Fig. 1: Phase separation of RGG domains on freestanding planar membranes results in liquid-like protein assemblies.

a Left: Schematic of the freestanding planar membrane array system. Inside a PDMS chamber, a transmission electron microscopy (TEM) grid with hexagonal holes is immersed in an aqueous buffer, and freestanding planar membranes are created within multiple hexagonal holes. Right: Cross-section of the freestanding membrane spanning a single hexagonal hole. Once proteins (histidine-tagged) are added to the aqueous buffer in the chamber, they bind to the membrane through histidine-nickel interactions and phase separate into protein-rich and protein-poor phases. b–d Representative images showing spinodal decomposition and the increase in domain size. The elapsed time since protein addition is indicated above each column. Scale bars, 50 µm. e Fusion events between different protein-rich domains on the membrane over time. Scale bar, 5 µm. White arrowheads indicate fusion events (b–e). f Top: Representative images showing fluorescence recovery after photobleaching (FRAP) of a protein-rich domain on the membrane. Bottom: Corresponding FRAP profile. Data are presented as mean values ± SD (n = 3). Gray lines represent each independent recovery curve. Scale bar, 5 µm. g Top: Schematic of four possible cases, from case 1 to 4, when phase-separating proteins are associated with membranes. Bottom: Representative images for each case. Scale bar, 50 µm. h Percentage of individual cases as a function of DGS-Ni-NTA concentration in the membrane and NaCl concentration in the aqueous buffer. More than 130 lipid membranes were analyzed from three independent experiments for each condition. Membrane composition: 75 mol% DOPC, 25 mol% DGS-Ni-NTA (b–d), 85 mol% DOPC, 15 mol% DGS-Ni-NTA (e, f). 0.5 mol% Texas Red-DHPE was added for all. Buffer: 25 mM HEPES, 100 mM NaCl, pH 7.4. 1 µM of his-RGG labeled with Atto 488 was used. Source data are provided as a Source Data file.