Seeing the machinery of live cells
Seeing the machinery of live cells
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DOI：
10.1126/science.280.5371.1954
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发表时间：
1998-06-19
期刊：
影响因子：
56.9
通讯作者：
Miyawaki, A
中科院分区：
文献类型：
--
作者：
Tsien, RY;Miyawaki, A
随着生物化学和遗传学揭示了更多负责生命的分子，我们需要了解这些分子在协调细胞反应时如何在空间和时间上相互作用。观察活细胞中荧光标记的大分子可以揭示这种动态，既可用于细胞如何处理信号的基础研究，也可用于候选药物的高通量筛选。水母维多利亚水母绿色荧光蛋白 (GFP) 的发展为活细胞成像提供了最近最强劲的推动力，该蛋白作为一种荧光标记，可通过基因融合融入蛋白质 (1)。 GFP 发出生理激活信号的最简单方法是将其与宿主蛋白连接，该宿主蛋白在刺激后从一个细胞区室易位到另一个细胞区室。例如，与糖皮质激素受体 (2) 融合的 GFP 在添加激素后从细胞质转移到细胞核。 GFP 与 β-arrestin (3)、蛋白激酶 C-γ 的 Cys1 结构域 (4) 和磷脂酶 C-γ 的 pleckstrin 同源结构域 (5) 的融合在 G 蛋白激活、二酰甘油形成和磷脂酰肌醇-3,4,5-三磷酸形成后分别移动到质膜。
As biochemistry and genetics uncover more of the molecules responsible for life, we need to see how these molecules interact with each other in space and time as they orchestrate cellular responses. Observation of fluorescently labeled macromolecules in live cells can reveal such dynamics, both for basic studies on how cells process signals and for high-throughput screening of candidate drugs.Live-cell imaging has received its strongest recent impetus from the development of the green fluorescent protein (GFP) of the jellyfish Aequorea victoria as a fluorescent label that can be incorporated into proteins by genetic fusion (1). The simplest way that GFP can signal physiological activation is when it is concatenated to a host protein that translocates from one cellular compartment to another upon stimulation. For example, GFP fused to glucocorticoid receptor (2) translocates from cytosol to nucleus upon hormone addition. GFP fusions to β-arrestin (3), the Cys1 domain from protein kinase C-γ (4), and the pleckstrin homology domain of phospholipase C-γ (5) move to the plasma membrane upon G-protein activation, diacylglycerol formation, and phosphatidylinositol-3, 4, 5-trisphosphate formation, respectively.