Farahani, Payam EYang, XiaoyuMesev, Emily VFomby, Kaylan ABrumbaugh-Reed, Ellen HBashor, Caleb JNelson, Celeste MToettcher, Jared E2023-07-212023-07-212023Farahani, Payam E, Yang, Xiaoyu, Mesev, Emily V, et al.. "pYtags enable spatiotemporal measurements of receptor tyrosine kinase signaling in living cells." <i>eLife,</i> 12, (2023) eLife Sciences Publications Ltd.: https://doi.org/10.7554/eLife.82863.https://hdl.handle.net/1911/114985Receptor 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.engExcept where otherwise noted, this work is licensed under a Creative Commons Attribution (CC BY) license.  Permission to reuse, publish, or reproduce the work beyond the terms of the license or beyond the bounds of Fair Use or other exemptions to copyright law must be obtained from the copyright holder.Journal articleelife-82863-v1https://doi.org/10.7554/eLife.82863