Journal: Nature Communications
Article Title: Chromatin organization drives the search mechanism of nuclear factors
doi: 10.1038/s41467-023-42133-5
Figure Lengend Snippet: a paSMT is carried out using highly inclined laminated optical sheet (HILO) microscopy (top left) by labelling the endogenous HaloTag-p53 with the photoactivatable dye PA-JF 549 (bottom left). Movies, acquired at a framerate of 100 fps highlight quasi-immobile chromatin-bound molecules (cyan arrowhead) and diffusing (purple arrowhead) ones (max proj = maximal projection over the entire movie; cyan dotted line indicates the cell nucleus, scale bar: 5 µm). b We use vbSPT to classify track segments into bound and diffusing components, and then focus on diffusing molecules, by computing diffusional anisotropy, by calculating the distributions of angles \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\theta$$\end{document} θ between consecutive jumps, and the fold-anisotropy metric, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f}_{180/0}$$\end{document} f 180 / 0 , calculated as the probability of observing a backward displacement \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$p(150^\circ \le \theta \le 210^\circ )$$\end{document} p ( 15 0 ∘ ≤ θ ≤ 21 0 ∘ ) over the probability of observing a forward displacement \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$p(-30^\circ \le \theta \le 30^\circ )$$\end{document} p ( − 3 0 ∘ ≤ θ ≤ 3 0 ∘ ) . c Different NFs display different diffusional anisotropy, with factors poorly localized in DNA-dense regions displaying lower anisotropy than factors enriched in DNA-dense regions. d Fold-anisotropy metric \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f}_{180/0}$$\end{document} f 180 / 0 as function of the distance run by the molecules. p53, CTCF, and H2B display high diffusional anisotropy at a spatial scale of ~100–150 nm, a signature of transient trapping of these molecules in traps of similar size ( n cells = 30, 30, 29, 14, 31, n angles = 59470, 62813, 180414, 26052, 26566 for HaloTag, p565, p53, CTCF, and Histone H2B respectively, error bars: s.e.m. estimated through boot-strapping). e Analysis of diffusional anisotropy in our SMT/mSIM data allows us to identify that the highest diffusional anisotropy occurs for molecules with slow instantaneous diffusion coefficients in regions at high chromatin density (same data as in Fig. ). Source data are provided as a Source Data file.
Article Snippet: The antibodies employed in this study were: mouse monoclonal anti-p53 DO-1 (Santa Cruz Biotechnology, cat. sc-126; 1:3000 dilution, incubated 1 h at RT), rabbit monoclonal anti-p21 (Abcam, cat. ab109520; 1:1000 dilution, incubated overnight at 4 °C), rabbit monoclonal anti-GAPDH (Abcam, cat. ab128915; 1:50,000 dilution, incubated 1 h at RT), rabbit monoclonal anti-NF-κB p65 (Cell Signaling cat. D14E12 XP®; dilution 1:1000), rabbit polyclonal anti-Histone H2B (Abcam cat. ab1790, dilution 1:5000), mouse monoclonal anti-HaloTag (Promega G921A, dilution 1:1000), mouse monoclonal anti-vinculin (Thermo-Fisher, cat. MA5-11690; 1:4000 dilution, incubated 1 h at RT).
Techniques: Microscopy, Diffusion-based Assay
![a paSMT is carried out using highly inclined laminated optical sheet (HILO) microscopy (top left) by labelling the endogenous HaloTag-p53 with the photoactivatable dye PA-JF 549 (bottom left). Movies, acquired at a framerate of 100 fps highlight quasi-immobile chromatin-bound molecules (cyan arrowhead) and diffusing (purple arrowhead) ones (max proj = maximal projection over the entire movie; cyan dotted line indicates the cell nucleus, scale bar: 5 µm). b We use vbSPT to classify track segments into bound and diffusing components, and then focus on diffusing molecules, by computing diffusional anisotropy, by calculating the distributions of angles \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\theta$$\end{document} θ between consecutive jumps, and the fold-anisotropy metric, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f}_{180/0}$$\end{document} f 180 / 0 , calculated as the probability of observing a backward displacement \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$p(150^\circ \le \theta \le 210^\circ )$$\end{document} p ( 15 0 ∘ ≤ θ ≤ 21 0 ∘ ) over the probability of observing a forward displacement \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$p(-30^\circ \le \theta \le 30^\circ )$$\end{document} p ( − 3 0 ∘ ≤ θ ≤ 3 0 ∘ ) . c Different NFs display different diffusional anisotropy, with factors poorly localized in DNA-dense regions displaying lower anisotropy than factors enriched in DNA-dense regions. d Fold-anisotropy metric \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${f}_{180/0}$$\end{document} f 180 / 0 as function of the distance run by the molecules. p53, CTCF, and <t>H2B</t> display high diffusional anisotropy at a spatial scale of ~100–150 nm, a signature of transient trapping of these molecules in traps of similar size ( n cells = 30, 30, 29, 14, 31, n angles = 59470, 62813, 180414, 26052, 26566 for HaloTag, p565, p53, CTCF, and Histone H2B respectively, error bars: s.e.m. estimated through boot-strapping). e Analysis of diffusional anisotropy in our SMT/mSIM data allows us to identify that the highest diffusional anisotropy occurs for molecules with slow instantaneous diffusion coefficients in regions at high chromatin density (same data as in Fig. ). Source data are provided as a Source Data file.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5952/pmc10575952/pmc10575952__41467_2023_42133_Fig2_HTML.jpg)