Biologically relevant properties of RABV P droplets.

A) Segregation of RNA-Cy5 (10- or 40-A nucleotides) at the concentration of 100 nM into non-fluorescent P droplets (P at 5 μM in presence of 5% PEG 8000 in 125 mM NaCl, 20 mM Tris-HCl pH 7.5) and exclusion of BSA-FITC at the concentration of 250 μg/ml (3.8 μM) from P droplets. Fluorescent RNA and BSA were added when LLPS was induced. Analysis was performed by fluorescence microscopy. Exclusion of BSA-FITC from P droplets was validated by confocal microscopy (right panel). Scale bars: 10 μm. B) Droplets formed by non-fluorescent P at the concentration of 20 μM in the presence of 5% PEG 8000 and 100 nM Cy5-RNAs in 125 mM NaCl, 20 mM Tris-HCl pH 7.5 were photobleached at different time-points post-mixing (less than 1 h or more than 4 h). Whole droplets as well as droplets subvolumes were photobleached. Images were acquired on a spinning disk confocal microscope. Scale bars: 5 μm. For the plots on the right, FRAP data were corrected for background, normalized to the minimum and maximum intensity. The mean is shown with error bars representing the SD. C) P at 5 μM (25 nM of fluorescent P protein) in 50 mM NaCl, 20 mM Tris-HCl pH 7.5 was incubated with either 5% PEG 8000, or 80 ng/μL (6μM) of synthetic 40-A nucleotides RNA, or 80 ng/μL of total RNAs extracted from BSR-T7/5 cells. Presence of droplets was assessed by fluorescence microscopy. Scale bar: 10 μm. D) Fluorescent liposomes (PE/PC/PS = 2/2/1 w/w, 100 μg/ml i.e. ~130μM of lipids containing 0.25% of BODIPY 500/510 C12-HPC) were incubated or not with P at 20 μM concentration (50 nM of fluorescent P protein) in 5% PEG 8000, 125 mM NaCl, 20 mM Tris-HCl pH 7.5 before analysis by confocal microscopy. Scale bars: 10 μm.