Abstract
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.
Highlights
Autophagy is an important catabolic pathway that preserves cellular homeostasis
Live cell imaging Live cell imaging is probably the best method to observe a defect in the homotypic fusion of ATG16L1 vesicles and it avoids
Fixed cells imaging To complement the live cell imaging strategy, we measured the sizes of ATG16L1 vesicles by microscopy on fixed samples to provide additional information regarding the maturation of ATG16L1 vesicles
Summary
(Macro) autophagy is a critical clearance pathway for organelles and long-lived proteins, including intracytoplasmic aggregateprone proteins that cause many neurodegenerative diseases, such as huntingtin in Huntington’s disease, and tau in Alzheimer’s disease [1]. It is not clear if these homotypic and heterotypic fusion events are sequential or parallel These data reveal that the SNARE-dependent fusion of distinct vesicles containing different autophagy proteins is required for optimal autophagosome biogenesis. We recently identified PICALM (CALM; phosphatidylinositol binding clathrin assembly protein), recently associated with Alzheimer’s disease, as an important regulator of both the homotypic fusion and the heterotypic fusion of autophagic precursors [7]. This is mostly based on live cell imaging and an in vitro fusion assay, which will be the methods presented below
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