在裂变酵母中结合单分子和膨胀显微镜,以在纳米结构层面上可视化蛋白质结构
Ilijana Vojnovic1,2, Oliver D Caspari1,3, Mehmet Ali Hoşkan1
1Department of Systems and Synthetic Microbiology, Max Planck Institute for Terrestrial Microbiology and LOEWE Center for Synthetic Microbiology (SYNMIKRO), Marburg, Germany.
Open biology
|February 6, 2024
概括
我们开发了SExY,这是一种用于裂变酵母成像的新型膨胀显微镜 (ExM) 方法. 这种强大的协议增强了光蛋白信号保留和同位素扩张,用于详细的细胞研究.
科学领域:
- 细胞生物学 细胞生物学
- 显微镜技术 显微镜技术
- 分子成像学分子成像学
背景情况:
- 扩展显微镜 (ExM) 增强了分辨率,但可以减少光信号.
- 现有的ExM协议在酵母细胞的蛋白质保留和同位素扩张方面面临挑战.
- 对酵母中少量蛋白质的高分辨率成像仍然很困难.
研究的目的:
- 开发一个强大的膨胀显微镜协议用于裂变酵母成像.
- 将扩展显微镜 (ExM) 与光激活局部化显微镜 (ExPALM) 结合起来.
- 为了优化蛋白质保留和同位素扩张,用于高分辨率的酵母细胞成像.
主要方法:
- 开发了一种结合ExM和ExPALM用于裂变酵母的新方案,称为SExY.
- 优化蛋白质保留到最初光蛋白信号的约50%.
- 实现了分裂酵母细胞的五倍,高度同位素扩张.
主要成果:
- 与proExM协议相比,SExY协议使光蛋白信号保留率翻了一番.
- 在示范分子标上证明了SExY,包括低丰度核蛋白 (cbp1,mis16) 和中心基组组素 (cnp1).
- 展示了该协议对遗传标记蛋白质的有效性,并减少了对外部染色的依赖.
结论:
- SExY协议为裂变酵母成像提供了信号保留和扩张的显著改进.
- SExY使低丰度蛋白质的高分辨率可视化成为可能,这对于理解细胞机制至关重要.
- 这种方法有利于依赖遗传标记的研究,并最大限度地减少来自外部染色的文物.
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