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Characterization of MORN2 stability and regulatory function in LC3-associated phagocytosis in macrophages
Maya Morita1, Mayu Kajiye1, Chiye Sakurai1
1Division of Molecular Biology, School of Life Sciences, Faculty of Medicine, Tottori University, Yonago, Tottori 683-8503, Japan.
Abstract:
Microtubule-associated protein A1/B1-light chain 3 (LC3)-associated phagocytosis (LAP) is a type of non-canonical autophagy that regulates phagosome maturation in macrophages. However, the role and regulatory mechanism of LAP remain largely unknown. Recently, the membrane occupation and recognition nexus repeat-containing-2 (MORN2) was identified as a key component of LAP for the efficient formation of LC3-recruiting phagosomes. To characterize MORN2 and elucidate its function in LAP, we established a MORN2-overexpressing macrophage line. At a steady state, MORN2 was partially cleaved by the ubiquitin-proteasome system. MORN2 overexpression promoted not only LC3-II production but also LAP phagosome (LAPosome) acidification during Escherichia coli uptake. Furthermore, the formation of LAPosomes containing the yeast cell wall component zymosan was enhanced in MORN2-overexpressing cells and depended on reactive oxygen species (ROS). Finally, MORN2-mediated LAP was regulated by plasma membrane-localized soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) such as SNAP-23 and syntaxin 11. Taken together, these findings demonstrate that MORN2, whose expression is downregulated via proteasomal digestion, is a limiting factor for LAP, and that membrane trafficking by SNARE proteins is involved in MORN2-mediated LAP.
Insights
Microtubule-associated protein A1/B1-light chain 3 (LC3)-associated phagocytosis (LAP) is regulated by MORN2, a key component. MORN2 overexpression enhances LAPosome formation and acidification, revealing its role in macrophage phagocytosis.
Area of Science:
- Cell Biology
- Immunology
- Molecular Biology
Background:
- Microtubule-associated protein A1/B1-light chain 3 (LC3)-associated phagocytosis (LAP) is a non-canonical autophagy pathway crucial for macrophage phagosome maturation.
- The precise regulatory mechanisms and functional significance of LAP remain incompletely understood.
- The membrane occupation and recognition nexus repeat-containing-2 (MORN2) has emerged as a critical factor for efficient LC3 recruitment in phagosomes.
Purpose of the Study:
- To characterize the function of MORN2 in LAP.
- To elucidate the regulatory mechanisms governing MORN2's role in phagocytosis.
- To investigate the impact of MORN2 overexpression on LAPosome formation and maturation.
Main Methods:
- Established a MORN2-overexpressing macrophage cell line for experimental analysis.
- Investigated MORN2 cleavage by the ubiquitin-proteasome system under steady-state conditions.
- Assessed LC3-II production and LAPosome acidification during Escherichia coli uptake.
- Examined LAPosome formation with zymosan and its dependence on reactive oxygen species (ROS).
- Analyzed the involvement of plasma membrane-localized SNARE proteins (SNAP-23, syntaxin 11) in MORN2-mediated LAP.
Main Results:
- MORN2 undergoes partial cleavage by the ubiquitin-proteasome system.
- MORN2 overexpression significantly enhanced LC3-II production and LAPosome acidification during bacterial uptake.
- Enhanced LAPosome formation was observed with zymosan, dependent on ROS.
- MORN2-mediated LAP was found to be regulated by SNARE proteins, including SNAP-23 and syntaxin 11.
Conclusions:
- MORN2 acts as a limiting factor for LAP, with its expression regulated by proteasomal degradation.
- Membrane trafficking mediated by SNARE proteins plays a crucial role in MORN2-dependent LAP.
- These findings provide significant insights into the molecular mechanisms governing LAP in macrophages.
