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
Related in: MedlinePlus
Mentions: 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. Indeed, the homotypic fusion of ATG16L1 vesicles increases their size, which can be easily measured using post-acquisition software, such as the one included in the Cellomics ArrayScan VTI HCS Reader: Spot Detector Bioapplication protocol version 3. In the example below, the size of ATG16L1 vesicles decrease in VAMP7 or Syntaxin 7 (Stx7) knockdown cells (Fig. 3), where fusion is abrogated.
Affiliation: Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, UK.