Solari Jacopo, Anquez Francois, Scherer Katharina M, Shimizu Thomas S
AMOLF Institute, Amsterdam, The Netherlands.
Laboratoire de Physique des Lasers, Atomes et Molécules, UMR CNRS 8523, Université Lille 1, Villeneuve d'Ascq, France.
Methods Mol Biol. 2018;1729:203-231. doi: 10.1007/978-1-4939-7577-8_18.
We describe two methods for high-resolution fluorescence imaging of the positioning and mobility of E. coli chemoreceptors fused to photoconvertible fluorescent proteins. Chemoreceptors such as Tar and Tsr are transmembrane proteins expressed at high levels (thousands of copies per cell). Together with their cognate cytosolic signaling proteins, they form clusters on the plasma membrane. Theoretical models imply that the size of these clusters is an important parameter for signaling, and recent PALM imaging has revealed a broad distribution of cluster sizes. We describe experimental setups and protocols for PALM imaging in fixed cells with ~10 nm spatial precision, which allows analysis of cluster-size distributions, and localized-photoactivation single-particle tracking (LPA-SPT) in live cells at ~10 ms temporal resolution, which allows for analysis of cluster mobility.
我们描述了两种用于对与光转换荧光蛋白融合的大肠杆菌化学感受器的定位和移动性进行高分辨率荧光成像的方法。诸如Tar和Tsr等化学感受器是高水平表达的跨膜蛋白（每个细胞数千个拷贝）。它们与其同源的胞质信号蛋白一起，在质膜上形成簇。理论模型表明，这些簇的大小是信号传导的一个重要参数，并且最近的光激活定位显微镜（PALM）成像揭示了簇大小的广泛分布。我们描述了用于固定细胞中具有约10 nm空间精度的PALM成像的实验设置和方案，这允许分析簇大小分布，以及用于活细胞中具有约10 ms时间分辨率的局部光激活单粒子追踪（LPA-SPT）的实验设置和方案，这允许分析簇的移动性。
