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Sökning: WFRF:(Florey Oliver)

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  • Aits, Sonja, et al. (författare)
  • Methods to Detect Loss of Lysosomal Membrane Integrity
  • 2019
  • Ingår i: Autophagy : Methods and Protocols - Methods and Protocols. - New York, NY : Springer New York. - 1064-3745. - 9781493988730 ; 1880, s. 315-329
  • Bokkapitel (refereegranskat)abstract
    • Loss of lysosomal membrane integrity, often referred to as lysosomal membrane permeabilization (LMP), occurs in many instances of cell death either as an initiating or as an amplifying event. Currently, the best method for detecting LMP is the galectin puncta formation assay which can be used for a broad range of sample types, both fixed and live, is easy to perform, and highly sensitive. This method, which is similar to the widely used LC3 puncta formation assay for autophagy, is based on the translocation of galectins to damaged lysosomes resulting in a change from uniform to punctate staining pattern. Here, we provide protocols for the galectin puncta formation assay in fixed and live cells and for an alternative assay based on fluorescent dextran release from damaged lysosomes, which can be performed in parallel
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  • Durgan, Joanne, et al. (författare)
  • Non-canonical autophagy drives alternative ATG8 conjugation to phosphatidylserine
  • 2021
  • Ingår i: Molecular Cell. - : Elsevier. - 1097-2765 .- 1097-4164. ; 81:9, s. 2031-2040
  • Tidskriftsartikel (refereegranskat)abstract
    • Autophagy is a fundamental catabolic process that uses a unique post-translational modification, the conjugation of ATG8 protein to phosphatidylethanolamine (PE). ATG8 lipidation also occurs during non-canonical autophagy, a parallel pathway involving conjugation of ATG8 to single membranes (CASM) at endolysosomal compartments, with key functions in immunity, vision, and neurobiology. It is widely assumed that CASM involves the same conjugation of ATG8 to PE, but this has not been formally tested. Here, we discover that all ATG8s can also undergo alternative lipidation to phosphatidylserine (PS) during CASM, induced pharmacologically, by LC3-associated phagocytosis or influenza A virus infection, in mammalian cells. Importantly, ATG8-PS and ATG8-PE adducts are differentially delipidated by the ATG4 family and bear different cellular dynamics, indicating significant molecular distinctions. These results provide important insights into autophagy signaling, revealing an alternative form of the hallmark ATG8 lipidation event. Furthermore, ATG8-PS provides a specific “molecular signature” for the non-canonical autophagy pathway.
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