Fig. 1

Predicting assembly mode of membraneless organelles by a FRET-based crowding sensor. a Representative images of mEGFP-HP1α and mEGFP-SRSF2 from the FRAP experiment. Scale bar, 5 μm. b Quantification of FRAP data for mEGFP-HP1α and mEGFP-SRSF2. The bleaching event occurred at t = 10 s. N = 12 cells per group. Data are represented as mean ± SEM. c, d SEC-MALS analysis of the purified mEGFP-HP1α protein (c) and mEGFP-SRSF2 protein (d). e Phase separation assay of 10 μM purified mEGFP-HP1α and mEGFP-SRSF2 protein in 10% PEG8000 (w/v) in vitro. Scale bar, 10 μm for HP1α and 2 μm for mEGFP-SRSF2. f Separation of HEK293T cell lysates by gel-filtration chromatography. Products were collected separately in 1.5-ml tubes from 6 ml to 19 ml. g SDS-PAGE analysis of separated HEK293T cell lysates stained with Coomassie brilliant blue and Western blot analysis of HP1α and SRSF2. h Schematic diagram of the structure and working principle of the sensor used to detect the degree of crowding. i Comparison of the YFP/CFP emission ratios of the H2B-fused Crowding sensor between cells at interphase and prometaphase. N = 80 cells per group from four independent experiments. Data are represented as mean ± SEM. j A color-coded representation of the emission ratio in (i). Scale bar, 5 μm. k Comparison of the YFP/CFP emission ratio of the H2B-fused Crowding sensor and optimized sensor to detect the degree of crowding containing random amino acid sequences of different lengths. N ≥ 60 cells per group from four independent experiments. Data are represented as mean ± SEM. l A color-coded representation of the emission ratio of the H2B-fused CYR090 sensor in (k). Scale bar, 5 μm. m, n Fluorescence emission spectra upon titration of the prokaryotically expressed and purified Crowding sensor (m) and CYR090 sensor (n) with Ficoll PM 70. The fluorescence emission spectra in (m) and (n) were normalized according to their donor fluorescence. o FRET ratio upon titration with Ficoll PM 70. N = 3 independent experiments. Data are represented as mean ± SD. The emission ratios in (i), (j), (k), (l) and (o) were normalized according to the corresponding control group. p Schematic diagram of YFP/CFP emission ratio image splitting method for dense phase and dilute phase. q Comparison of the degree of HP1α crowding in heterochromatin (dense phase) and nucleoplasm (dilute phase). A representative image of HP1α-CYR090 sensor fluorescence intensity (YFP) and corresponding color-coded image of the overall and separate YFP/CFP emission ratio. Scale bar, 5 μm. Higher ratio indicates higher degree of crowding. r Two curves in different colors showing relative trends in YFP fluorescence intensity (gray) and YFP/CFP emission ratio (orange) in (q). s Comparison of YFP/CFP emission ratio of HP1α-CYR090 sensor in different regions. N = 20 cells per group. Data are represented as mean ± SEM. t, u Comparison of the degree of SRSF2 crowding in nuclear speckles (dense phase) and the nucleoplasm (dilute phase). The images in (t) and (u) are similar to those in (q) and (r), except that the SRSF2-CYR090 sensor was used. v Comparison of YFP/CFP emission ratio of SRSF2-CYR090 sensor in different regions. N = 40 cells per group from two independent experiments. Data are represented as mean ± SEM. w Oligomeric molecule-mediated aggregation model (top), and polymeric molecule-mediated consolidation model (bottom)