Kindling fluorescent proteins for precise in vivo photolabeling
Kindling fluorescent proteins for precise in vivo photolabeling
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DOI：
10.1038/nbt778
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发表时间：
2003-02-01
影响因子：
46.9
通讯作者：
Lukyanov, KA
中科院分区：
文献类型：
--
作者：
Chudakov, DM;Belousov, VV;Lukyanov, KA
绿色荧光蛋白（GFP）的光漂白是一种广泛用于跟踪亚细胞结构和细胞内蛋白质运动的方法（1-3）。虽然光漂白是一种强大的技术，但它不能直接跟踪活细胞内物体的运动和速度。只有引入光敏荧光标记，才有可能直接跟踪。许多先前的研究已经报道了荧光蛋白的发射光谱中的光学诱导的变化（4-7）。然而，理想的光活化荧光标记物应该是能够“开启”的非荧光标记物（即，变成荧光）以响应特定波长、强度和持续时间的光的照射。在本报告中，我们产生了一个突变体Anemonia sulcata色蛋白asCP（8）。突变蛋白能够从非荧光到稳定的亮红色荧光形式（“点燃”）的独特的不可逆光转换。这种“点燃荧光蛋白”（KFP 1）可用于精确的体内光标记，以跟踪细胞，细胞器和蛋白质的运动。我们使用KFP 1在mRNA显微注射测定中用于体内细胞标记，以监测非洲爪蟾胚胎发育并跟踪哺乳动物细胞中的线粒体运动。
Photobleaching of green fluorescent protein (GFP) is a widely used approach for tracking the movement of subcellular structures and intracellular proteins(1-3). Although photobleaching is a powerful technique, it does not allow direct tracking of an object's movement and velocity within a living cell. Direct tracking becomes possible only with the introduction of a photoactivated fluorescent marker. A number of previous studies have reported optically induced changes in the emission spectra of fluorescent proteins(4-7). However, the ideal photoactivated fluorescent marker should be a nonfluorescent tag capable of "switching on" (i.e., becoming fluorescent) in response to irradiation by light of a particular wavelength, intensity, and duration. In this report, we generated a mutant of Anemonia sulcata chromoprotein asCP(8). The mutant protein is capable of unique irreversible photoconversion from the nonfluorescent to a stable bright-red fluorescent form ("kindling"). This "kindling fluorescent protein" (KFP1) can be used for precise in vivo photolabeling to track the movements of cells, organelles, and proteins. We used KFP1 for in vivo cell labeling in mRNA microinjection assays to monitor Xenopus laevis embryo development and to track mitochondrial movement in mammalian cells.