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Protein Dynamics in Living Cells01:19

Protein Dynamics in Living Cells

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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...
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Related Experiment Video

Updated: Aug 28, 2025

Real Time Monitoring of Intracellular Bile Acid Dynamics Using a Genetically Encoded FRET-based Bile Acid Sensor
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Fluorescent Biosensor for Measuring Ras Activity in Living Cells.

Ryan Weeks1,2, Xin Zhou1, Tina L Yuan3

  • 1Department of Pharmacology, University of California, San Diego, La Jolla, California 92093, United States.

Journal of the American Chemical Society
|September 19, 2022
PubMed
Summary

Scientists developed RasAR, a new tool to measure Ras protein activity in living cells. This reporter helps understand Ras regulation in cancer and evaluate new cancer drug effectiveness.

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Area of Science:

  • Molecular Biology
  • Cell Biology
  • Biochemistry

Background:

  • Ras GTPases are key regulators of cell growth, and their dysregulation is common in cancer.
  • Targeting Ras is a major goal in cancer therapy, but requires understanding its activity in living cells.
  • Current methods for measuring Ras activity in cells are limited.

Purpose of the Study:

  • To develop a novel reporter for quantitative, real-time measurement of Ras activity in living cells.
  • To investigate the spatiotemporal regulation of Ras isoforms and identify upstream regulators.
  • To assess the dynamics of Ras inhibition by covalent KRasG12C inhibitors.

Main Methods:

  • Development of a ratiometric Ras activity reporter (RasAR) for live-cell imaging.
  • Utilizing subcellularly targeted RasAR to study Ras isoform regulation.
  • Monitoring Ras activity in real-time following treatment with KRasG12C inhibitors.

Main Results:

  • RasAR enables quantitative, high-resolution measurement of Ras activity across all primary isoforms.
  • Src kinase was identified as an upstream inhibitor of HRas.
  • RasAR revealed persistent Ras activity for hours even with KRasG12C inhibitors like Sotorasib.

Conclusions:

  • RasAR is a versatile tool for interrogating Ras activity in live cells.
  • The reporter facilitates understanding of Ras biology and development of Ras-targeted cancer therapies.
  • Findings highlight the need for strategies to overcome residual Ras activity during inhibitor treatment.