细胞膜上信号转导蛋白的单分子成像与分析

doi:10.7503/cjcu20190663

摘要/Abstract

摘要：

结合本课题组的研究工作, 介绍了单分子荧光成像原理、荧光标记方法及数据分析方法, 并进一步综述了单分子荧光成像在几种重要的膜蛋白信号转导分子机制和相关药物研究中的进展.

关键词: 单分子荧光成像, 细胞膜受体, 全内反射荧光显微镜, 超分辨成像, 单分子分析

Abstract:

Membrane proteins play an important role in cell signal transduction. Structural and functional abnormalities of membrane proteins can cause a variety of diseases. The development of single-molecule fluorescence imaging technology has enabled researchers to study the location and dynamic behavior of single membrane proteins in living cell systems, and their interactions with other molecules. Single-molecule fluorescence imaging technology has been widely used to reveal the molecular mechanisms of signal transduction under physiological conditions in recent years. Based on the our research work, this paper introduces the imaging principle, fluorescent labeling method and data analysis method of single-molecule fluorescence imaging in the study of cell membrane proteins, and further reviews the research progress of single-molecule fluorescence imaging on mechanistic study of several important signal transduction membrane proteins and related drug development.

Key words: Single molecule fluorescence imaging, Cell membrane receptor, Total internal reflection fluorescence microscope, Super-resolution imaging, Single molecule analysis

中图分类号:

O657.3

TrendMD:

梁钰昕, 赵容, 梁馨月, 方晓红. 细胞膜上信号转导蛋白的单分子成像与分析. 高等学校化学学报, 2020, 41(6): 1127.

LIANG Yuxin, ZHAO Rong, LIANG Xinyue, FANG Xiaohong. Single-molecule Imaging and Analysis of Signal Transduction Proteins on Cell Membranes ^†. Chem. J. Chinese Universities, 2020, 41(6): 1127.

图/表 8

Fig.1 Principles of objective-type TIRFM

Fig.2 Architecture of CLDNN(A), the analysis accuracies of CLDNN and HMM to the synthesized data sets with different aSNRs(B) and the true distribution and outputted distributions of bleaching steps analyzed by different algorithms(C)[55] Copyright 2019, American Chemical Society.

Fig.4 A typical single-molecule image of TβRⅡ-GFP(A), distribution of the fluorescence intensity of diffraction-limited TβRⅡ-GFP spots, the solid curves show the fitting of Gaussian function and the two peaks represented TβRⅡ-GFP monomers and dimers, respectively(B), time courses of GFP emission after background correction show two-step bleaching(C)[12] Copyright 2009, the National Academy of Sciences of the United States of America.

Fig.5 A typical single-molecule fluorescence images of EGFP-Smad3 molecules docking on the cell membrane in the presence of TGF-β1(A), diffusion coefficient of membrane-docked EGFP-Smad3 molecules with the marked D value in HeLa cells in the presence of TGF-β1(B), cumulative histograms(solid) indicated the docking time of EGFP-Smad3 molecules in stimulated cells, the histograms were fitted with a single exponential function, the dotted lines are the fitting curves with the time constants τ(0.60 s)(C)[67] Copyright 2016, Springer Nature.

Fig.6 Triple-color live-cell confocal imaging of the cell co-expressing caveolin-1-EGFP, clathrin-DsRed and Myc-TβRⅠ, the boxed region was magnified(images to the right) to show the movement of a Myc-TβRⅠ, caveolin-1-EGFP and clathrin-DsRed triple-positive vesicle(arrows) from the lateral plasma membrane(white lines) to the cytoplasm[69] Copyright 2015, Springer Natare.

Fig.8 Aggregation states of β2AR-GFP under different conditions(A—C), the distributions of the fluorescence intensity of individual β2AR-GFP spots after the stimulation with 10 mmol/L carvedilol(A), isoproterenol(B), and propranolol(C), the fractions of one and two-step bleaching events of β2AR-GFP in the resting and drug-stimulated cells(D), before(E) and after(F) ligand stimulation in the presence of the siRNA targeting β-arrestin1(βarr1), or the siRNA targeting β-arrestin2(βarr2)[79] Copyright 2016, Royal Society of Chemistry.

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