Fluorescent protein ticker tape (FPTT): Multiplexed recording of transcriptional dynamics in living cells and in vivo.

Abstract

Recording and real-time imaging of promoter activities are critical for deciphering signaling cross-talk, but technologies for simultaneously capturing multiple transient events in living cells are lacking. Here, we design fluorescent protein-based ticker tapes (FPTT) for multiplexed, scalable, longitudinal recording of single-cell physiological activities by integrating multispectral monomeric fluorescent proteins with self-assembling protein fibers. FPTT logged dose-dependent, reversible endogenous cFos transcriptional histories in hippocampal neurons at 3-hour resolution over 8 days. We engineered FPTT variants for human nuclear factor κB (NF-κB), Janus kinase/signal transducer and activator of transcription 3 (STAT3), mechanistic target of rapamycin (mTOR), nuclear factor of activated T cells (NFAT), and adenosine 3',5'-monophosphate (cAMP) signaling. This expanded toolset enabled quantification of cFos and NF-κB cross-talk in neurons, tracking of STAT3/cAMP dynamics during mouse liver injury, discovery of unexpected NFAT/STAT3 cross-talk, and characterization of cell cycle-dependent oscillating mTOR dynamics. Last, we achieved simultaneous analysis of four major pathways during T cell activation. FPTT provides a versatile platform to investigate transcriptional histories and signaling interplay, with broad applications in developmental biology and disease modeling.