Skip to main content Accessibility help
×
Home

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

Published online by Cambridge University Press:  14 May 2019


Lauren Ann Metskas
Affiliation:
Structural and Computational Biology Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany Structural Studies Division, MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK
John A. G. Briggs
Affiliation:
Structural and Computational Biology Unit, European Molecular Biology Laboratory, 69117 Heidelberg, Germany Structural Studies Division, MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK
Corresponding
E-mail address:

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.


Type
Biological Applications
Creative Commons
This is an Open Access article, distributed under the terms of the Creative Commons Attribution licence (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted re-use, distribution, and reproduction in any medium, provided the original work is properly cited.
Copyright
Copyright © MRC Laboratory of Molecular Biology 2019

Footnotes

Present address: Biology and Biological Engineering Department, California Institute of Technology, Pasadena, CA 91106, USA.


References

Benchimol, MJ, Hsu, MJ, Schutt, CE, Hall, DJ, Mattrey, RF & Esener, SC (2013). Phospholipid/carbocyanine dye-shelled microbubbles as ultrasound-modulated fluorescent contrast agents. Soft Matter 9, 23842388.CrossRefGoogle ScholarPubMed
Blumenthal, R, Gallo, SA, Viard, M, Raviv, Y & Puri, A (2002). Fluorescent lipid probes in the study of viral membrane fusion. Chem Phys Lipids 116, 3955.CrossRefGoogle Scholar
Bright, NA, Gratian, MJ & Luzio, JP (2005). Endocytic delivery to lysosomes mediated by concurrent fusion and kissing events in living cells. Curr Biol 15, 360365.CrossRefGoogle ScholarPubMed
Bykov, YS, Cortese, M, Briggs, JAG & Bartenschlager, R (2016). Correlative light and electron microscopy methods for the study of virus–cell interactions. FEBS Lett 590, 18771895.CrossRefGoogle Scholar
Chang, Y-W, Chen, S, Tocheva, EI, Treuner-Lange, A, Löbach, S, Søgaard-Andersen, L & Jensen, GJ (2014). Correlated cryogenic photoactivated localization microscopy and cryo-electron tomography. Nat Methods 11, 737739.CrossRefGoogle ScholarPubMed
Chlanda, P, Mekhedov, E, Waters, H, Schwartz, CL, Fischer, ER, Ryham, RJ, Cohen, FS, Blank, PS & Zimmerberg, J (2016). The hemifusion structure induced by Influenza virus haemagglutinin is determined by physical properties of the target membranes. Nat Microbiol 1, 16050.CrossRefGoogle ScholarPubMed
Chlanda, P, Schraidt, O, Kummer, S, Riches, J, Oberwinkler, H, Prinz, S, Kräusslich, H-G & Briggs, JAG (2015). Structural analysis of the roles of influenza a virus membrane-associated proteins in assembly and morphology. J Virol 89, 89578966.CrossRefGoogle ScholarPubMed
Creemers, TMH, Lock, AJ, Subramaniam, V, Jovin, TM & Völker, S (2000). Photophysics and optical switching in green fluorescent protein mutants. Proc Natl Acad Sci USA 97, 29742978.CrossRefGoogle ScholarPubMed
Faro, AR, Adam, V, Carpentier, P, Darnault, C, Bourgeois, D & de Rosny, E (2010). Low-temperature switching by photoinduced protonation in photochromic fluorescent proteins. Photochem Photobiol Sci 9, 254262.CrossRefGoogle ScholarPubMed
Floyd, DL, Ragains, JR, Skehel, JJ, Harrison, SC & van Oijen, AM (2008). Single-particle kinetics of influenza virus membrane fusion. Proc Natl Acad Sci USA 105, 1538215387.CrossRefGoogle ScholarPubMed
Gui, L, Ebner, JL, Mileant, A, Williams, JA & Lee, KK (2016). Visualization and sequencing of membrane remodeling leading to influenza virus fusion. J Virol 90, 69486962.CrossRefGoogle ScholarPubMed
Kaufmann, R, Hagen, C & Grünewald, K (2014). Fluorescence cryo-microscopy: Current challenges and prospects. Curr Opin Chem Biol 20, 8691.CrossRefGoogle ScholarPubMed
Kopek, BG, Shtengel, G, Xu, CS, Clayton, DA & Hess, HF (2012). Correlative 3D superresolution fluorescence and electron microscopy reveal the relationship of mitochondrial nucleoids to membranes. Proc Natl Acad Sci USA 109, 61366141.CrossRefGoogle ScholarPubMed
Kreye, S, Malsam, J & Söllner, TH (2008). In vitro assays to measure SNARE-mediated vesicle fusion. Methods Mol Biol (Clifton, N.J.) 440, 3750.CrossRefGoogle ScholarPubMed
Kukulski, W, Schorb, M, Kaksonen, M & Briggs, JAG (2012 a). Plasma membrane reshaping during endocytosis is revealed by time-resolved electron tomography. Cell 150, 508520.CrossRefGoogle ScholarPubMed
Kukulski, W, Schorb, M, Welsch, S, Picco, A, Kaksonen, M & Briggs, JAG (2012 b). Precise, correlated fluorescence microscopy and electron tomography of lowicryl sections using fluorescent fiducial markers. Methods Cell Biol 111, 235257.CrossRefGoogle ScholarPubMed
Lakadamyali, M, Rust, MJ, Babcock, HP & Zhuang, X (2003). Visualizing infection of individual influenza viruses. Proc Natl Acad Sci USA 100, 92809285.CrossRefGoogle ScholarPubMed
Lakowicz, J (2006). Principles of Fluorescence Spectroscopy III. New York, NY: Springer Science+Business Media.CrossRefGoogle Scholar
Lebrun, M, Thelen, N, Thiry, M, Riva, L, Ote, I, Condé, C, Vandevenne, P, Di Valentin, E, Bontems, S & Sadzot-Delvaux, C (2014). Varicella-zoster virus induces the formation of dynamic nuclear capsid aggregates. Virology 454–455, 311327.CrossRefGoogle ScholarPubMed
Li, F, Sewald, X, Jin, J, Sherer, NM & Mothes, W (2014). Murine Leukemia virus gag localizes to the uropod of migrating primary lymphocytes. J Virol 88, 1054110555.CrossRefGoogle ScholarPubMed
Martinez, MG, Snapp, E-L, Perumal, GS, Macaluso, FP & Kielian, M (2014). Imaging the alphavirus exit pathway. J Virol 88, 69226933.CrossRefGoogle ScholarPubMed
Mastronarde, DN (2005). Automated electron microscope tomography using robust prediction of specimen movements. J Struct Biol 152, 3651.CrossRefGoogle ScholarPubMed
Moerner, WE & Orrit, M (1999). Illuminating single molecules in condensed matter. Science (New York, N.Y.) 283, 16701676.CrossRefGoogle ScholarPubMed
Muller-Reichert, T & Verkade, P (eds) (2017). Correlative Light and Electron Microscopy III, Vol. 140. Amsterdam, Netherlands: Elsevier.Google Scholar
Romero-Brey, I, Berger, C, Kallis, S, Kolovou, A, Paul, D, Lohmann, V & Bartenschlager, R (2015). NS5A domain 1 and polyprotein cleavage kinetics are critical for induction of double-membrane vesicles associated with hepatitis C virus replication. mBio 6, e00759.Google ScholarPubMed
Rust, MJ, Lakadamyali, M, Zhang, F & Zhuang, X (2004). Assembly of endocytic machinery around individual influenza viruses during viral entry. Nat Struct Mol Biol 11, 567573.CrossRefGoogle ScholarPubMed
Sartori, A, Gatz, R, Beck, F, Rigort, A, Baumeister, W & Plitzko, JM (2007). Correlative microscopy: bridging the gap between fluorescence light microscopy and cryo-electron tomography. J Struct Biol 160, 135145.CrossRefGoogle ScholarPubMed
Schindelin, J, Arganda-Carreras, I, Frise, E, Kaynig, V, Longair, M, Pietzsch, T, Preibisch, S, Rueden, C, Saalfeld, S, Schmid, B, Tinevez, J-Y, White, DJ, Hartenstein, V, Eliceiri, K, Tomancak, P & Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. Nat Methods 9, 676682.CrossRefGoogle ScholarPubMed
Schneider, CA, Rasband, WS & Eliceiri, KW (2012). NIH image to ImageJ: 25 years of image analysis. Nat Methods 9, 671675.CrossRefGoogle ScholarPubMed
Schorb, M, Gaechter, L, Avinoam, O, Sieckmann, F, Clarke, M, Bebeacua, C, Bykov, YS, Sonnen, AF-P, Lihl, R & Briggs, JAG (2017). New hardware and workflows for semi-automated correlative cryo-fluorescence and cryo-electron microscopy/tomography. J Struct Biol 197, 8393.CrossRefGoogle ScholarPubMed
Schwartz, CL, Sarbash, VI, Ataullakhanov, FI, McIntosh, JR & Nicastro, D (2007). Cryo-fluorescence microscopy facilitates correlations between light and cryo-electron microscopy and reduces the rate of photobleaching. J Microsc 227, 98109.CrossRefGoogle ScholarPubMed
Wolff, G, Hagen, C, Grünewald, K & Kaufmann, R (2016). Towards correlative super-resolution fluorescence and electron cryo-microscopy. Biol Cell 108, 245258.CrossRefGoogle ScholarPubMed
Zondervan, R, Kulzer, F, Orlinskii, SB & Orrit, M (2003). Photoblinking of Rhodamine 6G in poly(vinyl alcohol): radical dark state formed through the triplet. J Phys Chem A 107, 67706776.CrossRefGoogle Scholar

Altmetric attention score


Full text views

Full text views reflects PDF downloads, PDFs sent to Google Drive, Dropbox and Kindle and HTML full text views.

Total number of HTML views: 123
Total number of PDF views: 447 *
View data table for this chart

* Views captured on Cambridge Core between 14th May 2019 - 4th December 2020. This data will be updated every 24 hours.

Access
Open access
Hostname: page-component-b4dcdd7-lltvg Total loading time: 0.361 Render date: 2020-12-04T19:05:05.927Z Query parameters: { "hasAccess": "1", "openAccess": "1", "isLogged": "0", "lang": "en" } Feature Flags last update: Fri Dec 04 2020 19:00:26 GMT+0000 (Coordinated Universal Time) Feature Flags: { "metrics": true, "metricsAbstractViews": false, "peerReview": true, "crossMark": true, "comments": true, "relatedCommentaries": true, "subject": true, "clr": false, "languageSwitch": true }

Send article to Kindle

To send this article to your Kindle, first ensure no-reply@cambridge.org is added to your Approved Personal Document E-mail List under your Personal Document Settings on the Manage Your Content and Devices page of your Amazon account. Then enter the ‘name’ part of your Kindle email address below. Find out more about sending to your Kindle. Find out more about sending to your Kindle.

Note you can select to send to either the @free.kindle.com or @kindle.com variations. ‘@free.kindle.com’ emails are free but can only be sent to your device when it is connected to wi-fi. ‘@kindle.com’ emails can be delivered even when you are not connected to wi-fi, but note that service fees apply.

Find out more about the Kindle Personal Document Service.

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

Send article to Dropbox

To send this article to your Dropbox account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your <service> account. Find out more about sending content to Dropbox.

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

Send article to Google Drive

To send this article to your Google Drive account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your <service> account. Find out more about sending content to Google Drive.

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

Reply to: Submit a response


Your details


Conflicting interests

Do you have any conflicting interests? *