Methods to analyze SNARE-dependent vesicular fusion events that regulate autophagosome biogenesis.

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. Here we describe methods to analyze SNARE-dependent vesicular fusion events involving the homotypic and heterotypic fusion of autophagosome precursor structures. These two steps are essential for the maturation of small single-membrane autophagic precursors containing ATG16L1 and mATG9 proteins into double-membrane autophagosomes. The techniques described in this review are mostly based on live cell imaging, microscopy, and biochemistry using an in vitro fusion assay, and should help researchers to study autophagosome biogenesis.We are grateful to the Wellcome Trust (Principal Research Fellowship to DCR), a Wellcome Trust/MRC Strategic award in neurodegeneration, and a Wellcome Trust Strategic Grant to Cambridge Institute for Medical Research for funding.This is the final version. It was first published by Elsevier at http://www.sciencedirect.com/science/article/pii/S104620231400365