CaltechAUTHORS
  A Caltech Library Service

Fluorescence-Based Detection of Membrane Fusion State on a Cryo-EM Grid using Correlated Cryo-Fluorescence and Cryo-Electron Microscopy

Metskas, Lauren Ann and Briggs, John A. G. (2019) Fluorescence-Based Detection of Membrane Fusion State on a Cryo-EM Grid using Correlated Cryo-Fluorescence and Cryo-Electron Microscopy. Microscopy and Microanalysis, 25 (4). pp. 942-949. ISSN 1431-9276. https://resolver.caltech.edu/CaltechAUTHORS:20180927-114223095

[img] PDF - Submitted Version
Creative Commons Attribution.

6Mb
[img] PDF - Supplemental Material
Creative Commons Attribution.

430Kb

Use this Persistent URL to link to this item: https://resolver.caltech.edu/CaltechAUTHORS:20180927-114223095

Abstract

Correlated light and electron microscopy (CLEM) has become a popular technique for combining the protein-specific labeling of fluorescence with electron microscopy, both at room and cryogenic temperatures. Fluorescence applications at cryo-temperatures have typically been limited to localization of tagged protein oligomers due to known issues of extended triplet state duration, spectral shifts, and reduced photon capture through cryo-CLEM objectives. Here, we consider fluorophore characteristics and behaviors that could enable more extended applications. We describe how dialkylcarbocanine DiD, and its autoquenching by resonant energy transfer (RET), can be used to distinguish the fusion state of a lipid bilayer at cryo-temperatures. By adapting an established fusion assay to work under cryo-CLEM conditions, we identified areas of fusion between influenza virus-like particles and fluorescently labeled lipid vesicles on a cryo-EM grid. This result demonstrates that cryo-CLEM can be used to localize functions in addition to tagged proteins, and that fluorescence autoquenching by RET can be incorporated successfully into cryo-CLEM approaches. In the case of membrane fusion applications, this method provides both an orthogonal confirmation of functional state independent of the morphological description from cryo-EM and a way to bridge room-temperature kinetic assays and the cryo-EM images.


Item Type:Article
Related URLs:
URLURL TypeDescription
https://doi.org/10.1017/S1431927619000606DOIArticle
https://doi.org/10.1101/388579DOIDiscussion Paper
ORCID:
AuthorORCID
Metskas, Lauren Ann0000-0002-8073-6960
Briggs, John A. G.0000-0003-3990-6910
Alternate Title:Fluorescence-Based Detection of Fusion State on a Cryo-EM Grid using Correlated Cryo-Fluorescence and Cryo-Electron Microscopy
Additional Information:© 2019 Microscopy Society of America. Received 3 October 2018; revised 7 February 2019; accepted 16 April 2019. This research was technically supported by the Electron Microscopy Core Facility and the Protein Expression and Purification Core Facility at the European Molecular Biology Laboratory (EMBL, Heidelberg), and the EM facility at the MRC Laboratory of Molecular Biology. We thank M. Schorb, W. Hagen, W. Kukulski, E. Lemke, G. Paci, O. Avinoam, and Y. Bykov for helpful discussions and technical assistance with the Leica cryo-CLEM system, and M. Clarke for assistance with VLP preparations. This project was funded by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation programme (ERC-2014-CoG 648432—MEMBRANEFUSION) and the Medical Research Council (MC_UP_1201/16). JAGB is an inventor on filed patents that have been licensed by Leica Microsystems and commercialized in the Leica cryo-CLEM system used in this study. Author Contributions: L.A.M. performed the research and data analysis. L.A.M. and J.A.G.B. designed research, interpreted results, and wrote the manuscript.
Funders:
Funding AgencyGrant Number
European Research Council (ERC)648432
Medical Research Council (UK)MC_UP_1201/16
Subject Keywords:CLEM, cryo-EM, FRET, membrane fusion, resonant energy transfer
Issue or Number:4
Record Number:CaltechAUTHORS:20180927-114223095
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20180927-114223095
Official Citation:Metskas, L., & Briggs, J. (2019). Fluorescence-Based Detection of Membrane Fusion State on a Cryo-EM Grid using Correlated Cryo-Fluorescence and Cryo-Electron Microscopy. Microscopy and Microanalysis, 25(4), 942-949. doi:10.1017/S1431927619000606
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:89994
Collection:CaltechAUTHORS
Deposited By: George Porter
Deposited On:28 Sep 2018 15:03
Last Modified:03 Oct 2019 20:21

Repository Staff Only: item control page