Phase separation of Arabidopsis EMB1579 controls transcription, mRNA splicing, and development
Fig 2
EMB1579 forms dynamic bodies in the nucleus of Arabidopsis root cells and undergoes rapid phase separation in vitro.
(A) Micrographs of Arabidopsis primary root from pCAMBIA1301-proEMB1579::gEMB1579-TGFP; emb1579 plants. Root cells were revealed by staining with PI. Bar = 50 μm. (B) Micrographs of Arabidopsis root cells showing the subcellular localization of EMB1579. Nuclei were stained with Hoechst 33342. Bar = 5 μm. (C) Time-lapse images of root cells from pCAMBIA1301-proEMB1579::gEMB1579-TGFP; emb1579. White arrows indicate the time points that EMB1579 starts to disappear from the nucleus, whereas black arrows indicate the time points that EMB1579 starts to appear in the nucleus. Bar = 5 μm. (D) EMB1579 forms bodies within the nucleus of Arabidopsis root cells from the meristem zone and the elongation zone. Bar = 5 μm. (E) Histograms of size distribution of EMB1579 bodies in the nucleus from cells in the root meristem zone and the elongation zone. More than 200 bodies were measured in at least 12 nuclei. The average values are presented as mean ± s.e.m. Numerical data underlying this panel are available in S1 Data. (F) Images of an Arabidopsis nucleus before and after bleaching. The photobleached region is boxed. Bar = 5 μm. (G) Plot of fluorescence intensity before and after photobleaching. The blue curve represents the average value of fluorescence intensity of 18 bodies from 10 seedlings. All data are presented as mean ± s.e.m. Numerical data underlying this panel are available in S1 Data. (H) Images of an Arabidopsis nucleus showing the fusion of two EMB1579 bodies. Boxed region indicates two bodies that undergo fusion. Bar = 5 μm. (I) Analysis of the intrinsic disorder tendency and hydropathicity of the full-length EMB1579 protein. The intrinsic disorder (red line) was analyzed with IUPred2A. The scores are assigned between 0 and 1, and a score above 0.5 indicates disorder. Three long stretches of disordered regions are shaded in light blue. The hydropathicity score (blue line) was determined with ExPASy, which used the Kyte-Doolittle scale of amino acid hydropathicity with a sliding window size of 21. The scores were normalized from 0 to 1. A score above 0.5 indicates hydrophobicity. Numerical data underlying this panel are available in S1 Data. (J) Purified recombinant EMB1579 protein. The asterisks indicate EMB1579 protein bands. The lower band might be a degradation product of the full-length EMB1579. The original pictures are available in S1 Raw Images. (K) EMB1579 condensates visualized by DIC optics. The right panels show time-lapse images of the boxed region in the left panel. Condensates fuse to form a single large condensate. The arrows indicate the fusion events. Bars = 10 μm. (L) Phase diagram of EMB1579 condensate formation at the indicated protein and salt concentrations. The diagram was plotted after scoring the optically resolvable droplets at different protein/KCl concentrations. Red circles indicate the formation of condensates; black squares indicate no formation of condensates. (M) Images of EMB1579 droplets labeled with Oregon Green before and after photobleaching. Bar = 2 μm. (N) Plot of the changes in fluorescence intensity during the FRAP experiment. The blue curve represents the average value of fluorescence intensity from 15 bodies. Values are presented as mean ± s.e.m. Numerical data underlying this panel are available in S1 Data. (O) Half-FRAP of EMB1579 condensates. The left panel shows images of EMB1579 condensates labeled with Oregon Green before and after photobleaching during the half-bleach experiment. The right panel shows the kymograph analysis for the unbleached and bleached regions. Bar = 1 μm. EMB1579, EMBRYO DEFECTIVE 1579; DIC, differential interference contrast; FRAP, fluorescence recovery after photobleaching; TGFP, tandem copies of enhanced green fluorescent protein; PI, propidium iodide; WT, wild type.