Visualization, Quantification, and Statistical Evaluation of Dynamics for DNA-Binding Proteins in Bacteria by Single-Molecule Tracking

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.