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Related Concept Videos

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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FRET Imaging of Rho GTPase Activity with Red Fluorescent Protein-Based FRET Pairs.

Bryce T Bajar1, Xinmeng Guan2, Amy Lam3

  • 1Department of Biological Chemistry, Medical Scientist Training Program, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA.

Methods in Molecular Biology (Clifton, N.J.)
|February 11, 2022
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Summary

Fluorescence resonance energy transfer (FRET) enables real-time visualization of kinase activities in living cells. This study details FRET sensor preparation and imaging techniques for RhoA kinase activity.

Keywords:
FLIM-FRETFRETFluorescence lifetimeFluorescent proteinRho GTPaseRhoASensitized emission FRET

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Area of Science:

  • Biophysics
  • Cell Biology
  • Molecular Imaging

Background:

  • Fluorescence resonance energy transfer (FRET) and fluorescent proteins (FPs) are crucial for visualizing biological processes.
  • Advanced microscopy enables high spatiotemporal resolution imaging in living systems.
  • Kinase activities, such as RhoA kinase, are key targets for FRET-based studies.

Purpose of the Study:

  • To describe the preparation of FP-based FRET sensors for RhoA kinase.
  • To detail intensity- and lifetime-based FRET imaging methodologies.
  • To outline post-imaging data analysis for FRET studies.

Main Methods:

  • Expression of Förster or fluorescence resonance energy transfer (FRET) sensors in cells.
  • Utilization of advanced optical microscopy for FRET detection.
  • Measurement of changes in fluorescence intensity, lifetime, and anisotropy.

Main Results:

  • Successful preparation of FRET sensors for RhoA kinase.
  • Demonstration of intensity- and lifetime-based FRET imaging.
  • Established protocols for post-imaging data analysis.

Conclusions:

  • FRET is a powerful tool for real-time, noninvasive visualization of biological processes.
  • FP-based FRET sensors provide high spatiotemporal resolution for studying kinase activities.
  • The described methods facilitate the study of RhoA kinase in living cells.