Fig. 6
RGG-based synthetic organelles in protocells and living cells. Scale bars: 10 µm. a RGG-GFP-RGG forms stable compartments in model cell cytoplasm prepared from Xenopus laevis eggs. b Multiple, specific, SZ2-tagged cargos are simultaneously recruited into SZ1-RGG-RGG droplets in the presence of cytoplasm. Undiluted Xenopus egg cytoplasmic extract was mixed with SZ1-RGG-RGG (5 µM), GFP-SZ2 (1 µM), and RFP-SZ2 (1 µM). c RGG-GFP-RGG protein droplets form in cell-like structures, or protocells. Aqueous phase containing undiluted Xenopus egg cytoplasmic extract mixed with RGG-GFP-RGG protein was emulsified within a continuous mineral oil phase containing surfactant. d Protease-triggered phase separation in protocells. Aqueous phase containing Xenopus egg cytoplasmic extract mixed with 30 µM MBP-RGG-GFP-RGG protein and 0.5 µM TEV protease was encapsulated in emulsions. TEV activity liberates MBP from RGG-GFP-RGG, resulting in triggered formation of RGG-GFP-RGG droplets. e RGG-GFP-RGG forms synthetic membraneless organelles following transfection in multiple human cell lines: HEK293, HeLa, and U2OS. f Recruitment of exogenous cargo into synthetic organelles via SZ1/SZ2 interaction. RFP-SZ2 (cargo) plasmid was co-transfected with SZ1-RGG-GFP-RGG plasmid in HEK293 cells. g TEV expression reverses phase separation and disassembles synthetic organelles in HEK293 cells. RGG-x-GFP-RGG (x = TEV cut site) was co-transfected with RFP-TEV protease. In control experiments using RGG-GFP-RGG that lacks a TEV cut site, droplets form normally and are insensitive to co-expression of RFP-TEV protease
