Figure 1. Single-nucleosome imaging in... | Rockefeller University Press

Figure 1.

$Figure 1. Single-nucleosome imaging in living RPE-1 cells. (A) Expression of H2B-Halo in RPE-1 cells was confirmed by Western blotting with αH2B antibody (lane 1). In lane 2, parental RPE-1 cells show no H2B-Halo signals. (B) RPE-1 cells expressing H2B-Halo fluorescently labeled with TMR-HaloTag ligand (center). The left panel is DNA stained with DAPI. The merged image (DNA, blue; H2B-Halo, red) is shown at right. Putative inactive X chromosome, which is highly condensed, is marked with an arrowhead. Note that the TMR labeling pattern is very similar to the DNA staining one. (C) Scheme of oblique illumination microscopy. This illumination laser (green) can excite fluorescent molecules within a limited thin optical layer (red) of the nucleus and reduce background noise. (D) A small fraction of H2B-Halo was fluorescently labeled with TMR-HaloTag ligand (red). The labeled nucleosome movements can be tracked at super-resolution. (E) A single-nucleosome (H2B-Halo-TMR) image of a living RPE-1 nucleus after background subtraction. (F) Representative three trajectories of the tracked single nucleosomes. (G) MSD plots (±SD among cells) of single nucleosomes in living interphase (black) and FA-fixed (red) RPE-1 cells from 0.05 to 0.5 s. For comparison, MSD plots of single nucleosomes labeled with PA-mCherry (H2B-PA-mCherry) in living interphase RPE-1 cells (gray) are also shown. For each sample, n = 20–25 cells. N.S. (not significant, P = 0.47) and ***, P < 0.0001 (P = 1.5 × 10−11) for H2B-Halo versus FA-fixed cells by the Kolmogorov–Smirnov test. (H) MSD plots (±SD among cells) of single nucleosomes in living (black) and FA-fixed (red) RPE-1 cells in a longer tracking time range from 0.05 to 3 s. For each sample, n = 20 cells. In the MSD analyses for single nucleosomes, the originally calculated MSD was in two dimensions. To obtain 3D values, the original values of MSD were multiplied by 1.5 (4 to 6 Dt). The plots were fitted as a subdiffusive curve: MSD = 0.018t0.28 in a living cell; MSD = 0.003t0.01 in an FA-fixed cell. Rc (estimated radius of constraint of the nucleosome motion), 141 ± 19.2 nm (mean ± SD) in living cells; 56 ± 6.7 nm in FA-fixed cells. Their Rc values are significantly different: P = 2.2 × 10−10 by the Kolmogorov-Smirnov test.$

Single-nucleosome imaging in living RPE-1 cells. (A) Expression of H2B-Halo in RPE-1 cells was confirmed by Western blotting with αH2B antibody (lane 1). In lane 2, parental RPE-1 cells show no H2B-Halo signals. (B) RPE-1 cells expressing H2B-Halo fluorescently labeled with TMR-HaloTag ligand (center). The left panel is DNA stained with DAPI. The merged image (DNA, blue; H2B-Halo, red) is shown at right. Putative inactive X chromosome, which is highly condensed, is marked with an arrowhead. Note that the TMR labeling pattern is very similar to the DNA staining one. (C) Scheme of oblique illumination microscopy. This illumination laser (green) can excite fluorescent molecules within a limited thin optical layer (red) of the nucleus and reduce background noise. (D) A small fraction of H2B-Halo was fluorescently labeled with TMR-HaloTag ligand (red). The labeled nucleosome movements can be tracked at super-resolution. (E) A single-nucleosome (H2B-Halo-TMR) image of a living RPE-1 nucleus after background subtraction. (F) Representative three trajectories of the tracked single nucleosomes. (G) MSD plots (±SD among cells) of single nucleosomes in living interphase (black) and FA-fixed (red) RPE-1 cells from 0.05 to 0.5 s. For comparison, MSD plots of single nucleosomes labeled with PA-mCherry (H2B-PA-mCherry) in living interphase RPE-1 cells (gray) are also shown. For each sample, n = 20–25 cells. N.S. (not significant, P = 0.47) and ***, P < 0.0001 (P = 1.5 × 10⁻¹¹) for H2B-Halo versus FA-fixed cells by the Kolmogorov–Smirnov test. (H) MSD plots (±SD among cells) of single nucleosomes in living (black) and FA-fixed (red) RPE-1 cells in a longer tracking time range from 0.05 to 3 s. For each sample, n = 20 cells. In the MSD analyses for single nucleosomes, the originally calculated MSD was in two dimensions. To obtain 3D values, the original values of MSD were multiplied by 1.5 (4 to 6 Dt). The plots were fitted as a subdiffusive curve: MSD = 0.018t^0.28 in a living cell; MSD = 0.003t^0.01 in an FA-fixed cell. Rc (estimated radius of constraint of the nucleosome motion), 141 ± 19.2 nm (mean ± SD) in living cells; 56 ± 6.7 nm in FA-fixed cells. Their Rc values are significantly different: P = 2.2 × 10⁻¹⁰ by the Kolmogorov-Smirnov test.