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Protein Dynamics in Living Cells

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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Related Experiment Video

Updated: Mar 3, 2026

Visualization and Quantification of Endogenous Intra-Organelle Protein Interactions at ER-Mitochondria Contact Sites by Proximity Ligation Assays

Visualization and Quantification of Endogenous Intra-Organelle Protein Interactions at ER-Mitochondria Contact Sites by Proximity Ligation Assays

Published on: October 20, 2023

Imaging interorganelle membrane contact sites using dimerization-dependent fluorescent proteins.

Victoria H Williams¹, Chih-Hsuan Hsu¹, Gregory E Miner¹

¹Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, United States.

Methods in Enzymology

|March 1, 2026

Summary

Membrane contact sites (MCSs) are crucial for cell function but difficult to study. This protocol uses novel Contact-FP biosensors to visualize MCSs in live cells using advanced microscopy techniques.

Keywords:

Biosensors Endoplasmic reticulum Fluorescent proteins Lipid droplets Lysosomes Membrane contact sites Mitochondria Organelles Peroxisomes Plasma membrane

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Last Updated: Mar 3, 2026

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Area of Science:

Cell Biology
Microscopy
Molecular Biology

Background:

Membrane contact sites (MCSs) are vital for organelle function and implicated in diseases.
Studying MCSs is challenging due to their small size (10 nm) and limitations of light microscopy.
Dimerization-dependent fluorescent proteins offer a way to detect protein proximity.

Purpose of the Study:

To present a protocol for studying MCSs in live cells using organelle-targeted dimerization-dependent fluorescent proteins (Contact-FPs).
To enable visualization and analysis of MCSs with confocal or Airyscan microscopy.
To provide a versatile tool for studying single or multiple MCSs and inducing MCS formation.

Main Methods:

Development and application of Contact-FP biosensors targeting specific organelles.
Live-cell imaging using confocal and Airyscan microscopy.
Protocol includes transfection, imaging, and data analysis guidance.

Main Results:

Demonstration of Contact-FP utility for visualizing MCSs in live U-2 OS cells.
Protocol provides steps for studying single MCSs, multiple MCSs, and inducing MCSs.
Troubleshooting advice for transfection, imaging, and analysis is included.

Conclusions:

Contact-FP biosensors provide a powerful method for studying MCSs in live cells.
The protocol is adaptable for various cell types and research questions regarding organelle interactions.
This tool enhances the study of fundamental cellular processes and disease mechanisms.