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SUMMARY Protein clustering is pervasive in cell signaling, yet how signaling from higher-order assemblies differs from simpler forms of molecular organization is still poorly understood. We present an optogenetic approach to switch between oligomers and heterodimers with a single point mutation. We apply this system to study signaling from the kinase Zap70 and its substrate linker for activation of T cells (LAT), proteins that normally form membrane-localized condensates during T cell activation. We find that fibroblasts expressing synthetic Zap70:LAT clusters activate downstream signaling, whereas one-to-one heterodimers do not. We provide evidence that clusters harbor a positive feedback loop among Zap70, LAT, and Src-family kinases that binds phosphorylated LAT and further activates Zap70. Finally, we extend our optogenetic approach to the native T cell signaling context, where light-induced LAT clustering is sufficient to drive a calcium response. Our study reveals a specific signaling function for protein clusters and identifies a biochemical circuit that robustly senses protein oligomerization state., In brief Dine et al. study how different modes of molecular organization contribute to cell signaling using the kinase Zap70 and its substrate LAT as a model system. Optogenetic manipulation reveals that LAT:Zap70 clusters—but not dimers—trigger potent signaling via localized positive feedback among LAT, Zap70, and Src-family kinases., Graphical abstract

Paradigm-shifting research has revealed that certain intrinsically disordered proteins (IDPs) phase separate into spherical liquid droplets that act as membrane-less organelles in living cells. Here, we investigated protease activity as a strategy for triggering controlled droplet disassembly without changing temperature, salt concentration, or other variables that are difficult to manipulate in vivo. We selected a naturally occurring phase-separating IDP, the RGG domain from LAF-1, and produced a variant consisting of two RGG domains in tandem. This RGG tandem protein exhibits enhanced phase behavior - that is, it self-assembles into liquid droplets at lower protein concentration and higher temperature as compared to a single RGG domain. We incorporated a thrombin cleavage site between the two RGG domains of this construct and discovered that upon treatment of the RGG tandem droplets with thrombin, the droplet diameter shrank at a rate of 0.5 micrometers per hour, as measured by quantitative optical microscopy. This occurred because the single RGG domains that result upon cleavage of RGG tandem with thrombin have weaker phase behavior and do not phase separate under the same conditions as does RGG tandem. Through additional protein engineering, we generated a variant that releases a fluorescent reporter when the protein droplet disassembles upon treatment with protease. This is a modular platform for enzymatically-triggered disassembly and release of proteins from droplets. We envision applications of this concept in protein drug delivery, such as in release of antimicrobial peptides for wound healing, and in controlling the behavior of membrane-less organelles within cells in an inducible manner.