SMTracker: a tool for quantitative analysis, exploration and visualization of single-molecule tracking data reveals highly dynamic binding of B. subtilis global repressor AbrB throughout the genome

Abstract

Single-particle (molecule) tracking (SPT/SMT) is a powerful method to study dynamic processes in living cells at high spatial and temporal resolution. Even though SMT is becoming a widely used method in bacterial cell biology, there is no program employing different analytical tools for the quantitative evaluation of tracking data. We developed SMTracker, a MATLAB-based graphical user interface (GUI) for automatically quantifying, visualizing and managing SMT data via five interactive panels, allowing the user to interactively explore tracking data from several conditions, movies and cells on a track-by-track basis. Diffusion constants are calculated a) by a Gaussian mixture model (GMM) panel, analyzing the distribution of positional displacements in x- and y-direction using a multi-state diffusion model (e.g. DNA-bound vs. freely diffusing molecules), and inferring the diffusion constants and relative fraction of molecules in each state, or b) by square displacement analysis (SQD), using the cumulative probability distribution of square displacements to estimate the diffusion constants and relative fractions of up to three diffusive states, or c) through mean-squared displacement (MSD) analyses, allowing the discrimination between Brownian, sub- or superdiffusive behavior. A spatial distribution analysis (SDA) panel analyzes the subcellular localization of molecules, summarizing the localization of trajectories in 2D- heat maps. Using SMTracker, we show that the global transcriptional repressor AbrB performs highly dynamic binding throughout the Bacillus subtilis genome, with short dwell times that indicate high on/off rates in vivo. While about a third of AbrB molecules are in a DNA-bound state, 40% diffuse through the chromosome, and the remaining molecules freely diffuse through the cells. AbrB also forms one or two regions of high intensity binding on the nucleoids, similar to the global gene silencer H-NS in Escherichia coli, indicating that AbrB may also confer a structural function in genome organization.

The Bacillus subtilis dCMP deaminase ComEB acts as a dynamic polar localization factor for ComGA within the competence machinery

“…We employed SMT microscopy (Rosch, Oviedo‐Bocanegra, Fritz, & Graumann, ; Schenk et al, ) to investigate the mobility of ComGA and of ComEB. To this end, we generated fusions of the proteins to mV, which in our hands provides best results when tracking via YFP‐bleaching is employed.…”

BactMAP: An R package for integrating, analyzing and visualizing bacterial microscopy data

“…However, there are more types of analyses possible, some of which we slightly touched; for instance single molecule tracking, cell shape classification, analysis of cell lineages or visualization of localization microscopy. For advanced single molecule tracking, there are already software packages available, which can combine tracking analysis with single-cell outlines from other programs (e.g., iSBatch [Caldas et al, 2015] and SMTracker [Rösch, Oviedo-Bocanegra, Fritz, & Graumann, 2018]).…”

BactMAP: an R package for integrating, analyzing and visualizing bacterial microscopy data

“…For advanced single molecule tracking, there are already software packages available, which can combine tracking analysis with single cell outlines from other programs (e.g. ISBatch (Caldas et al, 2015) and SMTracker (Rösch et al, 2018).…”