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Single organelle dynamics linked to 3D structure by correlative live-cell imaging and 3D electron microscopy.

Authors
  • Fermie, Job1, 2
  • Liv, Nalan1
  • Ten Brink, Corlinda1
  • van Donselaar, Elly G1
  • Müller, Wally H3
  • Schieber, Nicole L4
  • Schwab, Yannick4
  • Gerritsen, Hans C2
  • Klumperman, Judith1
  • 1 Section Cell Biology, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands. , (Netherlands)
  • 2 Section Molecular Biophysics, Debye Institute for Nanomaterials Science, Utrecht University, Utrecht, The Netherlands. , (Netherlands)
  • 3 Section Cryo-EM, Department of Chemistry, Utrecht University, Utrecht, The Netherlands. , (Netherlands)
  • 4 Electron Microscopy Core Facility, EMBL Heidelberg, Heidelberg, Germany. , (Germany)
Type
Published Article
Journal
Traffic
Publisher
Wiley (Blackwell Publishing)
Publication Date
May 01, 2018
Volume
19
Issue
5
Pages
354–369
Identifiers
DOI: 10.1111/tra.12557
PMID: 29451726
Source
Medline
Keywords
Language
English
License
Unknown

Abstract

Live-cell correlative light-electron microscopy (live-cell-CLEM) integrates live movies with the corresponding electron microscopy (EM) image, but a major challenge is to relate the dynamic characteristics of single organelles to their 3-dimensional (3D) ultrastructure. Here, we introduce focused ion beam scanning electron microscopy (FIB-SEM) in a modular live-cell-CLEM pipeline for a single organelle CLEM. We transfected cells with lysosomal-associated membrane protein 1-green fluorescent protein (LAMP-1-GFP), analyzed the dynamics of individual GFP-positive spots, and correlated these to their corresponding fine-architecture and immediate cellular environment. By FIB-SEM we quantitatively assessed morphological characteristics, like number of intraluminal vesicles and contact sites with endoplasmic reticulum and mitochondria. Hence, we present a novel way to integrate multiple parameters of subcellular dynamics and architecture onto a single organelle, which is relevant to address biological questions related to membrane trafficking, organelle biogenesis and positioning. Furthermore, by using CLEM to select regions of interest, our method allows for targeted FIB-SEM, which significantly reduces time required for image acquisition and data processing. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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