Figure 2: Single-molecule analysis of TetR-Atto647N nuclear exploration.
From: Probing the target search of DNA-binding proteins in mammalian cells using TetR as model searcher
(a) Top Left: fluorescence image of NLS-LacI-YFP showing the binding locus position (white dashed circle) in the cell nucleus (the white continuous line indicates the nuclear envelope contour). Bottom left: individual fluorescent spots of microinjected TetR-Atto647N molecules. Top right: representation of individual TetR-Atto647N trajectories superimposed to the bright field image of the cell nucleus. Bottom right: positions of the quasi-immobile molecules at nonspecific sites (blue spots) and at the target locus (yellow spots). Scale bar, 5 μm. (b) From top to bottom: histograms of the instantaneous diffusion coefficient DInst for TetR-Atto647N in basal conditions (without Dox, N=10 cells, n=682 trajectories); in the presence of 2.5 μg ml−1 Dox (N=8 cells, n=623 trajectories); when TetR-Atto647N was co-injected with 10 × molar excess of tetO oligos (N=4 cells, n=572 trajectories); and on co-injection with 1,000 × molar excess of unlabelled TetRs (N=3 cells, n=460 trajectories). The colour bars indicate the fraction of proteins in the fast (red), intermediate (green) and slow (blue) population, numerical quantification is reported in Supplementary Note 4. The images on the right show the occupancy of the target locus by NLS-LacI-YFP (green) and TetR-Atto647N (red). Scale bar, 2 μm. (c) Individual MSD versus time curves for TetR-Atto647N. Red curves correspond to rapidly free diffusing proteins, green curves to proteins belonging to the intermediate population and blue curves to quasi-immobile molecules. Black lines are guide-to-eye representing the MSD for a diffusion coefficient of 10 (continuous line), 1 (dashed line) and 0.1 μm2 s−1 (dotted line).
