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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...

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Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells
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Subcellular localization-dependent changes in EGFP fluorescence lifetime measured by time-resolved flow cytometry.

Ali Vaziri Gohar1, Ruofan Cao, Patrick Jenkins

  • 1Molecular Biology Program, New Mexico State University, Las Cruces, NM 88003, USA.

Biomedical Optics Express
|September 7, 2013
PubMed
Summary

Time-resolved flow cytometry can track protein localization within cells. This method correlates enhanced green fluorescent protein (EGFP) fluorescence lifetime with subcellular protein distribution, aiding autophagy studies.

Keywords:
(140.3518) Lasers, frequency modulated(170.1530) Cell analysis(170.6920) Time-resolved imaging(260.2510) Fluorescence

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Area of Science:

  • Cell Biology
  • Biophysics
  • Biotechnology

Background:

  • Intracellular protein transport and localization are crucial for protein function.
  • Fluorescent proteins are commonly used to visualize protein dynamics.
  • Traditional methods like fluorescence microscopy are low-throughput for studying protein localization.

Purpose of the Study:

  • To evaluate time-resolved flow cytometry for high-throughput analysis of intracellular protein localization.
  • To correlate the fluorescence lifetime of enhanced green fluorescent protein (EGFP) with subcellular localization of human LC3 protein.
  • To assess the utility of this method in studying autophagy, a cellular process marked by LC3 localization to autophagosomes.

Main Methods:

  • Breast cancer cells expressing native EGFP and EGFP-LC3 fusion proteins were used.
  • Autophagy was induced via amino acid starvation.
  • Time-resolved flow cytometry measured fluorescence intensity and lifetime of EGFP variants (diffuse EGFP, punctate EGFP-LC3, diffuse EGFP-ΔLC3).
  • Confocal microscopy was used for low-throughput verification of EGFP-LC3 localization.

Main Results:

  • Changes in fluorescence lifetime of EGFP correlated with the subcellular localization of EGFP-LC3 fusion proteins.
  • Amino acid starvation induced autophagy-dependent LC3 localization, observable through altered EGFP-LC3 fluorescence.
  • Time-resolved flow cytometry results were comparable to confocal microscopy for verifying localization.

Conclusions:

  • Time-resolved flow cytometry offers a high-throughput approach to monitor intracellular protein localization.
  • Measuring fluorescence lifetime of EGFP fusion proteins is a viable method to assess subcellular protein distribution.
  • This technique provides a valuable tool for studying dynamic cellular processes like autophagy.