Project Summary/Abstract Biology is powered by the self-organization of protein activities in space and time, allowing cells to build emergent behaviors like motility and information-processing out of biochemical reactions. In multicellular systems, proper execution of these single-cell behaviors is critical for processes like development, immune surveillance, and wound healing. As a result, many human diseases have their origin in the dysregulation of protein activity dynamics, including cancer and autoimmune disorders. There is thus a pressing need to understand both the normal operation of the cell’s dynamic protein-based hardware and how it fails in diseased contexts. The proposed research describes an innovative strategy for installing “cellular radio” circuits into cells that generate a protein-based frequency-modulatable (FM) barcoded signal. This signal can then be used for visualizing, probing, and perturbing the protein activity dynamics of single cells in any complex biological setting. Our approach is enabled by our successful implementation of a genetically-encoded orthogonal patterning circuit (MinDE) in human cells that can produce an unprecedented breadth of cell-scale spatiotemporal protein dynamics and patterns. The fast oscillations of MinDE circuits generate a unique single-cell FM-barcoded fluorescent signal that can be locked on to and spectrally separated from other overlapping cells using frequency- domain image processing tools we have developed based on Fourier, Wavelet and Hilbert Transforms. Using protein-engineering and synthetic biology, we will develop a general platform for designing MinDE circuits that can be connected to any dynamic protein activity in the cell. This will allow for MinDE circuits that can read out and broadcast multiple protein activities simultaneously on a cell’s unique FM-barcoded signal, enabling us to unambiguously track how the internal state of individual cells changes as multicellular processes evolve; and MinDE circuits that can act as genetically-encoded control signals that perturb the dynamics of any target protein of interest, enabling dynamic profiling of key nodes of cell behavior by microscopy and high-throughput sequencing based assays. We will design and apply specific MinDE circuits to investigate oncogenic signaling dynamics and aberrant information processing in cancer cells and tumor organoids, asking how different upstream oncogenic driver mutations corrupt downstream signaling dynamics through ERK, mTOR, and PKA kinases. In parallel, we will generate MinDE circuits that stimulate ERK, mTOR, or PKA signaling at different timescales to define how temporal constraints on signal transmission are corrupted in different oncogenic backgrounds and their impact on tumor organoid development. While the applications in this proposal focus on oncogenic signaling, our platform is easily applied to any dynamic protein activity of interest. Our work will thus establish a new paradigm for understanding and engineering dynamic protein activities in biological systems, providing new insights into basic and translational biology with high potential for therapeutic applications.

Project Narrative Many human diseases arise from dysregulation of protein activity dynamics that drive single-cell behaviors underpinning critical multicellular functions. The proposed research will develop genetically-encoded “cellular radio stations”: synthetic protein circuits that generate a unique frequency-modulatable (FM) barcoded signal in the cell that can be used to unambiguously visualize, probe, and perturb single-cell protein activity dynamics in any complex biological setting. By applying this technology to investigate oncogenic signal processing across cancer cells and tumor organoids, we will advance our understanding of fundamental biology and identify new approaches for the diagnosis and treatment of human disease.