A novel biosensor for the spatiotemporal analysis of STING activation during innate immune responses to dsDNA

The cGAS-STING signalling pathway has a central role in the innate immune response to extrinsic and intrinsic sources of cytoplasmic dsDNA. At the core of this pathway is cGAS-dependent production of the intra- and extra-cellular messenger cGAMP, which activates STING and leads to IRF3-dependent expression of cytokines and interferons. Despite its relevance to viral and bacterial infections, cell death, and genome instability, the lack of specific live-cell reporters has precluded spatiotemporal analyses of cGAS-STING signalling. Here, we generate a fluorescent biosensor termed SIRF (STING-IRF3), which reports on the functional interaction between activated STING and IRF3 at the Golgi. We show that cells harbouring SIRF react in a time- and concentration-dependent manner both to STING agonists and to microenvironmental cGAMP. We demonstrate that the new biosensor is suitable for single-cell characterisation of immune responses to HSV-1 infection, mtDNA release upon apoptosis, or other sources of cytoplasmic dsDNA. Furthermore, our results indicate that STING signalling is not activated by ruptured micronuclei, suggesting that other cytosolic pattern recognition receptors underlie the interferon responses to chromosomal instability.