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Abstract Single-molecule tracking provides direct real-time measurements of protein dynamics in live cells with high spatiotemporal resolution. In the last decade, this method has been adopted by several labs to understand the dynamics of DNA replication, transcription, telomerase, chromatin remodeling, etc. in a variety of model systems (bacteria, yeast S. cerevisiae, cell lines, embryo). However, it has not been employed for the yeast Schizosaccharomyces pombe, despite being a valuable model system. Here, we present single-molecule tracking datasets for chromatin-bound histone H3 (Hht1) in live and fixed cells and freely diffusing GFP in S. pombe nuclei to benchmark the diffusion parameters (diffusion coefficient, mean squared displacement, fraction of bound molecules, residence time). These parameters will be used to differentiate chromatin-bound molecules from unbound (free) molecules of any protein. It will be a valuable resource for any lab that starts employing this method for studying protein dynamics. This dataset can also be used to validate the performance of various tracking software and as a training dataset for machine learning-based automated tracking.

Single-Molecule Tracking (SMT) is a powerful method to quantify protein dynamics in live cells. Recently, we have established a data analysis pipeline for estimating various biophysical parameters (mean squared displacement, diffusion coefficient, bound fraction, residence time, jump distances, jump angles, and track statistics) from the single-molecule time-lapse movies acquired from yeast Saccharomyces cerevisiae. We acquired the time-lapse movies using different time intervals (i.e. 15 ms, 200 ms, and 1000 ms) to extract the diffusion parameters (from 15 ms time interval movies) and residence time (from 200 ms and 1000 ms time interval movies). We tracked the single molecules from these movies using three MATLAB-based software packages (MatlabTrack, TrackIT, DiaTrack (Sojourner, and Spot-On)) to quantify various biophysical parameters. In this article, we have quantified the biophysical parameters of chromatin-bound histone H3 (Hht1), labeled using JF646 HaloTag Ligand (HTL), and shared a few raw time-lapse SMT movies for the same. Histone H3 is a chromatin-bound protein and it serves as a benchmark for the stably bound molecules for the SMT experiments. Hence, this dataset can be used by various researchers to quantify the biophysical parameters of chromatin-bound molecules (Histone H3). Any newly developed tracking software can use this dataset to validate the accuracy of its tracking algorithms.

Summary: Single-molecule tracking (SMT) is a powerful approach to quantify the biophysical parameters of protein dynamics in live cells. Here, we describe a protocol for SMT in live cells of the budding yeast Saccharomyces cerevisiae. We detail how to genetically engineer yeast strains for SMT, how to set up image acquisition parameters, and how different software programs can be used to quantify a variety of biophysical parameters such as diffusion coefficient, residence time, bound fraction, jump angles, and target-search parameters.For complete details on the use and execution of this protocol, please refer to Mehta et al. 1 and Ball et al..2 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.