Schuster BS , Reed EH , Parthasarathy R , Jahnke CN , Caldwell RM , Bermudez JG , Ramage H , Good MC , Hammer DA .

F32 GM119430 NIGMS NIH HHS , T32 GM007229 NIGMS NIH HHS , F32GM119430 U.S. Department of Health & Human Services | National Institutes of Health (NIH), DE-SC0007063 U.S. Department of Energy (DOE)

	Fig. 1. Phase separation of RGG-based IDP constructs. a Illustration of four-cell C. elegans embryo. LAF-1 is present in P granules, which contribute to germline specification. b Domain organization of LAF-1, RGG, RGG-RGG, and RGG-RGG-RGG. c Representative turbidity measurements show temperature-dependent phase behavior of RGG, RGG-RGG, and RGG-RGG-RGG at protein concentrations of 0.2 mg/mL (approximately 12 μM RGG domain concentration) in 150 mM NaCl buffer, pH 7.5. d Phase diagram of RGG-RGG as a function of salt and protein concentrations. Green markers indicate conditions at which optically resolvable droplets were observed. Inset: microscopy image of phase-separated RGG-RGG protein droplets. Scale bar: 10 µm. e Schematic illustrating design goals (i) inducible phase separation and (ii) cargo recruitment and triggered release
	Fig. 2. Protease-triggered assembly and disassembly of RGG-based protein droplets. a Schematic of protease-mediated disassembly of RGG-RGG droplets through reduction in valency. b Compared to untreated, the phase transition temperature of TEV protease-treated RGG-x-RGG (x = TEV cut site) is markedly reduced, matching that of single RGG. c Microscopy of TEV-triggered droplet dissolution. Time points 0, 20, 40, and 60 min after TEV addition (150 nM). Scale bar: 10 µm. d Analysis of droplet dissolution from time-lapse imaging: TEV cleavage rapidly reverses phase separation of RGG-x-RGG. Droplets formed by RGG-RGG lacking a cut site remain intact after adding TEV. TEV concentration: 150 nM. Representative individual traces (thin red lines) and population average (thick red line) for RGG-x-RGG, compared to population average for RGG-RGG droplets (thick gray line). Averages are of >750 droplets. e Protease treatment alters phase behavior of RGG-x-RGG-y-RGG (x = TEV protease cut site, y = HRV3C protease cut site). Treatment with TEV reduces phase transition temperature, however protein remains phase separated at room temperature. Upon treatment with both TEV and HRV3C, phase separation requires temperatures < 15 °C, consistent with the phase behavior of single RGG domain. f AND gate regulatory behavior of RGG-x-RGG-y-RGG revealed by optical microscopy. Upon treatment with TEV only, protein compartments remain intact and phase separated. Upon TEV treatment and addition of HRV3C protease, phase separation is reversed and droplets dissolve. TEV and HRV3C protease concentrations: 0.5 µM. Scale bar: 10 µm. g Schematic of protease-mediated droplet assembly by removal of a solubility-enhancing tag, MBP, from MBP-RGG-RGG. h Droplet assembly triggered by HRV3C protease monitored by time-lapse microscopy. HRV3C protease: 1 µM. Scale bar: 10 µm. i Analysis of droplet assembly from time-lapse videos. Traces of five representative individual droplets, along with population average (thick purple line) of >200 droplets. j Spectrophotometric assay of the kinetics of droplet assembly. Addition of HRV3C protease at t = 0 causes droplet assembly and a rapid increase in turbidity; the control, lacking protease, shows no increase in turbidity. For all experiments, initial concentrations of RGG-based constructs were 6 μMÂ
	Fig. 3. Permeability of RGG-RGG droplets to small molecules and macromolecules. a Schematic illustrating permeability of protein droplets to exogenous molecules: a molecule (red) diffusing in solution may become enriched in the droplet phase (left), partition equally between the two phases (middle), or be excluded from the droplets (right). b Rhodamine enriches in the droplets, whereas rhodamine-labeled dextrans exhibit size-dependent partitioning—larger molecules permeate the protein droplets less than smaller ones. Dextran 4.4 kDa enriches, while 10 kDa modestly enriches, and 70 kDa is largely excluded. c Partitioning of folded proteins into RGG-RGG droplets. None are strongly enriched and some are partially excluded from the RGG-RGG droplet phase. Intensity scale is the same for all micrographs. Scale bars: 10 µm
	Fig. 4. Targeted recruitment and enrichment of cargo proteins in RGG-based compartments. a Schematic of cargo recruitment into RGG-RGG liquid droplets. b Three strategies for recruitment of model cargo, RFP, that is normally excluded from RGG-RGG droplets: (i) recruitment via a single RGG domain, (ii) recruitment via two RGG domains, and (iii) recruitment via a high-affinity SYNZIP (SZ)-binding pair, in which SZ1 is attached to phase-separating RGG-RGG and its binding partner SZ2 is attached to RFP. Domain schematics and fluorescence micrographs of cargo enrichment are shown. Cargo protein concentration: 1 μM. RGG-RGG or SZ1-RGG-RGG concentration: 6 μM. c Quantification of cargo enrichment. Fluorescence intensity line scans of representative droplets show weak recruitment with a single RGG domain, vs. strong and uniform recruitment by two RGG domains or by SZ1/SZ2 binding. d Simultaneous recruitment of multiple cargo proteins, GFP-SZ2 and RFP-SZ2, into SZ1-RGG-RGG droplets. Merged image shows co-recruitment (yellow). Scale bars: 10 µm
	Fig. 5. Triggered cargo release from membraneless compartments. a Approach 1—protease-mediated release of covalently attached cargo concomitant with droplet dissolution. RGG-cargo-RGG construct; cargo is flanked by thrombin protease cleavage sites. b After thrombin treatment (25 nM) of RGG-GFP-RGG, droplets shrank and released the cargo, GFP, into solution. Thick curve: average droplet diameter vs. time for >400 droplets; thin traces: individual droplet shrinking. Inset below: micrographs 0.5, 1.5, and 2.5 h after addition of protease. Insert above: MALDI-TOF shows full-length cargo (GFP), along with the single RGG domains, released upon thrombin treatment. c Approach 2—protease-mediated release of tethered cargo; compartments remain intact. Cargo-SZ2 recruited into SZ1-RGG-RGG droplets. Cargo and SZ2 separated by TEV cut site. d Near-complete release of RFP cargo 8 h after treatment with TEV protease (0.5 μM) to remove SZ2 recruitment tag. SZ1-RGG-RGG droplets remain intact. e Kinetics of dual cargo release from droplets after TEV protease treatment to remove recruitment tag (average of >200 droplets; individual plots shown in Supplementary Fig. 5D). Cargos: GFP-SZ2 and RFP-SZ2 (1 μM each). f Two-step, multi-cargo, controlled release. Mixture of cargo2-SZ2 and SZ1-RGG-cargo1-RGG. Cargo 1 (GFP) flanked by thrombin cleavage sites; cargo 2 (RFP) and SZ2 separated by TEV cut site. g Initially, compartments contain both cargos, with colocalized green and red fluorescence. Upon treatment with TEV, RFP is released and red fluorescence is lost; protein droplets remain intact and display green fluorescence. Subsequent thrombin treatment liberates the GFP and disassembles the droplets. In all experiments, 5–10 μM RGG-RGG constructs are used. Scale bars: 10 µm
	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

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