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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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Dynamic assessment of PI4P metabolism using split GFP based sensors.

Samantha K A Manuel^1,2, Erika Bilenka¹, Monica Gupta¹

¹Department of Biochemistry and Molecular Biology, Miller School of Medicine, University of Miami, Coral Gables, FL, USA.

Scientific Reports

|August 21, 2025

Summary

Researchers developed a novel split GFP sensor for PI4P, offering high sensitivity for studying cellular metabolism over longer periods. This tool aids in understanding PI4P

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Area of Science:

Cellular Biology
Biochemistry
Molecular Biology

Background:

Phosphoinositides (PIXPs) are crucial for cellular signaling and metabolism.
Existing biosensors primarily target rapid cellular signaling events.
Tools for studying PI4P, particularly over extended metabolic timescales, are limited.

Purpose of the Study:

To develop and compare novel biosensors for phosphoinositide 4-phosphate (PI4P).
To address the limitations of existing tools for studying PI4P dynamics in cellular metabolism.
To create a sensitive and versatile sensor for PI4P applicable to various experimental setups.

Main Methods:

Development of a split GFP (sGFP) based sensor for PI4P (sGFP(PI4P)).
Comparison with a dimerization dependent RFP (ddRFP) based PI4P sensor.
Validation using inhibitors of cholesterol transport and PI4P synthesis in proliferation assays.
Assessment of sensor performance via FACS and in-cell lysate analysis.

Main Results:

The ddRFP(PI4P) sensor showed rapid response but lacked sensitivity at low expression levels.
The novel sGFP(PI4P) sensor demonstrated high sensitivity and spatial information at low expression levels.
sGFP(PI4P) offers accessible readout options (FACS, lysate) suitable for longer-term metabolic studies.
The utility of sGFP(PI4P) was confirmed in proliferation assays using specific inhibitors.

Conclusions:

The sGFP(PI4P) sensor represents a significant advancement for studying PI4P in cellular metabolism.
This new tool provides high sensitivity and versatility, overcoming limitations of previous PI4P sensors.
sGFP(PI4P) enables deeper investigation into PI4P's role in cellular functions beyond rapid signaling.