Correlated Cryo-Fluorescence and Cryo-Electron Microscopy can Identify Sites of Membrane Fusion

Abstract

Advances in cryo-electron microscopy have recently been coupled with correlated light and electron microscopy (CLEM), where fluorescence is used to locate regions of interest and characterize their molecular composition prior to data collection. While the goal of cryo-CLEM is typically the localization of a cell, organelle or protein complex of interest within a vitrified sample, recent advances of cryo-fluorescence microscopy into single-molecule regimes [Wolff et al., Biol. Cell. 2016] and the thin depth of samples suggest that fluorescence could also be used to probe function or dynamics similar to TIRF-based single-particle assays. In this way, cryo-CLEM has the ability to assist not only in efficient data collection, but also a description of functional state that might not be apparent by visualization alone. We have established that resonant energy transfer is possible at cryo temperatures, highlighting the potential of cryo-CLEM for applications requiring fluorophore autoquenching or FRET. Using autoquenching, we have adapted an influenza fusion assay for cryo-CLEM. During fusion between an influenza virus-like particle and a unilamellar vesicle, the two lipid bilayers enter into apposition before beginning lipid exchange, progressing to hemifusion and finally opening a fusion pore. By incorporating cryo-CLEM, fusion events are localized and identified based on the fluorescent signatures for these three stages, before being targeted for cryo-electron tomography. The fluorescence can then place fusion events from the cryo-CLEM experiment within either a bulk kinetic fusion assay time course completed with the same sample batch, or into published single-molecule time courses.