Edinburgh Cancer Research Centre, Institute of Genetics and Molecular Medicine, University of Edinburgh, Crewe Road South, Edinburgh EH4 2XR, Scotland, United Kingdom.
J Biol Chem. 2010 Apr 9;285(15):11607-16. doi: 10.1074/jbc.M110.102392. Epub 2010 Feb 5.
We report the development and application of photoactivatable Green Cherry (G(PA)C), the first genetically encoded "continuously red-photoactivatable green" two-color probe for live cell imaging. G(PA)C is unique in that it enables real-time tracking of selected subpopulations of proteins and organelles in the cell or of cells within tissues and whole organisms, with constant reference to the entire population of the probe. Using G(PA)C-zyxin as proof of utility, we obtained new insights into the dynamic movement of the cytoskeletal protein zyxin. We show that zyxin is continuously and rapidly recruited from the cytosol into established focal adhesions. It can also move rapidly within a given focal adhesion and "hop" between adjacent focal adhesions, emphasizing the dynamic nature of proteins within these structures. The in vivo utility of G(PA)C is exemplified by tracking hemocyte movements using a versatile transgenic Drosophila model engineered to express G(PA)C in tissues and cells of interest under the control of the GAL4-inducible promoter.
我们报告了光激活绿樱桃（G(PA)C）的开发和应用，这是第一个用于活细胞成像的遗传编码的“连续红-光激活绿”双色探针。G(PA)C 的独特之处在于，它能够实时跟踪细胞内选定的蛋白质和细胞器亚群或组织和整个生物体内的细胞，同时不断参考探针的整个群体。使用 G(PA)C-zyxin 作为效用的证明，我们获得了对细胞骨架蛋白 zyxin 动态运动的新见解。我们表明，zyxin 从细胞质中不断快速募集到已建立的焦点黏附处。它也可以在给定的焦点黏附处内快速移动，并在相邻的焦点黏附处之间“跳跃”，强调了这些结构中蛋白质的动态性质。G(PA)C 的体内效用通过使用多功能转基因果蝇模型进行跟踪，该模型在 GAL4 诱导型启动子的控制下在感兴趣的组织和细胞中表达 G(PA)C，从而得到了例证。
