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.
Highlights
With the advent of single-molecule localization microscopy, there is an increasing number of studies reporting the dynamics of single molecules at the millisecond range and at high optical resolution[1]
SMTracker contains 5 different panels (Fig. 1): (i) The import/exploration panel allows the user to interactively explore tracking data from several conditions, movies and cells on a track-by-track basis. (ii) The Gaussian mixture model (GMM) panel analyzes 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 infers the diffusion constants and relative fraction of molecules in each state. (iii) The squared displacement analysis (SQD) panel uses the cumulative probability distribution of squared displacements to estimate the diffusion constants and relative fractions of up to three diffusive states
The GMM and SQD analyses are closely related, each method has strengths and weaknesses in accurately inferring molecular properties under different experimental conditions, e.g. for different magnitudes of the diffusion constant, as shown by benchmarking both methods on computer-simulated single molecule tracking (SMT) data. (iv) The mean-square displacement (MSD) panel determines the mode of diffusion from a fit of the MSD plotted against the time lag between image frames, allowing the discrimination between Brownian, sub- or superdiffusive behavior. (v) The spatial distribution analysis (SDA) panel analyzes the subcellular localization of molecules, summarizing the localization of trajectories in 2D- and 3D-heat maps and in normalized distributions projected in x- and y-orientation
Summary
With the advent of single-molecule localization microscopy, there is an increasing number of studies reporting the dynamics of single molecules at the millisecond range and at high optical resolution[1]. We devised a GUI-based solution to quantify, explore and visualize the molecular kinetics and dynamics of single particles in bacterial cells This program fills a void for tools that provide access to different diffusive states of individual molecules, as often incurred by binding and unbinding to other subcellular structures. The software allows detailed access to single trajectories, to analyze the mode of diffusion via different approaches, the determination of binding kinetics (e.g. of protein-DNA interactions) and the 2-dimensional visualization of subcellular distributions of molecules and their tracks in a normalised cell. (v) The spatial distribution analysis (SDA) panel analyzes the subcellular localization of molecules, summarizing the localization of trajectories in 2D- and 3D-heat maps and in normalized distributions projected in x- and y-orientation This visualization allows distinguishing different localization patterns (cytoplasmic, nucleoid- or membrane-bound) arising from different biological conditions. Using SMTracker, we determined single molecule dynamics of AbrB, and found a surprising clustering of AbrB molecules on the nucleoids that may confer a more general role as chromosome-structuring element
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