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Updated: Nov 21, 2025
Isolation of Murine Peritoneal Macrophages to Carry Out Gene Expression Analysis Upon Toll-like Receptors Stimulation
Published on: April 29, 2015
ATG7 is dispensable for LC3-PE conjugation in thioglycolate-elicited mouse peritoneal macrophages
Nemanja Vujić1,2, Ivan Bradić1,2, Madeleine Goeritzer1,2
1Gottfried Schatz Research Center, Medical University of Graz, Graz, Austria.
Abstract:
Thioglycolate-elicited macrophages exhibit abundant conjugation of LC3 with PE (LC3-II). Among other autophagy-related (ATG) proteins, it is proposed that, like in yeast, both ATG5 and ATG7 are essential for LC3 conjugation. Using atg5-deficient (-/-) and atg7-/-macrophages, we provide evidence that loss of ATG5 but not of ATG7 resulted in LC3-II depletion. Accumulation of LC3-II in elicited atg7-/- macrophages in response to bafilomycin A1 validated these data. Furthermore, complete loss of ATG3 in atg7-/- macrophages demonstrated that ATG7 and ATG3 are dispensable for LC3-PE conjugation. In contrast to thioglycolate-elicited macrophages, naïve peritoneal and bone marrow-derived atg7-/- macrophages exhibited no LC3-II, even under inflammatory stimuli in vitro. Hence, the macrophage metabolic status dictates the level of LC3-PE conjugation with a supportive but nonessential role of ATG7, disclosing the eukaryotic exception from the LC3 lipidation model based on yeast data. Abbreviations: ATG: autophagy-related; BM: bone marrow; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; PE: phosphatidylethanolamine.
Insights
Autophagy-related protein 7 (ATG7) is not essential for microtubule-associated protein 1 light chain 3 (LC3) lipidation in macrophages. Macrophage metabolic status, not ATG7, dictates LC3-II levels, challenging the universal model.
Area of Science:
- Cell Biology
- Molecular Biology
- Immunology
Background:
- Microtubule-associated protein 1 light chain 3 (LC3) lipidation is crucial for autophagy.
- Autophagy-related proteins (ATGs), including ATG5 and ATG7, are thought to be essential for LC3 conjugation, based on yeast models.
- Thioglycolate-elicited macrophages show high levels of LC3-II, a lipidated form of LC3.
Purpose of the Study:
- To investigate the essentiality of ATG5 and ATG7 for LC3 lipidation in macrophages.
- To determine the role of ATG7 in LC3 conjugation in different macrophage states.
- To clarify the regulation of LC3 lipidation in macrophages and its divergence from yeast models.
Main Methods:
- Utilized autophagy-related gene-deficient (atg5-/- and atg7-/-) macrophages.
- Analyzed LC3-II levels in thioglycolate-elicited, naïve peritoneal, and bone marrow-derived macrophages.
- Assessed LC3-II accumulation in response to bafilomycin A1 and inflammatory stimuli in vitro.
- Investigated the role of ATG3 in ATG7-deficient macrophages.
Main Results:
- Loss of ATG5, but not ATG7, led to LC3-II depletion in elicited macrophages.
- LC3-II accumulated in elicited atg7-/- macrophages upon bafilomycin A1 treatment, confirming ATG7's non-essential role in this context.
- Naïve peritoneal and bone marrow-derived atg7-/- macrophages showed no LC3-II, even with inflammatory stimuli.
- ATG7 and ATG3 were dispensable for LC3-PE conjugation in atg7-/- macrophages.
Conclusions:
- Macrophage metabolic status significantly influences LC3-PE conjugation levels.
- ATG7 plays a supportive but non-essential role in LC3 lipidation in macrophages.
- These findings reveal a eukaryotic exception to the LC3 lipidation model derived from yeast studies.
