Quantifying transcription factor binding dynamics at the single-molecule level in live cells

Abstract

Progressive, technological achievements in the quantitative fluorescence microscopy field are allowing researches from many different areas to start unraveling the dynamic intricacies of biological processes inside living cells. From super-resolution microscopy techniques to tracking of individual proteins, fluorescence microscopy is changing our perspective on how the cell works. Fortunately, a growing number of research groups are exploring single-molecule studies in living cells. However, no clear consensus exists on several key aspects of the technique such as image acquisition conditions, or analysis of the obtained data. Here, we describe a detailed approach to perform single-molecule tracking (SMT) of transcription factors in living cells to obtain key binding characteristics, namely their residence time and bound fractions. We discuss different types of fluorophores, labeling density, microscope, cameras, data acquisition, and data analysis. Using the glucocorticoid receptor as a model transcription factor, we compared alternate tags (GFP, mEOS, HaloTag, SNAP-tag, CLIP-tag) for potential multicolor applications. We also examine different methods to extract the dissociation rates and compare them with simulated data. Finally, we discuss several challenges that this exciting technique still faces.

Live‐cell imaging reveals the interplay between transcription factors, nucleosomes, and bursting

“…The biphasic behavior in the semi-log plot indicates two populations of Gal4 with different binding rates (inset, Fig 4D). Multiple distributions have previously been observed for other TFs and have been attributed to nonspecific and specific binding (Chen et al, 2014;Ball et al, 2016;Presman et al, 2017). In agreement with this interpretation, the core histone H3 only showed a longer binding population and no shorter binding population ( Fig EV4B).…”

Single-molecule imaging reveals the interplay between transcription factors, nucleosomes, and transcriptional bursting

“…The biphasic behavior in the semi-log plot indicates two populations of Gal4 with different binding rates (inset, Figure 4D). Multiple distributions have previously been observed for other TFs and have been attributed to non-specific and specific binding (Ball et al, 2016;Chen et al, 2014;Presman et al, 2017). In agreement with this interpretation, the core histone H3 only showed a longer binding population and no shorter binding population ( Figure S4B).…”

Single molecule analysis of lamin dynamics

“…Here, we apply single molecule tracking (SMT) to quantitatively assess lamin and NET dynamics. A hallmark of SMT is its ability to characterize the trajectories of individual molecules with nanometer spatial resolution and millisecond temporal precision in the context of living cells [16,17]. Like other live-cell imaging methods, such as fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP), SMT employs an exogenously expressed protein fused to a fluorophore to measure protein dynamics.…”

“…Like other live-cell imaging methods, such as fluorescence correlation spectroscopy (FCS) and fluorescence recovery after photobleaching (FRAP), SMT employs an exogenously expressed protein fused to a fluorophore to measure protein dynamics. However, SMT offers the advantages of higher spatial resolution, discrimination between heterogeneous populations of molecules in the same cell, and is able to more easily assess the dynamics of relatively immobile molecules [17]. Previous studies have utilized SMT to study the dynamics of mRNAs, chromatin remodelers, and transcription factor binding events, among others [16][17][18][19][20][21][22][23].…”

“…However, SMT offers the advantages of higher spatial resolution, discrimination between heterogeneous populations of molecules in the same cell, and is able to more easily assess the dynamics of relatively immobile molecules [17]. Previous studies have utilized SMT to study the dynamics of mRNAs, chromatin remodelers, and transcription factor binding events, among others [16][17][18][19][20][21][22][23]. We use highly inclined and laminated optical sheet (HILO) illumination, sub-optimal transient transfection of Halotag-fused lamins and NETs, and low labelling concentrations of a Halotag ligand to perform time-lapse imaging of a variety of NE-associated proteins to determine the fractions of bound and unbound molecules as well as dwell times.…”