Fig. 2: A BPTAS-causing frameshift alters HMGB1 phase separation in vitro.
From: Aberrant phase separation and nucleolar dysfunction in rare genetic diseases
a, Graph plotting the intrinsic disorder of HMGB1. Red arrowhead shows the position of the BPTAS frameshift. The position of the IDR is highlighted with an orange bar and the position of HMG boxes with blue bars. b, Structures of WT and mutant HMGB1 predicted with AlphaFold2. Colours ranging from blue to orange depict the per-residue measure of local confidence (pLDDT) for the model. c, Representative images from droplet formation assays of eGFP–HMGB1 variants at the indicated concentrations. The experiment was repeated three times, with similar results obtained. d, Quantification of the relative amount of condensed protein at the indicated concentrations. Data displayed as the mean ± s.d. e, Relative fluorescence intensity of the bleached area from eGFP–HMGB1 condensates before and after photobleaching. Data displayed as the mean ± s.d. f, Scheme of co-droplet assays. g, Representative images of eGFP–HMGB1 proteins mixed with preassembled mCherry-labelled MED1-IDR, HP1α or NPM1 droplets. h,i, Quantification of eGFP (h) and 5′ FAM (i) fluorescence intensity in mCherry-labelled MED1-IDR, HP1α and NPM1 droplets mixed with full-length mEGFP–HMGB1 proteins (h) or 5′ FAM–HMGB1-IDR peptides (i). Fold change values between the mean intensities of WT and mutants (Mut.) are indicated above the plot. Median is shown as a line within the boxplot, which spans from the 25th to 75th percentiles. Whiskers depict a 1.5× interquartile range. P values are from two-tailed Welch’s t-test. **P < 1 × 10−2, ***P < 1 × 10−3, ****P < 1 × 10−4. Scale bars, 5 µm (c) and 10 µm (g).
