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Project: Membrane Fusion on EM Grids

Current cryo-CLEM methods suffer from one functional weakness: they are based upon tagging a specific protein or membrane, but cannot discriminate between subgroups based on functions of interest. In the Fusion on EM Grids project, we adapted fusion assays to the conditions of cryo-CLEM in order to specifically target viruses undergoing fusion reactions. Using these assays, we can distinguish between a tight membrane apposition and hemifusion, which are not easily distinguished by electron microscopy alone.

Metskas LA, Briggs JAG (2019) Fluorescence-Based Detection of Membrane Fusion State on a Cryo-EM Grid using Correlated Cryo-Fluorescence and Cryo-Electron Microscopy. Microsc Microanal 25, 942–949. doi:10.1017/S1431927619000606

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Figure: Top: cryo CLEM overlay of a fusion reaction between influenza virus-like particles (HK68 strain) and synthetic lipid vesicles.  Center: fluorescence intensity plots for yellow-boxed areas in the cryo CLEM images.  Letters correspond to features below.  Bottom: higher-magnification of features of interest on the grid, corresponding to adhesion, hemifusion, vesicles alone, and virus-like particles alone. Letters correspond to labels in the center fluorescence traces.  The hemifusion (D) has the strongest fluorescence intensity due to dequenching of the dye; the quenched vesicles have intermediate intensity (C, G), and unlabeled virus-like particles have no detectable fluorescence signal (H).

This tool will be very useful for the study of virus-host interactions, but we also learned a lot about fluorophore behavior in cryo CLEM that will assist future methods developments.

The back-story:

  • This work spanned three countries!  The project began in the Briggs lab at EMBL Heidelberg (Germany) with the labeling, VLP preparation, and fusion images.  Controls and finer characterization took place at MRC Laboratory of Molecular Biology (Cambridge, UK) following the Briggs lab move.  The manuscript was written after Dr. Metskas moved to a postdoc position at Caltech (Pasadena, USA).
  • This work was funded by an ERC grant to Dr. Briggs.
  • We owe a huge debt of thanks to the Kukulski group at the LMB for letting us use their Leica cryo CLEM to finish this work following our lab move.
  • We submitted first to Biophys J. as a letter.  The reviewers asked everything to be done inside cells (basically an entire second paper worth of experiments!), so we pulled the manuscript and re-submitted to Microscopy & Microanalysis, where it was accepted without additional large-scale experiments.
  • The paper was awarded Best Biological Sciences paper for 2019 in the journal, and was featured in Microscopy Today.
  • Time from first pipet tip to proof: 2 years 10 months.