Fig 4. RABV P alone phase separates in crowded environments.

A) Schematic representation of the organization of RABV P construct used. P contains an N-terminal domain (NTD) that associates with N in the N0-P complex and with the viral polymerase, two intrinsically disordered domains (IDD1 and IDD2), a dimerization domain (DD) and a C-terminal domain (CTD) that binds N associated with RNA. The cysteine at position 261 was replaced by a serine so that the resulting protein has only one cysteine residue in position 297. A StrepTagII was used to purify the protein. B) SDS PAGE analysis of purified P proteins. C) P-Strep protein expressed in E. coli at 10 μM concentration (320 μg/ml) (resp. full-length P protein expressed in Hi-5 cells at 5 μM) was incubated in absence or presence of 5% PEG 8000 in 125 mM NaCl, 20 mM Tris-HCl pH7.5. Droplets were imaged by differential interference contrast (DIC). Scale bar: 10 μm. D) P-Strep droplets were observed by fluorescence microscopy at different P concentrations in absence or presence of different molecular crowders in 50 mM NaCl, 20 mM Tris-HCl pH7.5. The mix contained 25 nM of P protein covalently labelled with Cy3 maleimide probe. Scale bars: 10 μm. E) Box plot representation of P-Strep droplets aspect ratios (formed at 5 μM with 5% PEG8000 as in D). Sample size: n = 936. The cross on the boxplot indicates the mean. F) Left panel: P-Strep droplets were observed by fluorescence microscopy at different concentrations in presence of 5% PEG 8000 and increasing concentrations of NaCl in 20 mM Tris-HCl pH 7.5 buffer. The mix contained 25 nM of P protein covalently labelled with Cy3 maleimide probe. Scale bars: 10 μm. Right panel: phase diagram at various P-Strep concentrations and with increasing concentrations of NaCl in presence of 5% PEG 8000. G) Fusion between P-Strep droplets ([P] = 20 μM in the presence of 3% PEG 8000, 125 mM NaCl, 20 mM Tris-HCl pH 7.5) imaged by time-lapse video-microscopy. Images were extracted from S2 Movie at the indicated times. Scale bar: 2 μm. H) Droplets formed by P-Strep at the concentration of 20 μM (50 nM of fluorescent P protein) in the presence of 5% PEG 8000 in 125 mM NaCl, 20 mM Tris-HCl pH 7.5 buffer were photobleached at different time-points post-mixing (less than 1 h or more than 3 h). Whole droplets as well as droplets subvolumes were photobleached. Images were acquired on a spinning disk microscope. For the plots on the right, FRAP data were background-corrected and normalized to the minimum and maximum intensity. The mean is shown with error bars representing the SD. Scale bars: 5 μm.