Correlative single‐molecule localization microscopy and electron tomography reveals endosome nanoscale domains

Many cellular organelles, including endosomes, show compartmentalization into distinct functional domains, which, however, cannot be resolved by diffraction‐limited light microscopy. Single molecule localization microscopy (SMLM) offers nanoscale resolution but data interpretation is often inconclus...

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Bibliographic Details
Published in:Traffic (Copenhagen, Denmark) Denmark), 2019-08, Vol.20 (8), p.601-617
Main Authors: Franke, Christian, Repnik, Urska, Segeletz, Sandra, Brouilly, Nicolas, Kalaidzidis, Yannis, Verbavatz, Jean‐Marc, Zerial, Marino
Format: Article
Language:English
Subjects:
Electron Microscope Tomography - methods
Electron microscopy
electron tomography
Endosomes
Endosomes - metabolism
Endosomes - ultrastructure
GTP-binding protein
Guanosine triphosphatases
HeLa Cells
Humans
Life Sciences
Localization
Microscopy
multicolor CLEM
Organelles
Rab5
rab5 GTP-Binding Proteins - metabolism
Rab5 protein
Single Molecule Imaging - methods
single‐molecule localization microscopy
super‐resolution microscopy
Tokuyasu cryosectioning
Tomography
Toolbox
Transferrin
Ultrastructure
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Summary:Many cellular organelles, including endosomes, show compartmentalization into distinct functional domains, which, however, cannot be resolved by diffraction‐limited light microscopy. Single molecule localization microscopy (SMLM) offers nanoscale resolution but data interpretation is often inconclusive when the ultrastructural context is missing. Correlative light electron microscopy (CLEM) combining SMLM with electron microscopy (EM) enables correlation of functional subdomains of organelles in relation to their underlying ultrastructure at nanometer resolution. However, the specific demands for EM sample preparation and the requirements for fluorescent single‐molecule photo‐switching are opposed. Here, we developed a novel superCLEM workflow that combines triple‐color SMLM (dSTORM & PALM) and electron tomography using semi‐thin Tokuyasu thawed cryosections. We applied the superCLEM approach to directly visualize nanoscale compartmentalization of endosomes in HeLa cells. Internalized, fluorescently labeled Transferrin and EGF were resolved into morphologically distinct domains within the same endosome. We found that the small GTPase Rab5 is organized in nanodomains on the globular part of early endosomes. The simultaneous visualization of several proteins in functionally distinct endosomal sub‐compartments demonstrates the potential of superCLEM to link the ultrastructure of organelles with their molecular organization at nanoscale resolution. Suborganelle compartmentalization cannot be resolved by diffraction‐limited light microscopy and interpreted without knowledge of the underlying ultrastructure. This work presents a novel superCLEM workflow that combines multicolor single‐molecule localization‐microscopy with electron tomography using semi‐thin Tokuyasu thawed cryosections to map fluorescent molecules on the ultrastructure of early endosomes. superCLEM reveals that the small GTPase Rab5 is organized in nanodomains largely devoid of cargo molecules Transferrin and EGF. This method opens new possibilities to perform structure‐function analysis of organelles at the nanoscale.
ISSN:1398-9219
1600-0854
DOI:10.1111/tra.12671