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Measurement of the mechanical behavior of yeast membrane sensors using single-molecule atomic force microscopy

Nature Protocols volume 5, pages 670–677 (2010)Cite this article

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Abstract

In Saccharomyces cerevisiae, surface stresses acting on the cell wall or plasma membrane are detected by a group of five membrane sensors: Wsc1, Wsc2, Wsc3, Mid2 and Mtl2. Here we present protocols to measure the mechanical properties of Wsc1 sensors in their native cellular environment, using the combination of genetic manipulations with single-molecule atomic-force microscopy (AFM). We describe procedures (i) for obtaining genetically modified sensors that are fully functional and suitable for AFM analysis, i.e., elongated Wsc1 derivatives terminated with a His-tag, and (ii) for detecting and stretching single Wsc1 sensors on the surface of living S. cerevisiae cells, using AFM tips functionalized with Ni2+-NTA groups. These procedures are multidisciplinary to implement and need competent researchers from at least two disciplines: molecular biology and nanotechnology. For experienced researchers in biological AFM, the entire protocol can be completed in ∼3 weeks.

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Figure 1: Schematic representation of the cell-wall integrity (CWI) signaling pathway and general structure of a Wsc-type sensor.
Figure 2: In vivo-recombination strategy to obtain an elongated Wsc1 sensor.
Figure 3: Detection and localization of single sensors.
Figure 4: Wsc1 is a nanospring that is sensitive to cell surface stress.

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References

  1. Klis, F.M., Boorsma, A. & De Groot, P.W. Cell wall construction in Saccharomyces cerevisiae. Yeast 23, 185–202 (2006).

    Article  CAS  PubMed  Google Scholar 

  2. Odds, F.C., Brown, A.J. & Gow, N.A. Antifungal agents: Mechanisms of action. Trends Microbiol. 11, 272–279 (2003).

    Article  CAS  PubMed  Google Scholar 

  3. Heinisch, J.J., Lorberg, A., Schmitz, H.-P. & Jacoby, J.J. The protein kinase C-mediated MAP kinase pathway involved in the maintenance of cellular integrity in Saccharomyces cerevisiae. Mol. Microbiol. 32, 671–680 (1999).

    Article  CAS  PubMed  Google Scholar 

  4. Levin, D.E. Cell wall integrity signaling in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 69, 62–291 (2005).

    Article  Google Scholar 

  5. Jung, U.S. & Levin, D.E. Genome-wide analysis of gene expression regulated by the yeast cell wall integrity signalling pathway. Mol. Microbiol. 34, 1049–1057 (1999).

    Article  CAS  PubMed  Google Scholar 

  6. Heinisch, J.J. Bakers yeast as a tool for the development of antifungal drugs which target cell integrity - an update. Expert Opin. Drug Discov. 3, 931–943 (2008).

    Article  CAS  PubMed  Google Scholar 

  7. Philip, B. & Levin, D.E. Wsc1 and Mid2 are cell surface sensors for cell wall integrity signaling that act through Rom2, a guanine nucleotide exchange factor for Rho1. Mol. Cell Biol. 21, 271–280 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Straede, A. & Heinisch, J.J. Functional analyses of the extra- and intracellular domains of the yeast cell wall integrity sensors Mid2 and Wsc1. FEBS Lett. 581, 4495–4500 (2007).

    Article  CAS  PubMed  Google Scholar 

  9. Müller, D.J., Helenius, J., Alsteens, D. & Dufrêne, Y.F. Force probing surfaces of living cells to molecular resolution. Nat. Chem. Biol. 5, 383–390 (2009).

    Article  PubMed  Google Scholar 

  10. Dufrêne, Y.F. Towards nanomicrobiology using atomic force microscopy. Nat. Rev. Microbiol. 6, 674–680 (2008).

    Article  PubMed  Google Scholar 

  11. Hinterdorfer, P., Baumgartner, W., Gruber, H.J., Schilcher, K. & Schindler, H. Detection and localization of individual antibody-antigen recognition events by atomic force microscopy. Proc. Natl. Acad. Sci. USA 93, 3477–3481 (1996).

    Article  CAS  PubMed  Google Scholar 

  12. Rief, M., Gautel, M., Oesterhelt, F., Fernandez, J.M. & Gaub, H.E. Reversible unfolding of individual titin immunoglobulin domains by AFM. Science 276, 1109–1112 (1997).

    Article  CAS  PubMed  Google Scholar 

  13. Oesterhelt, F. et al. Unfolding pathways of individual bacteriorhodopsins. Science 288, 143–146 (2000).

    Article  CAS  PubMed  Google Scholar 

  14. Dupres, V. et al. Nanoscale mapping and functional analysis of individual adhesins on living bacteria. Nat. Methods 2, 515–520 (2005).

    Article  CAS  PubMed  Google Scholar 

  15. Helenius, J., Heisenberg, C.P., Gaub, H.E. & Müller, D.J. Single-cell force spectroscopy. J. Cell Sci. 121, 1785–91 (2008).

    Article  CAS  PubMed  Google Scholar 

  16. Dupres, V. et al. The yeast Wsc1 cell surface sensor behaves like a nanospring in vivo. Nat. Chem. Biol. 5, 857–862 (2009).

    Article  CAS  PubMed  Google Scholar 

  17. Vogel, V. & Sheetz, M. Local force and geometry sensing regulate cell functions. Nat. Rev. Mol. Cell Biol. 7, 265–275 (2006).

    Article  CAS  PubMed  Google Scholar 

  18. Arvanitidis, A. & Heinisch, J.J. Studies on the function of yeast phosphofructokinase subunits by in vitro mutagenesis. J. Biol. Chem. 269, 8911–8918 (1994).

    CAS  PubMed  Google Scholar 

  19. Gietz, D., St. Jean, A., Woods, R.A. & Schiestl, R.H. Improved method for high efficiency transformation of intact yeast cells. Nucleic Acids Res. 20, 1425–1425 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Klebe, R.J., Harriss, J.V., Sharp, Z.D. & Douglas, M.G. A general method for polyethylene-glycol-induced genetic transformation of bacteria and yeast. Gene 25, 333–341 (1983).

    Article  CAS  PubMed  Google Scholar 

  21. Berben, G., Dumont, J., Gilliquet, V., Bolle, P.A. & Hilger, F. The YDp plasmids: A uniform set of vectors bearing versatile gene disruption cassettes for Saccharomyces cerevisiae. Yeast 7, 475–477 (1991).

    Article  CAS  PubMed  Google Scholar 

  22. Francius, G. et al. Stretching polysaccharides on live cells using single molecule force spectroscopy. Nat. Protoc. 4, 939–946 (2009).

    Article  CAS  PubMed  Google Scholar 

  23. Verbelen, C., Gruber, H.J. & Dufrêne, Y.F. The NTA-His6 bond is strong enough for AFM single-molecular recognition studies. J. Mol. Recognit. 20, 490–494 (2007).

    Article  CAS  PubMed  Google Scholar 

  24. Lee, G. et al. Nanospring behaviour of ankyrin repeats. Nature 440, 246–249 (2006).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by the National Foundation for Scientific Research (FNRS), the Université catholique de Louvain (Fonds Spéciaux de Recherche), the Région wallonne, the Federal Office for Scientific, Technical and Cultural Affairs (Interuniversity Poles of Attraction Programme) and the Research Department of the Communauté française de Belgique (Concerted Research Action). Y.F.D. and D.A. are Senior Research Associate and Research Fellow of the FRS-FNRS. Work at the University of Osnabrück was funded by the Deutsche Forschungsgemeinschaft (DFG) within the framework of the SFB431.

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Author notes

  1. Jürgen J Heinisch, Vincent Dupres and Yves F Dufrêne: These authors contributed equally to this work.

Authors and Affiliations

  1. Fachbereich Biologie/Chemie, Universität Osnabrück, Osnabrück, Germany

    Jürgen J Heinisch

  2. Unité de Chimie des Interfaces, Université Catholique de Louvain, Louvain-la-Neuve, Belgium

    Vincent Dupres, David Alsteens & Yves F Dufrêne

Authors
  1. Jürgen J Heinisch
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  2. Vincent Dupres
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  3. David Alsteens
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  4. Yves F Dufrêne
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Contributions

J.J.H., V.D., D.A. and Y.F.D. designed the experiments, analyzed the data and wrote the article. J.J.H. carried out the genetic manipulations whereas V.D. and D.A. collected the AFM data.

Corresponding authors

Correspondence to Jürgen J Heinisch or Yves F Dufrêne.

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Heinisch, J., Dupres, V., Alsteens, D. et al. Measurement of the mechanical behavior of yeast membrane sensors using single-molecule atomic force microscopy. Nat Protoc 5, 670–677 (2010). https://doi.org/10.1038/nprot.2010.19

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