Noninvasive imaging of 5-HT3 receptor trafficking in live cells -: From biosynthesis to endocytosis
Noninvasive imaging of 5-HT3 receptor trafficking in live cells -: From biosynthesis to endocytosis
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DOI：
10.1074/jbc.m407467200
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发表时间：
2004-12-17
影响因子：
4.8
通讯作者：
Vogel, H
中科院分区：
文献类型：
--
作者：
Ilegems, E;Pick, HM;Vogel, H
通过多色荧光共聚焦显微镜在活细胞中在时间和空间上解析离子型 5-HT3 受体 (5-HT3R) 生命周期的连续阶段。将增强的青色荧光蛋白插入到大的细胞内环中，产生了在配体结合特异性和通道活性方面功能齐全的荧光5-HT3R，这首次实现了属于配体门控离子通道超家族的受体的细胞内生物发生、膜靶向和配体介导的内化的完整实时可视化和记录。转染开始后约3小时，在内质网中可检测到新表达受体的荧光信号。在此阶段，受体亚基组装形成活性配体结合位点，如通过荧光 5-HT3R 特异性拮抗剂的结合现场证明的那样。新的蛋白质合成被化学阻断后，内质网和高尔基池中的 5-HT3R 群体几乎定量地移动到细胞表面，表明有效的受体折叠和组装。细胞内 5-HT3 受体以囊泡样结构沿着微管运输到细胞表面，速度通常低于 1 mum/s，并以与富含肌动蛋白的结构域重叠的特征簇分布插入质膜中。在暴露于细胞外激动剂后几分钟内观察到细胞表面 5-HT3 受体的内化。我们对受体、其同源配体和特定细胞器标记精心设计的光谱可区分荧光标记的使用可以被视为一种通用方法，允许亚细胞洞察膜受体运输的动态过程。
Sequential stages in the life cycle of the ionotropic 5-HT3 receptor (5-HT3R) were resolved temporally and spatially in live cells by multicolor fluorescence confocal microscopy. The insertion of the enhanced cyan fluorescent protein into the large intracellular loop delivered a fluorescent 5-HT3R fully functional in terms of ligand binding specificity and channel activity, which allowed for the first time a complete real- time visualization and documentation of intracellular biogenesis, membrane targeting, and ligand-mediated internalization of a receptor belonging to the ligand-gated ion channel superfamily. Fluorescence signals of newly expressed receptors were detectable in the endoplasmic reticulum about 3 h after transfection onset. At this stage receptor subunits assembled to form active ligand binding sites as demonstrated in situ by binding of a fluorescent 5-HT3R-specific antagonist. After novel protein synthesis was chemically blocked, the 5-HT3R populations in the endoplasmic reticulum and Golgi cisternae moved virtually quantitatively to the cell surface, indicating efficient receptor folding and assembly. Intracellular 5-HT3 receptors were trafficking in vesicle-like structures along microtubules to the cell surface at a velocity generally below 1 mum/s and were inserted into the plasma membrane in a characteristic cluster distribution overlapping with actin-rich domains. Internalization of cell surface 5-HT3 receptors was observed within minutes after exposure to an extracellular agonist. Our orchestrated use of spectrally distinguishable fluorescent labels for the receptor, its cognate ligand, and specific organelle markers can be regarded as a general approach allowing subcellular insights into dynamic processes of membrane receptor trafficking.