title: Macroautophagic cargo sequestration assays.
abstract: Macroautophagy, the process responsible for bulk sequestration and lysosomal degradation of cytoplasm, is often monitored by means of the autophagy-related marker protein LC3. This protein is linked to the phagophoric membrane by lipidation during the final steps of phagophore assembly, and it remains associated with autophagic organelles until it is degraded in the lysosomes. The transfer of LC3 from cytosol to membranes and organelles can be measured by immunoblotting or immunofluorescence microscopy, but these assays provide no information about functional macroautophagic activity, i.e., whether the phagophores are actually engaged in the sequestration of cytoplasmic cargo and enclosing this cargo into sealed autophagosomes. Moreover, accumulating evidence suggest that macroautophagy can proceed independently of LC3. There is therefore a need for alternative methods, preferably effective cargo sequestration assays, which can monitor actual macroautophagic activity. Here, we provide an overview of various approaches that have been used over the last four decades to measure macroautophagic sequestration activity in mammalian cells. Particular emphasis is given to the so-called "LDH sequestration assay", which measures the transfer of the autophagic cargo marker enzyme LDH (lactate dehydrogenase) from the cytosol to autophagic vacuoles. The LDH sequestration assay was originally developed to measure macroautophagic activity in primary rat hepatocytes. Subsequently, it has found use in several other cell types, and in this article we demonstrate a further validation and simplification of the method, and show that it is applicable to several cell lines that are commonly used to study autophagy.
authors.affiliation: Centre for Molecular Medicine Norway, Nordic EMBL Partnership, University of Oslo and Oslo University Hospital, N-0318 Oslo, Norway.
authors.forename: Per O
authors.initials: PO
authors.lastname: Seglen
authors.affiliation: Centre for Molecular Medicine Norway, Nordic EMBL Partnership, University of Oslo and Oslo University Hospital, N-0318 Oslo, Norway.
authors.forename: Morten
authors.initials: M
authors.lastname: Luhr
authors.affiliation: Centre for Molecular Medicine Norway, Nordic EMBL Partnership, University of Oslo and Oslo University Hospital, N-0318 Oslo, Norway; Department of Cancer Prevention and the Department of Urology, Oslo University Hospital, N-0424 Oslo, Norway.
authors.forename: Ian G
authors.initials: IG
authors.lastname: Mills
authors.affiliation: Centre for Molecular Medicine Norway, Nordic EMBL Partnership, University of Oslo and Oslo University Hospital, N-0318 Oslo, Norway.
authors.forename: Frank
authors.initials: F
authors.lastname: Sætre
authors.affiliation: Centre for Molecular Medicine Norway, Nordic EMBL Partnership, University of Oslo and Oslo University Hospital, N-0318 Oslo, Norway.
authors.forename: Paula
authors.initials: P
authors.lastname: Szalai
authors.affiliation: Centre for Molecular Medicine Norway, Nordic EMBL Partnership, University of Oslo and Oslo University Hospital, N-0318 Oslo, Norway. Electronic address: k.n.engedal@ncmm.uio.no.
authors.forename: Nikolai
authors.initials: N
authors.lastname: Engedal
mesh_terms: D001343:Autophagy / Q000235:genetics*; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D007770:L-Lactate Dehydrogenase / Q000276:immunology / Q000378:metabolism; D008247:Lysosomes / Q000235:genetics / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000276:immunology / Q000378:metabolism; D008967:Molecular Biology / Q000379:methods*
chemical_list: C477012:MAP1LC3A protein, human; D008869:Microtubule-Associated Proteins; D007770:L-Lactate Dehydrogenase
keywords: Autophagy; Cargo sequestration; LC3; LDH; Lactate dehydrogenase; Macroautophagy