Zentrum für Synthetische Mikrobiologie (SYNMIKRO), AG Graumann, Marburg, Germany.
Methods Mol Biol. 2024;2819:189-223. doi: 10.1007/978-1-0716-3930-6_10.
All DNA-binding proteins in vivo exist as a population of freely diffusing molecules and of DNA-bound molecules. The molecules bound to DNA can be split into specifically/tightly and nonspecifically bound proteins. Single-molecule tracking (SMT) is a method allowing to visualize protein dynamics in living cells, revealing their behavior in terms of mode of motion, diffusion coefficient/speed, change of dwell times, and unveiling preferred subcellular sites of dwelling. Bleaching-type SMT or fluorescent protein-tagged SMT involves rapid laser-induced bleaching of most fluorophore-labeled molecules. The remaining single fluorescent proteins are then continuously tracked. The trajectories of several fluorescent molecules per cell for a population of cells are analyzed and combined to permit a robust analysis of average behavior of single molecules in live cells, including analyses of protein dynamics in mutant cells or cells exposed to changes in environmental conditions.In this chapter, we describe the preparation of Bacillus subtilis cells, the recording of movies of those cells expressing a monomeric variant of a yellow fluorescent protein (mNeonGreen) fused to a protein of choice, and the subsequent curation of the movie data including the statistical analysis of the protein dynamics. We present a short overview of the analysis program SMTracker 2.0, highlighting its ability to analyze SMT data by non-expert scientists.
所有体内的 DNA 结合蛋白都以自由扩散分子和 DNA 结合分子的形式存在。与 DNA 结合的分子可以分为特异性/紧密结合的蛋白质和非特异性结合的蛋白质。单分子追踪（SMT）是一种能够在活细胞中可视化蛋白质动力学的方法，揭示其运动模式、扩散系数/速度、停留时间变化等方面的行为，并揭示其优先的亚细胞栖息部位。漂白型 SMT 或荧光蛋白标记的 SMT 涉及快速激光诱导的大多数荧光标记分子的漂白。然后，连续追踪剩余的单个荧光蛋白。对单个细胞中的几个荧光分子的轨迹进行分析，并将其组合起来，以允许对活细胞中单分子的平均行为进行稳健的分析，包括对突变细胞或暴露于环境条件变化的细胞中的蛋白质动力学的分析。在这一章中，我们描述了枯草芽孢杆菌细胞的制备，表达与所选蛋白质融合的单体变体黄色荧光蛋白（mNeonGreen）的细胞的电影记录，以及随后对电影数据的管理，包括蛋白质动力学的统计分析。我们简要概述了 SMTracker 2.0 分析程序，强调了它能够由非专家科学家分析 SMT 数据的能力。
