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Live-cell measurements of kinase activity in single cells using translocation reporters

Abstract

Although kinases are important regulators of many cellular processes, measuring their activity in live cells remains challenging. We have developed kinase translocation reporters (KTRs), which enable multiplexed measurements of the dynamics of kinase activity at a single-cell level. These KTRs are composed of an engineered construct in which a kinase substrate is fused to a bipartite nuclear localization signal (bNLS) and nuclear export signal (NES), as well as to a fluorescent protein for microscopy-based detection of its localization. The negative charge introduced by phosphorylation of the substrate is used to directly modulate nuclear import and export, thereby regulating the reporter's distribution between the cytoplasm and nucleus. The relative cytoplasmic versus nuclear fluorescence of the KTR construct (the C/N ratio) is used as a proxy for the kinase activity in living, single cells. Multiple KTRs can be studied in the same cell by fusing them to different fluorescent proteins. Here, we present a protocol to execute and analyze live-cell microscopy experiments using KTRs. We describe strategies for development of new KTRs and procedures for lentiviral expression of KTRs in a cell line of choice. Cells are then plated in a 96-well plate, from which multichannel fluorescent images are acquired with automated time-lapse microscopy. We provide detailed guidance for a computational analysis and parameterization pipeline. The entire procedure, from virus production to data analysis, can be completed in 10 d.

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Figure 1: Schematic representation of KTRs.
Figure 2: General schematic showing how live-cell measurements of kinase activity are performed using kinase translocation reporters (KTRs).
Figure 3: NES and bNLS consensus sequences.
Figure 4: Schematic outlining image and downstream analysis workflow.

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Acknowledgements

We thank the members of Covert lab for advice and critical commentary on the manuscript, in particular K. Lane for supplying the images of primary macrophages in Supplementary Figure 1. We gratefully acknowledge funding from several sources, including a Distinguished Investigator award and a Discovery Center grant from the Paul Allen Family Foundation, as well as an NIH Pioneer Award (5DP1LM01150-05) to M.W.C., a Human Frontier Science Program (HFSP) postdoctoral fellowship (LT000529/ 2012-L) to S.R., the Nakajima Foundation Scholarship to T.K., a DOE CSGF grant (DE-FG02-97ER25308) to D.N.M., and a Systems Biology Center grant (P50 GM107615).

Author information

Affiliations

Authors

Contributions

S.R. developed the reporter strategy and designed the experiments. T.K. and D.N.M. developed the computational pipelines and prepared the protocols. J.J.H. and S.A. developed the general workflow for the modeling. T.K., S.J., and M.W.C. wrote the manuscript, with input from all authors.

Corresponding authors

Correspondence to Sergi Regot or Markus W Covert.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 KTR translocation in different cell lines.

Time relative to stimulation is shown on each images.

(A) HeLa cells expressing JNK KTR were stimulated with anisomycin 50 ng/ml.

(B) HEK293 cells expressing JNK KTR were stimulated with anisomycin 50 ng/ml.

(C) Raw 264.7 cells expressing JNK KTR were stimulated with LPS 100 ng/ml.

(D) PC-12 cells expressing ERK KTR were stimulated with NGF 25 ng/ml.

(E) Primary Bone Marrow-derived Macrophages (BMDMs) isolated from mice expressing JNK KTR were stimulated with LPS 100 ng/ml.

Supplementary Figure 2 KTR translocation and the estimated active JNK concentration.

(A) Heatmap illustrating the experimental C/N ratio, simulated C/N ratio and predicted active JNK concentration upon IL-1β stimulation. Each row denotes a color coded individual time course of an indicated property for a single cell. This is the final output from the jupyter notebook demonstration. (B) The population average traces of the C/N ratio (red), active JNK concentration (blue) and total phosphorylated KTR concentration (green). Note that the C/N ratio is acquired by direct analysis of the KTR image data, while the JNK and KTR concentrations are calculated by further analysis using a computational mechanistic model of KTR phosphorylation and dephosphorylation as well as nuclear or cytoplasmic translocation. The concentrations are normalized by minimum and maximum values.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1 and 2, Supplementary Methods, Supplementary Tables 1–3, Supplementary Notes 1 and 2, and Supplementary Data. (PDF 8160 kb)

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Kudo, T., Jeknić, S., Macklin, D. et al. Live-cell measurements of kinase activity in single cells using translocation reporters. Nat Protoc 13, 155–169 (2018). https://doi.org/10.1038/nprot.2017.128

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