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

Area of Science:

• Molecular Biology
• Cell Biology
• Biophysics

Background:

• In vivo, DNA-binding proteins exist as free or DNA-bound molecules, with bound proteins categorized as specifically or nonspecifically associated.
• Single-molecule tracking (SMT) is a powerful technique for visualizing protein dynamics within living cells.

Purpose of the Study:

• To detail the methodology for preparing Bacillus subtilis cells for SMT experiments.
• To describe the process of recording and curating movie data for SMT analysis.
• To introduce the SMTracker 2.0 software for analyzing protein dynamics and facilitating research by non-expert scientists.

Main Methods:

• Utilizing fluorescent protein-tagged SMT, specifically employing a monomeric yellow fluorescent protein (mNeonGreen) fused to a protein of interest.
• Implementing bleaching-type SMT involving rapid laser-induced bleaching of fluorophore-labeled molecules.
• Recording and analyzing time-lapse movies of Bacillus subtilis cells to capture single-molecule trajectories.

Main Results:

• Demonstrated the ability to track individual fluorescent protein-tagged molecules in live Bacillus subtilis.
• Enabled the analysis of protein diffusion coefficients, dwell times, and subcellular localization patterns.
• Showcased the utility of SMTracker 2.0 in robustly analyzing SMT data for average molecular behavior.

Conclusions:

• SMT provides detailed insights into protein dynamics, including diffusion and binding behaviors, in living cells.
• The described methods and SMTracker 2.0 software democratize the analysis of SMT data for a broader scientific audience.
• This approach facilitates the study of protein dynamics in various cellular contexts, including mutant strains and altered environmental conditions.