Macroautophagic cargo sequestration assays

Autophagy of cytoplasmic bulk cargo does not require LC3

“…It should be noted that in addition to the GABARAPs, we have found that macroautophagy in LNCaP cells requires the RB1CC1/FIP200 protein (and undoubtedly many more). 5 Although our density gradient fractionations showed some overlap between LC3 and sequestered LDH at the early hours after cycloheximide treatment, 6 they cannot really tell whether there is ever any physical association of LC3 with the phagophores that perform macroautophagic cargo sequestration. If there is, it is not implausible that LC3 could perform some auxiliary function, 10 e.g., picking up defective proteins, aggregates, or organelles, thus adding a selective element to an otherwise nonselective macroautophagy, given the well-established role of LC3 as a recruiter of cargo receptors in selective autophagy.…”

“…4 We routinely use LDH (lactate dehydrogenase) for this purpose, since it is a cytosolic enzyme present in all cells, and is degraded exclusively by the autophagic-lysosomal pathway. 4 Our macroautophagic cargo sequestration assay, which can be adapted to any cell type, 5 measures the net transfer of LDH into sedimentable autophagic vacuoles in the presence of leupeptin or bafilomycin A 1 to prevent LDH degradation in amphisomes and lysosomes. Cells are usually incubated in an amino acid-and serum-free buffer to ensure that the maximally attainable autophagic activity (autophagic capacity) is measured.…”

A Novel Role of Listeria monocytogenes Membrane Vesicles in Inhibition of Autophagy and Cell Death

“…Two days later, the medium was replaced with 1.5 ml fresh medium and 500 μl MVs, 500 μl PBS (vehicle-control), or 500 μl medium (used for subtraction of background LDH sedimentation; Seglen et al, 2015) was added. After 3 h, Torin1 (50 nM) or DMSO (0.1%) was added, and after an additional hour BafA1 (100 nM) or DMSO (0.1%) was added.…”

“…After 3 h, Torin1 (50 nM) or DMSO (0.1%) was added, and after an additional hour BafA1 (100 nM) or DMSO (0.1%) was added. Following an additional 2 h incubation, the cells were harvested with the use of Accumax (Sigma), and the autophagic sequestration rates were determined as previously described (Seglen et al, 2015), with minor modifications. Briefly, the cells were washed twice in 10% sucrose/1% BSA followed by re-suspension in 400 μl 10% sucrose/0.2% BSA and selective electro-disruption of the plasma membrane by a single discharge of 2 kV/cm and 1.2 μF in a 1 × 1 × 5 cm electrode chamber coupled to an in-house power generator.…”

Assessing the progression of autophagy pathways in different organisms and tissues

“…The section on biochemical methods offers four articles: Article 2, Peidu Jiang and Noboru Mizushima, ''LC3-and p62-based biochemical methods for the analysis of autophagy progression in mammalian cells'' [14]; Article 3, Kevin Moreau and co-workers, ''Methods to analyze SNARE-dependent vesicular fusion events that regulate autophagosome biogenesis'' [15]; Article 4, Per Seglen and co-workers, ''Macroautophagic cargo sequestration assays'' [16]; and Article 5, Bettina Zens and co-workers, ''In vitro systems for Atg8 lipidation [17]. The fluorescence microscopy section provides two views: Article 6, Eleftherios Karanasios and Nicholas T. Ktistakis, ''Live-cell imaging for the assessment of the dynamics of autophagosome formation: Focus on early steps'' [18]; Article 7, Ann-Katrin Thost and co-workers, ''Fluorescence-based imaging of autophagy progression by human WIPI protein detection'' [19].…”