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

Research output: Contribution to journalArticleScientificpeer-review

Abstract

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.
Original languageEnglish
JournalMethods in Enzymology
Volume587
Pages (from-to)331-349
Number of pages19
ISSN0076-6879
DOIs
Publication statusPublished - 2017
MoE publication typeA1 Journal article-refereed

Bibliographical note

Chapter Nineteen

Fields of Science

  • 1182 Biochemistry, cell and molecular biology
  • autophagy
  • phagophore
  • high-pressure freezing
  • freeze substitution
  • electron tomography

Cite this

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title = "Ultrastructural characterization of phagophores using electron tomography on cryoimmobilized and freeze substituted samples: Ultrastructure of autophagosomes",
abstract = "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.",
keywords = "1182 Biochemistry, cell and molecular biology, autophagy, phagophore, high-pressure freezing, freeze substitution, electron tomography",
author = "Joanna Biazik and Helena Vihinen and Jokitalo, {Eija Sofia} and Eeva-Liisa Eskelinen",
note = "Chapter Nineteen",
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TY - JOUR

T1 - Ultrastructural characterization of phagophores using electron tomography on cryoimmobilized and freeze substituted samples

T2 - Ultrastructure of autophagosomes

AU - Biazik, Joanna

AU - Vihinen, Helena

AU - Jokitalo, Eija Sofia

AU - Eskelinen, Eeva-Liisa

N1 - Chapter Nineteen

PY - 2017

Y1 - 2017

N2 - 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.

AB - 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.

KW - 1182 Biochemistry, cell and molecular biology

KW - autophagy

KW - phagophore

KW - high-pressure freezing

KW - freeze substitution

KW - electron tomography

U2 - 10.1016/bs.mie.2016.09.063

DO - 10.1016/bs.mie.2016.09.063

M3 - Article

VL - 587

SP - 331

EP - 349

JO - Methods in Enzymology

JF - Methods in Enzymology

SN - 0076-6879

ER -