12 Jan 2023
by Richa

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:

  • What is the impact of different autophagy conditions on the exact Phospholipid and cargo contents of autophagosomes/autophagic vesicles?

  • Optimize a method for the isolation of unmanipulated autophagic vesicles at large quantities for rapid lipid and cargo profiling.

Key methods:

  • anti-Atg8 antibody-based FACS mediated isolation approach to purify intact native autophagic vesicles from bafilomycin A1 (accumulates autophagic vesicles) treated HeLa cell homogenates.

  • negative stain electron microscopy to visualize intact vesicles, and proteinase K digestion assay for p62/SQSTM1 to evaluate intactness

  • quantitative lipidomics and proteomics analyses to identify PLs and cargo proteins of autophagic vesicles

Major takeaways:

  • Antibody binding in cell homogenates and FACS enrichment based pipeline can isolate intact autophagic vesicles

  • Size evaluation of isolated vesicles: diameters range from 340 to 1,150 nm

  • phosphatidylcholine (PC) enriched: accounts for 42% of all detected PLs in autophagic vesicles. Also contain sphingomyelin (SM). Phosphatidylglycerol (PG) appears to be excluded.

  • Protein composition of autophagic vesicles (dataset EV1): identified >4000 proteins. Composition varied more upon proteasome inhibition than starvation.

  • Approach does not distinguish between autophagosomes and autolysosomes.