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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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Related Experiment Video

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Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations
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A live-imaging protocol for tracking receptor dynamics in single cells.

Yibin Huang1, Toshimasa Takahashi1, Herbert Gaisano2

  • 1Department of Geriatric and General Medicine, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan.

STAR Protocols
|May 5, 2022
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Summary

This study presents a super-resolution microscopy method to track two membrane receptors simultaneously in single cells. The protocol enables detailed analysis of receptor dynamics and cellular responses to ligands.

Keywords:
Cell BiologyCell cultureMicroscopySignal TransductionSingle Cell

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Area of Science:

  • Cell Biology
  • Microscopy Techniques
  • Molecular Imaging

Background:

  • Adjacent membrane receptors exhibit varied cellular responses upon ligand stimulation.
  • Understanding these differential dynamics is crucial for deciphering cellular signaling pathways.
  • Existing methods may lack the resolution or multiplexing capability to track multiple receptors concurrently.

Purpose of the Study:

  • To develop and validate a super-resolution microscopy protocol for simultaneous tracking of two distinct membrane-bound receptors in live single cells.
  • To provide a detailed methodology encompassing transfection, imaging, and data analysis for receptor dynamics studies.
  • To demonstrate the protocol's applicability to investigate receptor endocytosis.

Main Methods:

  • Super-resolution microscopy (e.g., STORM, PALM) for high-resolution imaging of membrane receptors.
  • Electroporation for efficient cell transfection and receptor labeling.
  • Single-cell imaging techniques to capture receptor dynamics in real-time.
  • Establishment of a comprehensive data analysis pipeline for quantitative assessment of receptor behavior.

Main Results:

  • Successful implementation of a protocol for dual-receptor tracking in single cells using super-resolution microscopy.
  • Demonstration of the protocol's utility in analyzing the endocytosis dynamics of LOX-1 and AT1 receptors in CHO-K1 cells.
  • Validation of the method's potential for studying various membrane receptors across different cell lines.

Conclusions:

  • The developed super-resolution microscopy protocol offers a powerful tool for dissecting the complex dynamics of multiple membrane receptors.
  • This methodology facilitates a deeper understanding of receptor-ligand interactions and downstream cellular signaling.
  • The protocol is adaptable and broadly applicable to diverse cell types and receptor systems in cell biology research.