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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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Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules

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Near-instant surface-selective fluorogenic protein quantification using sulfonated triarylmethane dyes and fluorogen

Qi Yan¹, Brigitte F Schmidt, Lydia A Perkins

¹Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA. bruchez@cmu.edu.

Organic & Biomolecular Chemistry

|December 19, 2014

Summary

This study introduces a novel method for tracking cell surface receptors using a genetically encoded tag and a special dye. This technique enables rapid and precise analysis of dynamic cellular processes like receptor recycling.

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Area of Science:

Cell biology
Molecular signaling
Biochemistry

Background:

G-protein coupled receptor (GPCR) endocytosis and recycling are crucial for cellular signaling.
Existing methods for analyzing these dynamic processes are limited in speed and quantification.

Purpose of the Study:

To develop an improved, rapid, and quantitative method for analyzing GPCR endocytosis and recycling.
To enable precise imaging and analysis of dynamic receptor trafficking events.

Main Methods:

Genetic fusion of a fluorogen activating protein (FAP) to a GPCR.
Utilizing a sulfonated malachite green (MG) analog for selective cell surface labeling.
Employing fluorescence microscopy and flow cytometry for analysis.

Main Results:

The developed dye selectively labels cell surface receptors without crossing the plasma membrane.
High-affinity binding to FAP-GPCR fusion constructs was observed.
Rapid activation of tagged surface receptors within seconds of dye addition.

Conclusions:

The FAP-MG labeling approach provides a powerful tool for quantitative analysis of dynamic receptor endocytosis and recycling.
This method significantly advances the study of GPCR trafficking and signaling.