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Receptor tyrosine kinases (RTKs) are major signaling hubs in metazoans, playing crucial roles in cell proliferation, migration, and differentiation. However, few tools are available to measure the activity of a specific RTK in individual living cells. Here, we present pYtags, a modular approach for monitoring the activity of a user-defined RTK by live-cell microscopy. pYtags consist of an RTK modified with a tyrosine activation motif that, when phosphorylated, recruits a fluorescently labeled tandem SH2 domain with high specificity. We show that pYtags enable the monitoring of a specific RTK on seconds-to-minutes time scales and across subcellular and multicellular length scales. Using a pYtag biosensor for epidermal growth factor receptor (EGFR), we quantitatively characterize how signaling dynamics vary with the identity and dose of activating ligand. We show that orthogonal pYtags can be used to monitor the dynamics of EGFR and ErbB2 activity in the same cell, revealing distinct phases of activation for each RTK. The specificity and modularity of pYtags open the door to robust biosensors of multiple tyrosine kinases and may enable engineering of synthetic receptors with orthogonal response programs. 

Cells receive enormous amounts of information from their environment. How they act on this information—by migrating, expressing genes, or relaying signals to other cells—comprises much of the regulatory and self-organizational complexity found across biology. The “parts list” involved in cell signaling is generally well established, but how do these parts work together to decode signals and produce appropriate responses? This fundamental question is increasingly being addressed with optogenetic tools: light-sensitive proteins that enable biologists to manipulate the interaction, localization, and activity state of proteins with high spatial and temporal precision. In this review, we summarize how optogenetics is being used in the pursuit of an answer to this question, outlining the current suite of optogenetic tools available to the researcher and calling attention to studies that increase our understanding of and improve our ability to engineer biology.