Abstract
Over the past five years, atomic force microscopy (AFM)-based approaches have evolved into a powerful multiparametric tool set capable of imaging the surfaces of biological samples ranging from single receptors to membranes and tissues. One of these approaches, force–distance curve-based AFM (FD-based AFM), uses a probing tip functionalized with a ligand to image living cells at high-resolution and simultaneously localize and characterize specific ligand–receptor binding events. Analyzing data from FD-based AFM experiments using appropriate probabilistic models allows quantification of the kinetic and thermodynamic parameters that describe the free-energy landscape of the ligand–receptor bond. We have recently developed an FD-based AFM approach to quantify the binding events of single enveloped viruses to surface receptors of living animal cells while simultaneously observing them by fluorescence microscopy. This approach has provided insights into the early stages of the interaction between a virus and a cell. Applied to a model virus, we probed the specific interaction with cells expressing viral cognate receptors and measured the affinity of the interaction. Furthermore, we observed that the virus rapidly established specific multivalent interactions and found that each bond formed in sequence strengthened the attachment of the virus to the cell. Here we describe detailed procedures for probing the specific interactions of viruses with living cells; these procedures cover tip preparation, cell sample preparation, step-by-step FD-based AFM imaging and data analysis. Experienced microscopists should be able to master the entire set of protocols in 1 month.
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Acknowledgements
We thank our colleagues and collaborators for sharing exciting experiments and discussions. This protocol owes much to previous work from the labs of S. Seung (MIT; ΔGRabies propagation), D. Trono (École polytechnique fédérale de Lausanne; lentivirus production) and H. Gruber (Johannes Kepler University Linz; cantilever functionalization). We thank K. Yonehara (Aarhus University) and members of the B. Roska lab (Friedrich Miescher Institute), particularly J. Jüttner and K. Balint, for their valuable help in modifying the virus production protocols to suit our needs. We thank K. Conzelmann (Ludwig-Maximilans University), S. Finke. (Friedrich-Loeffler Institute) and B. Roska for kindly providing stocks of ΔGRabies. The plasmid pAAV-EF1α-FLEX-TVA-mCherry was a gift from N. Uchida (Harvard University) and the plasmids pRRLSIN.cppt.PGK-GFP.WPRE, pMD2.G and pCMV-dR8.74 were gifts from D. Trono. We thank M. Mohr (ETH Zurich) for assistance in sub-cloning the pRRLSIN.cppt.EF1α plasmid. This work was supported by the National Foundation for Scientific Research (FNRS), the Université catholique de Louvain (Fonds Spéciaux de Recherche), the 'MOVE-IN Louvain' incoming post-doc fellowship programme, the Swiss National Science Foundation (SNF; grant no. 310030B_160225) and NCCR Molecular Systems Engineering. D.A. is a research associate at the FRS-FNRS.
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R.N., M.D., D.J.M. and D.A. designed and performed the experiments. R.N., M.D., M.K., A.C.D., P.R.L., D.J.M. and D.A. wrote the paper.
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D.J.M. and D.A. have applied for a patent for the chamber enabling AFM and optical microscopy under cell-culture conditions (EP15002176.4). The other authors declare no competing financial interests.
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Newton, R., Delguste, M., Koehler, M. et al. Combining confocal and atomic force microscopy to quantify single-virus binding to mammalian cell surfaces. Nat Protoc 12, 2275–2292 (2017). https://doi.org/10.1038/nprot.2017.112
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DOI: https://doi.org/10.1038/nprot.2017.112
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