Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins
Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins
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DOI：
10.1038/28190
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发表时间：
1998-07-09
期刊：
影响因子：
64.8
通讯作者：
Rothman, JE
中科院分区：
文献类型：
--
作者：
Miesenböck, G;De Angelis, DA;Rothman, JE
在神经系统中，信息通常是由细胞的集合而不是由单个单元携带的。光学指示剂（1）提供了一种强有力的手段来揭示这种分布的活性，特别是当基于蛋白质且可编码在DNA中时（2-4）：可编码探针可以通过遗传操作引入细胞、组织或转基因生物体，在解剖学或功能上限定的细胞群中选择性表达，并且理想地原位记录，而不需要外源辅因子，我们已经通过结构定向组合诱变开发了绿色荧光蛋白（“pHluorins”）的pH敏感突变体，目的是利用分泌囊泡（5，6）内的酸性pH来监测囊泡胞吐和再循环。当连接到一个囊泡膜蛋白，pHluorins被分类为分泌和突触囊泡，并报告在个别的突触终扣，以及分泌和融合孔“闪烁”的单个分泌颗粒的传输。
In neural systems, information is often carried by ensembles of cells rather than by individual units. Optical indicators(1) provide a powerful means to reveal such distributed activity, particularly when protein-based and encodable in DNA(2-4): encodable probes can be introduced into cells, tissues, or transgenic organisms by genetic manipulation, selectively expressed in anatomically or functionally defined groups of cells, and, ideally, recorded in situ, without a requirement for exogenous cofactors, Here we describe sensors for secretion and neurotransmission that fulfill these criteria We have developed pH-sensitive mutants of green fluorescent protein ('pHluorins') by structure-directed combinatorial mutagenesis, with the aim of exploiting the acidic pH inside secretory vesicles(5,6) to monitor vesicle exocytosis and recycling. When linked to a vesicle membrane protein, pHluorins were sorted to secretory and synaptic vesicles and reported transmission at individual synaptic boutons, as well as secretion and fusion pore 'flicker' of single secretory granules.