Ultrastructural characterization of phagophores using electron tomography on cryoimmobilized and freeze substituted samples: Ultrastructure of autophagosomes

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Sammanfattning

Electron tomography (ET) has significantly contributed to recent findings regarding the biogenesis of the phagophore, an organelle which initiates autophagic sequestration. The information obtained from 1.9 nm slices through the tomograms have revealed that during biogenesis the phagophore is in contact with the membranes of apposing organelles to form tubular connections and membrane contact sites (MCSs). The most reported and established tubular connections occur between the phagophore and the endoplasmic reticulum (ER). However, as the phagophore continues to grow and expand, connections and MCSs have also been reported to occur between the phagophore and several other organelles in a possible attempt to utilise lipids for membrane expansion from alternative sources. Since the lifespan of the phagophore is only a few minutes and membrane connections and MCSs are very dynamic, capturing these two events requires precision during fixation. Up to date there is no quicker alternative for sample preservation in transmission electron microscopy than cryoimmobilization. In this report we describe our protocol for cryoimmobilization using high-pressure freezing and freeze substitution, and report our first findings on phagophore morphology using this approach.
Originalspråkengelska
TidskriftMethods in Enzymology
Volym587
Sidor (från-till)331-349
Antal sidor19
ISSN0076-6879
DOI
StatusPublicerad - 2017
MoE-publikationstypA1 Tidskriftsartikel-refererad

Bibliografisk information

Chapter Nineteen

Vetenskapsgrenar

  • 1182 Biokemi, cell- och molekylärbiologi

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