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Genetically Targeted Ratiometric and Activated pH Indicator Complexes (TRApHIC) for Receptor Trafficking
Biochemistry ( IF 2.9 ) Pub Date : 2018-01-16 00:00:00 , DOI: 10.1021/acs.biochem.7b01135 Lydia A. Perkins , Qi Yan 1 , Brigitte F. Schmidt , Dmytro Kolodieznyi , Saumya Saurabh 2 , Mads Breum Larsen 3 , Simon C. Watkins 3 , Laura Kremer 4 , Marcel P. Bruchez
Biochemistry ( IF 2.9 ) Pub Date : 2018-01-16 00:00:00 , DOI: 10.1021/acs.biochem.7b01135 Lydia A. Perkins , Qi Yan 1 , Brigitte F. Schmidt , Dmytro Kolodieznyi , Saumya Saurabh 2 , Mads Breum Larsen 3 , Simon C. Watkins 3 , Laura Kremer 4 , Marcel P. Bruchez
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Fluorescent protein-based pH sensors are useful tools for measuring protein trafficking through pH changes associated with endo- and exocytosis. However, commonly used pH-sensing probes are ubiquitously expressed with their protein of interest throughout the cell, hindering our ability to focus on specific trafficking pools of proteins. We developed a family of excitation ratiometric, activatable pH responsive tandem dyes, consisting of a pH sensitive Cy3 donor linked to a fluorogenic malachite green acceptor. These cell-excluded dyes are targeted and activated upon binding to a genetically expressed fluorogen-activating protein and are suitable for selective labeling of surface proteins for analysis of endocytosis and recycling in live cells using both confocal and superresolution microscopy. Quantitative profiling of the endocytosis and recycling of tagged β2-adrenergic receptor (B2AR) at a single-vesicle level revealed differences among B2AR agonists, consistent with more detailed pharmacological profiling.
中文翻译：
遗传性靶向比率和活化的pH指示剂配合物（TRApHIC），用于受体运输。
基于荧光蛋白的pH传感器是用于通过与胞吞作用和胞吐作用相关的pH改变来测量蛋白质运输的有用工具。但是，常用的pH敏感探针会随其感兴趣的蛋白质在整个细胞中普遍表达，这阻碍了我们专注于特定的蛋白质运输库的能力。我们开发了一系列激发比率式，可激活的pH响应串联染料，该染料由与荧光孔雀石绿受体相连的pH敏感Cy3供体组成。这些细胞排除的染料在与遗传表达的氟激活蛋白结合后被靶向并被激活，适用于使用共聚焦和超分辨率显微镜对表面蛋白进行选择性标记，以分析内吞作用并在活细胞中回收。
更新日期：2018-01-16
中文翻译：
遗传性靶向比率和活化的pH指示剂配合物（TRApHIC），用于受体运输。
基于荧光蛋白的pH传感器是用于通过与胞吞作用和胞吐作用相关的pH改变来测量蛋白质运输的有用工具。但是，常用的pH敏感探针会随其感兴趣的蛋白质在整个细胞中普遍表达，这阻碍了我们专注于特定的蛋白质运输库的能力。我们开发了一系列激发比率式，可激活的pH响应串联染料，该染料由与荧光孔雀石绿受体相连的pH敏感Cy3供体组成。这些细胞排除的染料在与遗传表达的氟激活蛋白结合后被靶向并被激活，适用于使用共聚焦和超分辨率显微镜对表面蛋白进行选择性标记，以分析内吞作用并在活细胞中回收。
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