Yamashiro Sawako, Mizuno Hiroaki, Watanabe Naoki
Department of Pharmacology, Kyoto University Faculty of Medicine, Kyoto, Japan.
Methods Cell Biol. 2015;125:43-59. doi: 10.1016/bs.mcb.2014.10.013. Epub 2015 Jan 8.
Single-molecule speckle (SiMS) microscopy has been a powerful method to analyze actin dynamics in live cells by tracking single molecule of fluorescently labeled actin. Recently we developed a new SiMS method, which is easy-to-use for inexperienced researchers and achieves high spatiotemporal resolution. In this method, actin labeled with fluorescent DyLight dye on lysines is employed as a probe. Electroporation-mediated delivery of DyLight-actin (DL-actin) into cells enables to label cells with 100% efficiency at the optimal density. DL-actin labels cellular actin filaments including formin-based structures with improved photostability and brightness compared to green fluorescent protein-actin. These favorable properties of DL-actin extend time window of the SiMS analysis. Furthermore, the new SiMS method enables nanometer-scale displacement analysis with a low localization error of ±8-8.5 nm. With these advantages, our new SiMS microscopy method will help researchers to investigate various actin remodeling processes. In this chapter, we introduce the methods for preparation of DL-actin probes, electroporation to deliver DL-actin, the SiMS imaging and data analysis.
单分子散斑（SiMS）显微镜技术一直是通过追踪荧光标记肌动蛋白的单分子来分析活细胞中肌动蛋白动力学的有力方法。最近，我们开发了一种新的SiMS方法，该方法对缺乏经验的研究人员来说易于使用，并能实现高时空分辨率。在这种方法中，用荧光DyLight染料标记赖氨酸的肌动蛋白被用作探针。通过电穿孔介导将DyLight-肌动蛋白（DL-肌动蛋白）递送至细胞，能够以最佳密度100%有效地标记细胞。与绿色荧光蛋白-肌动蛋白相比，DL-肌动蛋白标记包括基于formin的结构在内的细胞肌动蛋白丝，具有更高的光稳定性和亮度。DL-肌动蛋白的这些有利特性扩展了SiMS分析的时间窗口。此外，新的SiMS方法能够进行纳米级位移分析，定位误差低至±8-8.5 nm。凭借这些优势，我们新的SiMS显微镜方法将有助于研究人员研究各种肌动蛋白重塑过程。在本章中，我们介绍了DL-肌动蛋白探针的制备方法、电穿孔递送DL-肌动蛋白、SiMS成像和数据分析。
