Visualization of accessible cholesterol using a GRAM domain-based biosensor.

Area of Science:

• Cell Biology
• Biochemistry
• Neuroscience

Background:

• Cholesterol is vital for cell membrane integrity and signaling.
• Dysregulation of cellular cholesterol distribution is linked to diseases, including neurodegenerative disorders.
• Monitoring accessible cholesterol in live cells is challenging due to limited tools.

Purpose of the Study:

• To engineer a sensitive biosensor for visualizing accessible cholesterol in live cells.
• To investigate the regulation of accessible cholesterol levels in various cell types.
• To develop methods for ultrasensitive detection of plasma membrane accessible cholesterol.

Main Methods:

• Engineered a biosensor (GRAM-W) using the GRAM domain of GRAMD1b, a cholesterol-sensing element.
• Visualized accessible cholesterol distribution in real-time across diverse cell types (e.g., human keratinocytes, iPSC-derived neurons).
• Combined GRAM-W with a dimerization-dependent fluorescent protein (ddFP) for enhanced detection.

Main Results:

• Successfully visualized accessible cholesterol distribution in live cells using GRAM-W.
• Demonstrated differential reliance on cholesterol biosynthesis and uptake for maintaining accessible cholesterol.
• Established a strategy for ultrasensitive detection of accessible plasma membrane cholesterol.

Conclusions:

• The GRAM-W biosensor provides a novel tool for real-time monitoring of accessible cholesterol.
• This technology offers insights into cellular cholesterol homeostasis and its dysregulation in disease.
• The developed tools will advance research into the molecular mechanisms of cholesterol distribution regulation.