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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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Updated: May 31, 2025

A Protocol for Real-time 3D Single Particle Tracking

A Protocol for Real-time 3D Single Particle Tracking

Published on: January 3, 2018

Single-particle tracking reveals heterogeneous PIEZO1 diffusion.

Alan T Ly¹, J Alfredo Freites², Gabriella A Bertaccini¹

¹Department of Physiology & Biophysics, UC Irvine, Irvine, California; Sue and Bill Gross Stem Cell Research Center, UC Irvine, Irvine, California.

Biophysical Journal

|January 23, 2025

Summary

This summary is machine-generated.

Mechanically activated ion channel PIEZO1 exhibits heterogeneous diffusion in live cells. Membrane composition and activation state significantly influence PIEZO1 mobility, revealing insights into its plasma membrane dynamics.

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Area of Science:

Cell Biology
Biophysics
Membrane Protein Dynamics

Background:

The mechanically activated ion channel PIEZO1 plays a crucial role in various physiological processes.
PIEZO1 is activated by mechanical cues and its mobility in the plasma membrane is essential for its function.

Purpose of the Study:

To investigate the diffusion dynamics of endogenous PIEZO1 in live cells.
To determine how plasma membrane composition and channel activation affect PIEZO1 mobility.

Main Methods:

Single-particle tracking (SPT) of tdTomato-tagged PIEZO1 using total internal reflection fluorescence microscopy.
Manipulation of membrane cholesterol levels and addition of margaric acid.
Treatment with PIEZO1 agonist Yoda1 and inhibitor GsMTx-4.

Main Results:

PIEZO1 displayed heterogeneous diffusion, categorized as mobile and immobile subpopulations.
Cholesterol depletion and Yoda1 treatment increased PIEZO1 mobility.
GsMTx-4 treatment and cholesterol supplementation decreased PIEZO1 mobility.
Mobile PIEZO1 puncta exhibited anomalous subdiffusion.

Conclusions:

Plasma membrane composition and PIEZO1 activation state critically regulate its diffusion.
Understanding PIEZO1 mobility is key to elucidating its role in cellular mechanotransduction.
These findings provide a foundation for studying how cellular processes influence PIEZO1 activity.