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
