Fluorescent protein-based ticker tapes for multiplexed recordings of transcriptional histories in single cells in culture and in vivo

Abstract

Recording and imaging of promoter activities in real time is critical for deciphering cellular states and dynamic signaling crosstalk, but technologies capable of simultaneously capturing multiple transient events in living cells are lacking. Here, we custom-design fluorescent protein-based ticker tapes (FPTT) for multiplexed and longitudinal recording of physiological activities in single cells. FPTT integrates multi-spectral monomeric fluorescent proteins with self-assembling protein fibers, enabling massively parallel analysis of signaling dynamics under varying cellular conditions. Using FPTT, we were able to log dose-dependent and reversible transcriptional histories of endogenous cFos signaling in primary hippocampus neurons at a 3-hour temporal resolution via biological timestamps and an extended recording time of over 8 days. Furthermore, we expanded the imaging toolset by engineering genetically encoded FPTT variants for human NF{kappa}B, JAK/STAT3, mTOR, NFAT and cAMP signaling, allowing for the quantification of potential crosstalk between cFos and NF{kappa}B pathways in neurons, multiplexed recording of STAT3- and cAMP-specific promoters during drug-induced liver injury in mice, as well as simultaneous analysis of up to four major signaling pathways involved in experimental T-cell activation. This platform advances single-cell analysis by providing a versatile tool to investigate transcriptional histories and signaling interplay across diverse biological contexts, with broad applications in developmental biology and disease modeling.