遗传密码的扩展使得活细胞和超分辨率成像能够对特定位置标记的细胞蛋白进行超分辨率成像
Chayasith Uttamapinant1, Jonathan D Howe1, Kathrin Lang1
1Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, United Kingdom.
Journal of the American Chemical Society
|April 2, 2015
概括
这项研究提出了一种使用非自然氨基酸精确标记细胞内的蛋白质的新方法. 这种技术增强了超高分辨率显微镜,揭示了像核活性纤维这样的详细细胞结构.
科学领域:
- 细胞生物学 细胞生物学
- 生物化学 生物化学
- 显微镜的使用方法
背景情况:
- 超高分辨率成像需要密集的,特定位置的蛋白质标记与明亮的光灯.
- 遗传密码扩展和生物对角化学允许特定地点的蛋白质标签.
- 高效的非自然氨基酸结合和特定的细胞内标签仍然是高分辨率成像的挑战.
研究的目的:
- 为超分辨率显微镜的细胞内蛋白质开发一种高效,特定于位点的,密集的光标记方法.
- 为了克服非自然氨基酸整合效率和细胞内标签特异性的局限性.
- 为了可视化活细胞内的亚衍射超结构特征.
主要方法:
- 用基因工程改造的细胞骨蛋白质 (β-actin,vimentin) 在特定的部位结合了bicyclo[6.1.0]nonyne-lysine.
- 利用氨酸-光合物用于活细胞中工程蛋白质的选择性光标记.
- 采用超分辨率辐射定位显微镜 (STORM) 进行高分辨率成像.
主要成果:
- 成功地产生了细胞骨蛋白质,其中包含了特定地点的bicyclo[6.1.0]nonyne-lysine.
- 在细胞蛋白质组内实现了工程蛋白质的选择性光标记.
- 创建了密集标记的细胞骨超结构,使STORM能够对亚衍射特征进行成像,包括核活性纤维.
结论:
- 这项工作建立了一种有效的方法,用于高密度的细胞内蛋白质的特定位置,活细胞光标记.
- 开发的技术显著提升了用于可视化细胞超结构的超分辨率成像能力.
- 这种方法克服了以前的局限性,为细胞内详细的超结构分析铺平了道路.
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