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

Christian Franke,Sandra Segeletz,Yannis Kalaidzidis,Marino Zerial,Jean‐Marc Verbavatz,Urska Repnik,Nicolas Brouilly

doi:10.1111/tra.12671

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.

Highlights

Cellular organelles have a characteristic size, shape and a morphologically distinguishable subdomain organization.[1,2,3,4,5,6] Such subdomains are Christian Franke, Urska Repnik, Sandra Segeletz, and Nicolas Brouilly contributed to this study.distinct in protein and lipid composition and provide the microenvironment necessary for coordinated biochemical reactions
epidermal growth factor (EGF) remains bound to its receptor and is sorted onto intraluminal vesicles (ILVs), whereas LDL is released from its receptor into the lumen of the endosome
Our results show for the first time the compartmentalization of EGF, Tfn and Rab[5] simultaneously in relation to the 3D organelle ultrastructure with nanometer resolution in both imaging modes, demonstrating the applicability of our superCLEM workflow to complex biological questions at the suborganelle level

Summary

| INTRODUCTION

Cellular organelles have a characteristic size, shape and a morphologically distinguishable subdomain organization.[1,2,3,4,5,6] Such subdomains are Christian Franke, Urska Repnik, Sandra Segeletz, and Nicolas Brouilly contributed to this study. Correlative light electron microscopy (CLEM) approaches deploying SMLM can be used to investigate the localization of proteins at the nanoscale in the context of the ultrastructural reference space.[30,31,32,33] Yet, single-molecule based CLEM approaches have mostly been limited to one high-resolved color, either a fluorescent protein or an organic dye This is largely due to the difficulty of combining multiple photo-switchable markers in a single CLEM workflow with SMLM imaging constraints.[27,34,35,36] some dual-color SMLM based CLEM approaches have been reported,[37,38] they have never been applied to structurally frail structures like endosomes, which are susceptible to ultrastructural damage. Our results show for the first time the compartmentalization of EGF, Tfn and Rab[5] (or LDL) simultaneously in relation to the 3D organelle ultrastructure with nanometer resolution in both imaging modes, demonstrating the applicability of our superCLEM workflow to complex biological questions at the suborganelle level

| RESULTS

| DISCUSSION

Findings

| MATERIAL AND METHODS