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: The last method used to study the homotypic fusion of ATG16L1 vesicles is an in vitro fusion assay between different fluorescent pools of ATG16L1 vesicles. In this assay, the fused vesicles appear yellow (as the consequence of green and red colocalization) that can be easily quantified (Fig. 4). In the example below, we used the SNARE inhibitor NEM to show a decrease in yellow vesicles (fused vesicles), indicating a defect in the homotypic fusion of ATG16L1 vesicles (Fig. 4).
Affiliation: Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, UK.