24949979

PMC4097317

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.

CDC Public Access

5DP1LM01150-05/DP/NCCDPHP CDC HHS/United States
DP1 LM011510/LM/NLM NIH HHS/United States
DP1 OD006413/OD/NIH HHS/United States
P50 GM107615/GM/NIGMS NIH HHS/United States
P50GM107615/GM/NIGMS NIH HHS/United States
R21 5R21AI104305-02/AI/NIAID NIH HHS/United States
R21 AI104305/AI/NIAID NIH HHS/United States