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 sub-domains 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-colour 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 labelled Transferrin and EGF were resolved into morphologically distinct domains within the same endosome. We found that the small GTPase Rab5 is organized in nano-domains 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. This article is protected by copyright. All rights reserved.
PubMed ID: 31206952
Citation: Traffic. 2019 Jun 17. doi: 10.1111/tra.12671.
Date Published: 17th Jun 2019
Authors: C. Franke, U. Repnik, S. Segeletz, N. Brouilly, Y. Kalaidzidis, J. M. Verbavatz, Marino Zerial
Created: 27th Jun 2019 at 13:35