流程，通过使用全内反射荧光显微镜（TIRFM）测量G蛋白偶联受体（GPCR）和代谢型谷氨酸受体3（mGluR 3）的药物激活动力学。本教程指南包含一个名为smDynamicsAnalyzer的开源软件，使用该软件，只需在构建指定的文件夹结构后遵循工作流程即可轻松分析SMT数据集（https：//github.com/masataka-yanagawa/IgorPro8-smDynamicsAnalyzer）。
Single-molecule imaging (SMI) is a powerful method to measure the dynamics of membrane proteins on the cell membrane. The single-molecule tracking (SMT) analysis provides information about the diffusion dynamics, the oligomer size distribution, and the particle density change. The affinity and on/off-rate of a protein—protein interaction can be estimated from the dual-color SMI analysis. However, it is difficult for trainees to determine quantitative information from the SMI movies. The present protocol guides the detailed workflows to measure the drug-activated dynamics of a G protein-coupled receptor (GPCR) and metabotropic glutamate receptor 3 (mGluR3), by using the total internal reflection fluorescence microscopy (TIRFM). This tutorial guidance comprises an open-source software named smDynamicsAnalyzer, with which one can easily analyze the SMT dataset by just following the workflows after building a designated folder structure (https://github.com/masataka-yanagawa/IgorPro8-smDynamicsAnalyzer).