Imaging complex protein metabolism in live organisms by stimulated Raman scattering microscopy with isotope labeling.
Imaging complex protein metabolism in live organisms by stimulated Raman scattering microscopy with isotope labeling.
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DOI：
10.1021/cb500787b
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发表时间：
2015-03-20
影响因子：
4
通讯作者：
Min W
中科院分区：
文献类型：
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作者：
Wei L;Shen Y;Xu F;Hu F;Harrington JK;Targoff KL;Min W
蛋白质代谢包括合成和降解，是一个高度复杂的过程，在整个生理和病理过程中起着不可或缺的调节作用。近几十年来，广泛的努力，使用的方法，如放射自显影，质谱和荧光显微镜，已致力于蛋白质代谢的研究。然而，蛋白质代谢的非侵入性和全局可视化已被证明是非常具有挑战性的，特别是在活体系统中。最近，受激拉曼散射（SRS）显微镜加上代谢标记的氘代氨基酸（D-AAs）被证明用于成像新合成的蛋白质在培养的细胞系。在这里，我们显着概括这一概念，开发一个全面的标记和成像平台，用于复杂蛋白质代谢的实时可视化，包括两个时间定义的人群的合成，降解和脉冲追踪分析。首先，优化氘标记效率，允许以高时空分辨率对单个活细胞内的蛋白质合成动力学进行延时成像。其次，通过跟踪归因于预先存在的蛋白质的甲基（CH 3）分布，该平台还使我们能够绘制活细胞内的蛋白质降解。第三，使用两个子集的结构和光谱不同的D-AA，我们实现了双色脉冲追逐成像，通过观察突变亨廷顿蛋白的聚集体形成。最后，超越简单的细胞系，我们证明了脑组织，斑马鱼和小鼠体内蛋白质合成的成像能力。因此，所提出的标记和成像平台将是一个有价值的工具，研究复杂的蛋白质代谢，具有高灵敏度，分辨率和生物相容性的广泛的系统，从细胞到模型动物和可能的人类。
Protein metabolism, consisting of both synthesis and degradation, is highly complex, playing an indispensable regulatory role throughout physiological and pathological processes. Over recent decades, extensive efforts, using approaches such as autoradiography, mass spectrometry, and fluorescence microscopy, have been devoted to the study of protein metabolism. However, noninvasive and global visualization of protein metabolism has proven to be highly challenging, especially in live systems. Recently, stimulated Raman scattering (SRS) microscopy coupled with metabolic labeling of deuterated amino acids (D-AAs) was demonstrated for use in imaging newly synthesized proteins in cultured cell lines. Herein, we significantly generalize this notion to develop a comprehensive labeling and imaging platform for live visualization of complex protein metabolism, including synthesis, degradation, and pulse–chase analysis of two temporally defined populations. First, the deuterium labeling efficiency was optimized, allowing time-lapse imaging of protein synthesis dynamics within individual live cells with high spatial–temporal resolution. Second, by tracking the methyl group (CH3) distribution attributed to pre-existing proteins, this platform also enables us to map protein degradation inside live cells. Third, using two subsets of structurally and spectroscopically distinct D-AAs, we achieved two-color pulse–chase imaging, as demonstrated by observing aggregate formation of mutant hungtingtin proteins. Finally, going beyond simple cell lines, we demonstrated the imaging ability of protein synthesis in brain tissues, zebrafish, and mice in vivo. Hence, the presented labeling and imaging platform would be a valuable tool to study complex protein metabolism with high sensitivity, resolution, and biocompatibility for a broad spectrum of systems ranging from cells to model animals and possibly to humans.