Summary- paper 3:
Lipid and protein content profiling of isolated native autophagic vesicles
Daniel Schmitt, Süleyman Bozkurt, Pascale Henning-Domres, Heike Huesmann, Stefan Eimer, Laura Bindila, Christian Behrends, Emily Boyle, Florian Wilfling, Georg Tascher, Christian Münch, Christian Behl, Andreas Kern
EMBO Reports, 2022
Questions/gaps addressed:
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What is the impact of different autophagy conditions on the exact Phospholipid and cargo contents of autophagosomes/autophagic vesicles?
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Optimize a method for the isolation of unmanipulated autophagic vesicles at large quantities for rapid lipid and cargo profiling.
Key methods:
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anti-Atg8 antibody-based FACS mediated isolation approach to purify intact native autophagic vesicles from bafilomycin A1 (accumulates autophagic vesicles) treated HeLa cell homogenates.
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negative stain electron microscopy to visualize intact vesicles, and proteinase K digestion assay for p62/SQSTM1 to evaluate intactness
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quantitative lipidomics and proteomics analyses to identify PLs and cargo proteins of autophagic vesicles
Major takeaways:
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Antibody binding in cell homogenates and FACS enrichment based pipeline can isolate intact autophagic vesicles
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Size evaluation of isolated vesicles: diameters range from 340 to 1,150 nm
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phosphatidylcholine (PC) enriched: accounts for 42% of all detected PLs in autophagic vesicles. Also contain sphingomyelin (SM). Phosphatidylglycerol (PG) appears to be excluded.
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Protein composition of autophagic vesicles (dataset EV1): identified >4000 proteins. Composition varied more upon proteasome inhibition than starvation.
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Approach does not distinguish between autophagosomes and autolysosomes.