Dynamic assessment of PI4P metabolism using split GFP based sensors

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.