pYtags enable spatiotemporal measurements of receptor tyrosine kinase signaling in living cells

Payam E. Farahani, Xiaoyu Yang, Emily V. Mesev, Kaylan A. Fomby, Ellen H. Brumbaugh-Reed, Caleb J. Bashor, Celeste M. Nelson, Jared E. Toettcher

Research output: Contribution to journalArticlepeer-review


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.

Original languageEnglish (US)
Article numbere82863
StatePublished - 2023

All Science Journal Classification (ASJC) codes

  • General Immunology and Microbiology
  • General Biochemistry, Genetics and Molecular Biology
  • General Neuroscience


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