A new class of biosensors for detecting signaling dynamics without live-cell microscopy

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PROJECT SUMMARY Every cell exists in a complex and changing environment. To deal with their complex surroundings, cells have evolved diverse systems to sense external cues (such as nutrients, stresses, or communication molecules from neighboring cells) and store this information in an internal representation. Yet the details of this internal representation are still mysterious. What patterns of protein activity do cells use to represent information about their environment? How are these patterns generated, and what fates do they control? Growth factor signaling is an important model system for understanding principles of cell signaling, where activation of cell surface receptors is coupled to activation of a membrane-localized protein Ras, the kinase Erk and various downstream genes. Growth factor signaling plays crucial roles in embryo development (where growth factors trigger cells to differentiate), adult tissue regeneration (where it controls various aspects of wound healing), and cancer (where mutations in growth factor signaling genes drive uncontrolled growth and tumorigenesis). Owing to its importance, growth factor signaling is intensely studied at increasingly high resolution. Biosensors are now available to monitor Erk activity in real-time and in living cells, enabling the experimentalist to trace fluctuations in growth factor signaling from one cell to another across a tissue and in different cellular contexts. Studies using Erk biosensors have revealed previously-unappreciated complexity in growth factor signaling activity. Instead of simply turning from off to on upon stimulation, Erk may pulse on and off rapidly in cells, or even exhibit traveling waves of activity that propagate across entire swaths of tissue. Yet the field does not yet understand whether Erk pulses lead cells to adopt distinct functional states, nor how the pulses themselves are generated by biochemical networks inside or between cells. This state of affairs is not unique to Erk: pulses have also been widely observed in many other signaling pathways and are generally poorly understood. The current proposal aims to provide new tools for studying signaling pulses to aid their study in cultured cells and in living animals. We have invented a new technology – a prototype gene circuit that acts as an Erk “pulse detector” – which will allow researchers to study Erk pulses without live imaging. This technology addresses an important need: currently, pulses can only be detected by high resolution microscopy of living cells, limiting contexts where they can be studied. Here, we propose to develop our imaging-free biosensor for rapid deployment in mouse and human cell lines, to expand its design to other pathways and signaling dynamics, and to establish transgenic animals expressing the biosensor for studies in many tissues where microscopy is difficult or impossible to perform. Successful completion of this work will produce a new class of biosensors to shed light on complex signaling with potential impact on human disease.