Quantifying transcription factor binding dynamics at the single-molecule level in live cells.
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.
Laboratory of Receptor Biology and Gene Expression, Building 41, 41 Library Drive, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Diego M
DM
Presman
Laboratory of Receptor Biology and Gene Expression, Building 41, 41 Library Drive, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
David A
DA
Ball
Laboratory of Receptor Biology and Gene Expression, Building 41, 41 Library Drive, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Ville
V
Paakinaho
Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, VA 20147, USA.
Jonathan B
JB
Grimm
Janelia Research Campus, Howard Hughes Medical Institute, 19700 Helix Drive, Ashburn, VA 20147, USA.
Luke D
LD
Lavis
Laboratory of Receptor Biology and Gene Expression, Building 41, 41 Library Drive, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Tatiana S
TS
Karpova
Laboratory of Receptor Biology and Gene Expression, Building 41, 41 Library Drive, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. Electronic address: hagerg@exchange.nih.gov.
Gordon L
GL
Hager
D000818:Animals; D000944:Antigens, Differentiation, B-Lymphocyte / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D004847:Epithelial Cells / Q000378:metabolism* / Q000648:ultrastructure; D005786:Gene Expression Regulation; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D056945:Hep G2 Cells; D000949:Histocompatibility Antigens Class II / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000706:statistics & numerical data*; D007700:Kinetics; D061986:MCF-7 Cells; D051379:Mice; D011485:Protein Binding; D011965:Receptors, Glucocorticoid / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D000072760:Single Molecule Imaging / Q000379:methods*
D000944:Antigens, Differentiation, B-Lymphocyte; D000949:Histocompatibility Antigens Class II; D011965:Receptors, Glucocorticoid; D011993:Recombinant Fusion Proteins; C045084:invariant chain; D049452:Green Fluorescent Proteins
DNA binding; Dynamics; Fluorescence microscopy; Glucocorticoid receptor; Single-molecule tracking; Transcription factor