Using GRAM Domain-Based Biosensors to Monitor Accessible Cholesterol Distribution in Live Cells.

Area of Science:

• Cell Biology
• Biochemistry
• Molecular Biology

Background:

• Cholesterol is essential for cellular membrane integrity and signaling, with highest concentrations in the plasma membrane (PM).
• Aberrant cholesterol distribution is implicated in cardiovascular and neurological diseases.
• Limited tools exist for visualizing accessible cholesterol in live cells, hindering research.

Purpose of the Study:

• To develop novel biosensors for visualizing accessible cholesterol in live cells.
• To enable monitoring of cholesterol dynamics within the plasma membrane.
• To provide new tools for studying cholesterol distribution and its role in disease.

Main Methods:

• Utilized biosensors based on the GRAM domain of GRAMD1s/Asters (GRAMD1a, GRAMD1b, GRAMD1c).
• Developed two GRAM domain variants (GRAM-H, GRAM-W) with differing cholesterol affinities.
• Coupled biosensors with dimerization-dependent fluorescent protein (ddFP) for flow cytometry analysis.

Main Results:

• Successfully visualized accessible cholesterol dynamics in the plasma membrane using GRAM domain-based biosensors.
• Demonstrated detection of both increases and decreases in accessible PM cholesterol.
• Enabled population-level monitoring of accessible PM cholesterol via flow cytometry.

Conclusions:

• GRAM domain-based biosensors offer a novel method for visualizing accessible cholesterol.
• These tools enhance understanding of cholesterol distribution and transport mechanisms.
• The developed biosensors complement existing techniques and open new avenues for research into cholesterol-related disorders.