title: High-sensitivity measurements of multiple kinase activities in live single cells.
abstract: Increasing evidence has shown that population dynamics are qualitatively different from single-cell behaviors. Reporters to probe dynamic, single-cell behaviors are desirable yet relatively scarce. Here, we describe an easy-to-implement and generalizable technology to generate reporters of kinase activity for individual cells. Our technology converts phosphorylation into a nucleocytoplasmic shuttling event that can be measured by epifluorescence microscopy. Our reporters reproduce kinase activity for multiple types of kinases and allow for calculation of active kinase concentrations via a mathematical model. Using this technology, we made several experimental observations that had previously been technicallyunfeasible, including stimulus-dependent patterns of c-Jun N-terminal kinase (JNK) and nuclear factor kappa B (NF-κB) activation. We also measured JNK, p38, and ERK activities simultaneously, finding that p38 regulates the peak number, but not the intensity, of ERK fluctuations. Our approach opens the possibility of analyzing a wide range of kinase-mediated processes in individual cells.
authors.affiliation: Department of Bioengineering, Stanford University, Stanford, CA 94305, USA. Electronic address: sregot@stanford.edu.
authors.forename: Sergi
authors.initials: S
authors.lastname: Regot
authors.affiliation: Department of Bioengineering, Stanford University, Stanford, CA 94305, USA.
authors.forename: Jacob J
authors.initials: JJ
authors.lastname: Hughey
authors.affiliation: Department of Bioengineering, Stanford University, Stanford, CA 94305, USA.
authors.forename: Bryce T
authors.initials: BT
authors.lastname: Bajar
authors.affiliation: Department of Bioengineering, Stanford University, Stanford, CA 94305, USA.
authors.forename: Silvia
authors.initials: S
authors.lastname: Carrasco
authors.affiliation: Department of Bioengineering, Stanford University, Stanford, CA 94305, USA. Electronic address: mcovert@stanford.edu.
authors.forename: Markus W
authors.initials: MW
authors.lastname: Covert
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015374:Biosensing Techniques / Q000379:methods*; D048031:JNK Mitogen-Activated Protein Kinases / Q000737:chemistry / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010770:Phosphotransferases / Q000378:metabolism*; D016415:Sequence Alignment; D059010:Single-Cell Analysis
chemical_list: D010770:Phosphotransferases; D048031:JNK Mitogen-Activated Protein Kinases