Single-molecule tracking of transcription protein dynamics in living cells: seeing is believing, but what are we seeing?

Abstract

A universe of transcription factors (TFs), cofactors, as well as chromatin remodeling and modifying enzymes combine or compete on chromatin to control transcription. Measuring quantitatively how these proteins dynamically interact is required in order to formulate models with predictive ability to elucidate transcription control mechanisms. Single molecule tracking (SMT) provides a powerful tool towards this goal: it is a fluorescence microscopy approach that measures the location and mobility of individual TF molecules, as well as their rates of association with and dissociation from chromatin in the physiological context of the living cell. Here we review SMT principles, and discuss key TF properties uncovered by live-cell SMT, such as fast turnover (seconds), and formation of clusters that locally increase activity.

Figure 1

(a) Stages of the transcription cycle. Transcription occurs in a series of steps, starting with the binding of sequence-specific Transcription Factors (TFs), that recruit co-activators, chromatin remodelers (for instance depositing histone variants such as H2A.Z, marked with Z) and eventually members of the Pre-Initiation Complex (PIC). Upon formation of the complete PIC containing Pol II, transcription starts, marked by Ser5 phosphorylation of the CTD of Pol II. Upon release from the promoter-proximal pause (marked by phosphorylation of the CTD at Ser2), productive elongation of RNA transcripts. (b) Single-Molecule Tracking workflow. A Transcription Factor (TF) of interest is fused to the self-labeling tag HaloTag. Cells containing the fusion protein are labeled with a HaloTag ligand conjugated to a photoactivatable fluorophore, and unbound ligands are washed out. Upon a low intensity pulse of blue light excitation, ~1–20 TF molecules turn fluorescent (example image shows individual molecules of H2B-HaloTag labeled with PA-JF646; cell nucleus outlined in yellow). The motion of those molecules is subsequently recorded until they leave the focal plane or photobleach; a new cycle is initiated by another pulse of blue light that turns on a fresh subset of molecules. Subsequent analysis of molecule trajectories (x,y positions over time) enables classifying TF molecules into different mobility populations.