Methods to analyze SNARE-dependent vesicular fusion events that regulate autophagosome biogenesis.
Bottom Line: Autophagy is an important catabolic pathway that preserves cellular homeostasis.The formation of autophagosomes is a complex process requiring the reorganization of membranes from different compartments.These two steps are essential for the maturation of small single-membrane autophagic precursors containing ATG16L1 and mATG9 proteins into double-membrane autophagosomes.
Affiliation: Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, UK.Show MeSH
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Mentions: Live cell imaging is probably the best method to observe a defect in the homotypic fusion of ATG16L1 vesicles and it avoids potential artifacts due to cell preparation such as fixation or lysis. We identified SNAREs, which primarily mediate vesicle fusion, as important determinants of this process (VAMP7, Syntaxin7, Syntaxin8 and Vti1B). Using a SNARE inhibitor called NEM (N-Ethyl maleimide), that inhibits an ATPase involved in SNAREs-dependent fusion, we observed a clear defect in the homotypic fusion of ATG16L1 vesicles (Fig. 2). Moreover, when we stimulated autophagy by rapamycin treatment or by amino acid and serum starvation, we observed an increase in the rate of homotypic fusion (Fig. 2).
Affiliation: Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, UK.