Protein supersaturation powers innate immune signaling.

Innate immunity protects us in youth but turns against us as we age. The reason for this tradeoff is unclear. Seeking a thermodynamic basis, we focused on death fold domains (DFDs), whose ordered polymerization has been stoichiometrically linked to innate immune signal amplification. We hypothesized that soluble ensembles of DFDs function as phase change batteries that store energy via supersaturation and subsequently release it through nucleated polymerization. Using imaging and FRET-based cytometry to characterize the phase behaviors of all 109 human DFDs, we found that the hubs of innate immune signaling networks encode large nucleation barriers that are intrinsically insulated from cross-pathway activation. We showed via optogenetics that supersaturation drives signal amplification and that the inflammasome is constitutively supersaturated in vivo. Our findings reveal that the soluble "inactive" states of adaptor DFDs function as essential, yet impermanent, kinetic barriers to inflammatory cell death, suggesting a thermodynamic driving force for aging.

DAmFRET measures saturating concentrations and toxicities of protein phase transitions in vivo.

Protein phase transitions broadly govern protein function and dysfunction. However, analyzing the consequences of specific phase transitions in cells is hindered by the low throughput and limited resolution of fluorescence microscopy, and this problem is compounded for proteins with complex phase behavior such as those implicated in age-associated neurodegenerative diseases. As one solution to this problem, we incorporated an orthogonally fluorescence proxy of total protein expression to adjust for effective cell volume differences in a flow cytometric assay for protein self-association-Distributed Amphifluoric FRET (DAmFRET)-thereby allowing the intracellular saturating concentrations of different proteins to be precisely compared in single experiments. We further found that the effective cell volume decreased in cells experiencing proteotoxicity, which provided a simple way to assign toxicity to specific phases of ectopically expressed proteins.