A3 Refereed book chapter or chapter in a compilation book
Ultrastructural Characterization of Phagophores Using Electron Tomography on Cryoimmobilized and Freeze Substituted Samples
Authors: J. Biazik, H. Vihinen, E. Jokitalo, E.-L. Eskelinen
Editors: Anna Marie Pyle, David W. Christianson
Publishing place: Cambridge
Publication year: 2017
Journal: Methods in Enzymology
Book title : Molecular Characterization of Autophagic Responses, Part A
Journal name in source: MOLECULAR CHARACTERIZATION OF AUTOPHAGIC RESPONSES, PT A
Journal acronym: METHOD ENZYMOL
Series title: Methods in Enzymology
Number in series: 587
Volume: 587
First page : 331
Last page: 349
Number of pages: 19
ISBN: 978-0-12-809675-8
ISSN: 0076-6879
DOI: https://doi.org/10.1016/bs.mie.2016.09.063
Abstract
Electron tomography 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. 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 utilize 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.
Electron tomography 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. 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 utilize 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.
UTU Research Portal