Autophagosome marker, LC3, is released extracellularly via several distinct pathways

Area of Science:

• Cell Biology
• Molecular Biology
• Biochemistry

Background:

• Autophagy is crucial for maintaining intracellular homeostasis through the secretion of cytoplasmic components.
• The precise mechanisms governing autophagy-mediated secretion, particularly the release of specific proteins like LC3, remain incompletely understood.

Purpose of the Study:

• To develop and utilize an ultrasensitive detection system to analyze the conditions triggering the secretion of LC3 (microtubule-associated protein 1A/1B-light chain 3).
• To investigate the role of autophagosome formation and other mechanisms in LC3 release into the extracellular space.

Main Methods:

• Development of an ultrasensitive detection system using HiBiT tag/NanoBiT technology for quantifying extracellular HiBiT-fused LC3.
• Analysis of HiBiT-LC3 release in wild-type, ATG5/FIP200 knockout cells, and cells with specific LC3 mutations (C-terminal truncation ΔG, K51A/L53A mutation).
• Treatment with bafilomycin A1 and detergent to modulate autophagosome-lysosome fusion and membrane association, respectively.

Main Results:

• Extracellular HiBiT-LC3 was detected in culture supernatants, requiring detergent for release, indicating dissociation from lipid membranes.
• Bafilomycin A1 treatment increased extracellular HiBiT-LC3, and this increase was reduced in ATG5 or FIP200 knockout cells, suggesting dependence on autophagosome formation.
• LC3 release was partially observed in knockout cells and was reduced but not abolished by LC3 mutations, indicating the existence of both autophagy-dependent and -independent release pathways.

Conclusions:

• The developed HiBiT tag/NanoBiT system provides a sensitive tool to study autophagy-mediated secretion.
• LC3 secretion is primarily dependent on autophagosome formation but can also occur through autophagy-independent mechanisms.
• Multiple pathways contribute to the extracellular release of LC3, highlighting the complexity of this cellular process.