Abstract
We describe an engineered family of highly antigenic molecules based on GFP-like fluorescent proteins. These molecules contain numerous copies of peptide epitopes and simultaneously bind IgG antibodies at each location. These 'spaghetti monster' fluorescent proteins (smFPs) distributed well in neurons, notably into small dendrites, spines and axons. smFP immunolabeling localized weakly expressed proteins not well resolved with traditional epitope tags. By varying epitope and scaffold, we generated a diverse family of mutually orthogonal antigens. In cultured neurons and mouse and fly brains, smFP probes allowed robust, orthogonal multicolor visualization of proteins, cell populations and neuropil. smFP variants complement existing tracers and greatly increase the number of simultaneous imaging channels, and they performed well in advanced preparations such as array tomography, super-resolution fluorescence imaging and electron microscopy. In living cells, the probes improved single-molecule image tracking and increased yield for RNA-seq. These probes facilitate new experiments in connectomics, transcriptomics and protein localization.
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Acknowledgements
We thank the Cell Culture, Vivarium, Fly Facility, Histology, Electron Microscopy, Media and Molecular Biology Shared Resources, and the Fly Light Project Team, at Janelia. N. Betley, A. Hantman, J. Colonell, J.-C. Rah, S. Sengupta, M. Baird and G. Tervo provided helpful discussions. H. Su and H. Kimura helped with reagents, B. Karsh assisted with image alignment for immunoEM and AT, and H. Rouault helped with statistical analysis. Members of the Looger lab and M. Jefferies provided helpful feedback during the project. A. Hantman and K. Ritola (Janelia) provided the mTagBFP2 virus. This work was supported by the Howard Hughes Medical Institute.
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Contributions
S.V., G.M.R. and L.L.L. conceived of the project. L.L.L. performed molecular modeling and designed sequences. S.V. and B.D.P. constructed the clones. S.V. and M.E.W. performed experiments in cultured neurons. M.E.W. performed hippocampal neuron work. B.M.H. and C.R.G. performed four-color labeling experiments. E.B.B. performed AT experiments. C.M.S. and X.Z. designed STORM experiments, and C.M.S. performed STORM imaging and analyzed data. J.J.M. and R.P. performed biophysical characterization. A.N. performed fly experiments. W.-P.L. and Y.W. performed EM. T.J.S. and B.P.E. performed single-molecule imaging. T.T. and G.L.H. performed pulldown experiments. S.V. and L.L.L. led the project.
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Supplementary Text and Figures
Supplementary Figures 1–18 and Supplementary Tables 1 and 2 (PDF 47159 kb)
Supplementary Video 1
Single-molecule tracking of H2B molecules labelled with EGFP, Halo-tag and Alexa488 substrate, and smFP_FLAG and anti-FLAG antibody (AVI 3130 kb)
Supplementary Video 2
Movie showing 41 silver enhanced immunogold labelled sections expressing smFP_FLAG (MOV 36148 kb)
Supplementary Video 3
3D reconstruction of smFP_FLAG immunogold labelled dendritic segment (AVI 13473 kb)
Supplementary Video 4
Movie showing 30 silver enhanced immunogold labelled sections expressing smFP_FLAG and smFP_myc (MOV 43546 kb)
Supplementary Video 5
3D reconstruction of smFP_FLAG and smFP_myc immunogold labelled dendritic segment (AVI 9003 kb)
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Viswanathan, S., Williams, M., Bloss, E. et al. High-performance probes for light and electron microscopy. Nat Methods 12, 568–576 (2015). https://doi.org/10.1038/nmeth.3365
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DOI: https://doi.org/10.1038/nmeth.3365
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