Fig. 1: The killswitch arrests nucleolar dynamics.
From: Probing condensate microenvironments with a micropeptide killswitch
a, Schematic of fsHMGB1 (top). NoLS, nucleolar localization sequence; fs, frameshifted. Bottom, live-cell fluorescence microscopy images of U2OS cells expressing ectopic mEGFP–HMGB1 proteins and RFP–FIB1. The contour of the nucleus is highlighted with a dashed line. Scale bar, 10 µm. The experiment was repeated independently five times with similar results. b, FRAP analysis of mEGFP–HMGB1 in nucleoli. Data are mean ± s.d. c, Cell viability of U2OS cells expressing mEGFP–HMGB1 proteins. n = 3 biological replicates. Data are mean ± s.d. P values were calculated using two-way analysis of variance (ANOVA) followed by Dunnett’s multiple-comparison test versus fsHMGB1(full length). RLU, relative light units. d, Schematic of the FRAP assay. The proportion of the signal that recovers in the experimental time window is a proxy for the mobile fraction of protein in the bleached condensate. The proportion of the signal that does not recover is a proxy for the immobile fraction of protein in the bleached condensate. e, The amino acid sequences of the killswitch variants in the tested mEGFP–HMGB1 proteins (left). Middle, quantification of the mobile and immobile fractions of mEGFP–HMGB1 proteins in nucleoli. Data are mean ± s.d. Right, mean GFP fluorescence of bleached nucleoli. n is the number of nucleoli tested, from at least two independent experiment series. P values were calculated using two-way ANOVA (FRAP plots) or one-way ANOVA (GFP intensities) followed by Dunnett’s multiple-comparison test versus fsHMGB1(full length). *P < 0.05, ***P < 10–3, ****P < 10−4. Exact P values are listed in the ‘Statistics and reproducibility’ section of the Methods. NS, not significant. f, In vitro droplet formation by purified, recombinant GFP-tagged killswitch peptides. The experiment was repeated independently twice with similar results. Scale bar, 5 µm. g, Quantification of the relative amount of protein in droplets. Data are mean ± s.d. n = 10 images from two independent experiments. csat, saturation concentration; NA, not available.
