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

Abstract

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.

Stochastic activation and bistability in a Rab GTPase regulatory network

“…We demonstrate that this minimal biochemical system is able to act in a switch-like, bistable manner. Furthermore, we found conditions that allow the system to form spatial patterns of Rab5 activation, which share features of Rab domains found on endosomes in vivo (23,24). nucleotide exchange by CF488A-Rab5, which can be followed by an increase of fluorescence intensity on the membrane surface.…”

“…In our system, RabGAP-5 acts as a global inhibitor, rather than a long-ranged diffusing inhibitor, resulting in simpler spatiotemporal activation pattern. Despite the large difference in scales, the Rab5 patches we observed experimentally might originate from a similar mechanism as endosomal Rab domains (23,24).…”

Ultrastructural and dynamic studies of the endosomal compartment in Down syndrome

“…Experiments resulting in the enlargement of early endosomes, achieved via transfection of Rab5-Q79L, clearly show the clusters of high intensity of endosomal proteins, such as EEA1, at the endosomal limiting membrane [33,55,63,64]. Likewise, the protein microdomains can be observed when endosomes are imaged using super-resolution microscopy [30]. Nevertheless, it is unlikely that the clusters of EEA1-positive puncta shown in the insets of Fig.…”

Unraveling nanotopography of cell surface receptors

“…Two-color SMLM experiments were performed on a Nikon Eclipse Ti microscope, which is specified elsewhere in detail 44 . Contrary to the case of dTRABI analysis, the FWHM was set as a free fit parameter, but in the limits of 275-750 nm, both for Alexa Fluor 647 antibody and mEOS2 fusion protein localizations.…”

“…Electron microscopy (EM), with its ability to provide information about ultrastructural details, was the key method for microvilli characterization in the pioneering works 41 . However, EM cannot visualize specific proteins efficiently, since the labelling densities are limited by ligand/antigen accessibility, steric hindrance and electron repulsion [42][43][44] . A rapid development of SR light microscopy techniques enabled the visualization of three-dimensional objects with high precision, with the use of highly specific and frequently efficient labelling methods and on living cells (e.g., refs.…”