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Transcriptional readout of neuronal activity via an engineered Ca2+-activated protease

Significance Molecular reporters that “remember” the activity history of a cell and give a corresponding signal that can be read out by imaging, sequencing, or other modalities are valuable tools for the study of neural networks. Here, we develop a reporter that drives stable expression of any transgene (such as a fluorescent protein) in response to cellular calcium activity during a user-defined time window. As calcium is a universal proxy for neural activity, our reporter provides a stable “snapshot” of neural activity which can be used for subsequent imaging, analysis, or manipulation of behavior-associated neural networks. A core feature of our reporter is an engineered calcium-activated protease, which cuts a protein sequence to reveal a transcription factor when cellular calcium levels rise. Molecular integrators, in contrast to real-time indicators, convert transient cellular events into stable signals that can be exploited for imaging, selection, molecular characterization, or cellular manipulation. Many integrators, however, are designed as complex multicomponent circuits that have limited robustness, especially at high, low, or nonstoichiometric protein expression levels. Here, we report a simplified design of the calcium and light dual integrator FLARE. Single-chain FLARE (scFLARE) is a single polypeptide chain that incorporates a transcription factor, a LOV domain–caged protease cleavage site, and a calcium-activated TEV protease that we designed through structure-guided mutagenesis and screening. We show that scFLARE has greater dynamic range and robustness than first-generation FLARE and can be used in culture as well as in vivo to record patterns of neuronal activation with 10-min temporal resolution.