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Related Concept Videos

Protein Dynamics in Living Cells01:19

Protein Dynamics in Living Cells

Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
Reporter Genes02:11

Reporter Genes

Reporter genes are a type of protein-coding gene that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually produce visually identifiable characteristics like fluorescence and luminescence when expressed along with the gene of interest. Thus, reporter genes “report” the presence or absence of genes of interest in an organism, determine the gene expression pattern, or track the physical location of a DNA segment or protein in the cell.
Commonly used reporter...

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Visualizing Protein Kinase A Activity In Head-fixed Behaving Mice Using In Vivo Two-photon Fluorescence Lifetime Imaging Microscopy
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Visualization of JNK activity dynamics with a genetically encoded fluorescent biosensor.

Matthew Fosbrink1, Nwe-Nwe Aye-Han, Raymond Cheong

  • 1Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Proceedings of the National Academy of Sciences of the United States of America
|March 10, 2010
PubMed
Summary

Researchers developed JNKAR1, a fluorescent biosensor, to track JNK pathway activity in real-time within living cells. This tool reveals JNK signaling

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Area of Science:

  • Cellular signaling and molecular biology
  • Biophysics and biosensor development

Background:

  • The JNK (c-Jun N-terminal kinase) pathway is crucial for cellular responses to stress and cytokines, regulating apoptosis, proliferation, differentiation, and inflammation.
  • Understanding the dynamic information flow and signal processing of the JNK pathway in living cells is essential but challenging.

Purpose of the Study:

  • To engineer a genetically encoded fluorescent protein-based biosensor for detecting endogenous JNK activity in living cells.
  • To characterize the dynamic properties and cellular localization of JNK signaling.

Main Methods:

  • Development of JNKAR1 (JNK activity reporter), a Förster Resonance Energy Transfer (FRET)-based biosensor utilizing fluorescent proteins.
  • Detection of JNK activity in response to stress (ribotoxic, osmotic) and cytokine (TNF-alpha) stimuli in living cells.
  • Quantitative single-cell analysis to assess JNK activity kinetics, localization, and dynamic behaviors.

Main Results:

  • JNKAR1 successfully detected stress- and cytokine-induced JNK activity, showing a 15–30% increase in the yellow-to-cyan emission ratio due to phosphorylation-dependent FRET.
  • JNK activity was observed in the cytoplasm, nucleus, mitochondria, and plasma membrane with rapid kinetics following ribotoxic stress.
  • Anisomycin-induced JNK activity exhibited ultrasensitivity, sustainability, and bimodality, characteristic of bistable systems.

Conclusions:

  • The JNKAR1 biosensor provides a robust tool for real-time monitoring of endogenous JNK activity in various cellular compartments.
  • The study reveals complex dynamic behaviors of the JNK cascade at the single-cell level, including ultrasensitivity and bistability.
  • JNKAR1 lays the foundation for further investigation into JNK signaling properties and cellular responses in live-cell contexts.