Protocol for quantifying LC3B FRET biosensor activity in living cells using a broad-to-sensitive data analysis...

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Autophagy is a fundamental cellular process involved in degradation and recycling of cellular components.
• Quantifying autophagy initiation is crucial for understanding cellular homeostasis and disease pathogenesis.
• Existing methods for assessing autophagy initiation can be complex or lack precision.

Purpose of the Study:

• To present a comprehensive protocol for quantifying autophagy initiation.
• To utilize the microtubule-associated protein 1 light chain 3 beta (LC3B) Förster's resonance energy transfer (FRET) biosensor for this purpose.
• To provide a robust image analysis pipeline applicable to FRET/FLIM imaging.

Main Methods:

• Detailed protocol for cell seeding and transfection.
• FRET/FLIM (fluorescence lifetime imaging microscopy) imaging techniques.
• Image analysis pipeline for FRET sensor data.

Main Results:

• Successful implementation of a protocol to quantify autophagy initiation.
• Demonstration of LC3B FRET biosensor utility in assessing autophagy status.
• Development of an adaptable image analysis pipeline for FRET/FLIM data.

Conclusions:

• The presented protocol offers a reliable method for quantifying autophagy initiation.
• This approach is versatile and applicable across various physiological and disease contexts.
• The analysis pipeline can be extended to other genetically encoded FRET sensors.