ArticleNature protocols2018
Live-cell measurements of kinase activity in single cells using translocation reporters.
Article in Nature protocols, 2018. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 75 papers.
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Who cites it
75 citing papers in PubMed.
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- Synthetic budding morphogenesis by optogenetic receptor tyrosine kinase signaling.bioRxiv : the preprint server for biology · 2026Article
- Real-Time Visualization of Isoform-Specific RAF-KRAS Interactions in Living Cells Using FRET-BRET Hybrid Biosensors.Advanced science (Weinheim, Baden-Wurttemberg, Germany) · 2026Article
- Signal control during tissue regeneration in adult animals.Nature reviews. Molecular cell biology · 2026Review
- Temozolomide increases the generation of cell heterogeneity in ERK activity in glioma cells.Journal of molecular medicine (Berlin, Germany) · 2026Article
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- Perturbing H-NS function reveals roles in restricting virulence heterogeneity and pathogen adaptation.bioRxiv : the preprint server for biology · 2026Article
- Integrated single cell transcriptomics analysis for unraveling heterogeneity and plasticity of root cells for sustainable and regenerative agriculture.Frontiers in plant science · 2026Review
- Coiled-coil interactions drive the ectopic condensation of overexpressed MAD1 to promote mitotic slippage in cancer cells.iScience · 2025Article
- Recent advances in spying on cell signaling with fluorescent biosensors.Current opinion in cell biology · 2025Review
- A 2D cell segmentation protocol for monitoring multiple STAT signaling pathways by fluorescence microscopy.STAR protocols · 2025Article
- Responsive DNA artificial cells for contact and behavior regulation of mammalian cells.Nature communications · 2025Article
- Genetically encoded protein oscillators for FM streaming of single-cell data.bioRxiv : the preprint server for biology · 2025Article
- A Kolmogorov metric embedding for live cell microscopy signaling patterns.Bioinformatics advances · 2025Article
- A concise guide to fluorescent cell cycle reporters for live-cell imaging.Frontiers in cell and developmental biology · 2025Review
15 more citing papers are in PubMed but not listed here.
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Authors and funding
7 authors.
Funding
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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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.