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Stretching polysaccharides on live cells using single molecule force spectroscopy

Nature Protocols volume 4, pages 939–946 (2009)Cite this article

Abstract

The knowledge of molecular mechanisms underlying the adhesive and mechanical properties of cell surface-associated molecules is a key to understanding their functions. In this context, single-molecule force spectroscopy (SMFS) has recently offered new opportunities for probing the adhesion and mechanics of polysaccharides and proteins on live cells. Here we present a protocol that we have used to analyze polysaccharide chains of different nature on the bacterium Lactobacillus rhamnosus GG. We describe procedures (i) for functionalizing atomic force microscopy (AFM) tips with Pseudomonas aeruginosa–I or concanavalin A lectins, (ii) for stretching specific polysaccharide molecules on live bacteria using SMFS with lectin tips and (iii) for mapping the localization, adhesion and extension of individual polysaccharide chains. We also discuss data treatment, emphasizing how to gain insight into the elasticity of the stretched macromolecules using the extended freely jointed chain model. Even though the presented protocol is for L. rhamnosus, it can be easily modified for other cell types. For users having expertise in the field, the entire protocol can be completed in about 5 d.

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Figure 1: Stretching single-polysaccharide molecules on Lactobacillus rhamnosus GG (LGG) using SMFS with lectin-modified tips.
Figure 2: Mapping the localization, adhesion and extension of individual polysaccharide chains.
Figure 3: The CMPG5413 mutant strain impaired in cell adherence and polysaccharide production displays very different polymer properties.
Figure 4: Stretching single mannoproteins on the yeast Saccharomyces cerevisiae.

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Acknowledgements

We wish to thank our colleagues and collaborators for sharing exciting experiments and discussions. 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), the Research Department of the Communauté française de Belgique (Concerted Research Action) and Fonds voor Wetenschappelijk Onderzoek-Vlaanderen (FWO). D.A. and Y.F.D. are Research Fellow and Senior Research Associate, respectively at the FRS-FNRS.

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Author notes

  1. Grégory Francius and David Alsteens: These authors contributed equally to this work.

Authors and Affiliations

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

    Grégory Francius, David Alsteens, Vincent Dupres & Yves F Dufrêne

  2. Centre of Microbial and Plant Genetics and INPAC, K.U. Leuven, Leuven, Belgium

    Sarah Lebeer, Sigrid De Keersmaecker & Jos Vanderleyden

  3. Institute for Biophysics, Johannes Kepler University of Linz, Linz, Austria

    Hermann J Gruber

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  1. Grégory Francius
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  2. David Alsteens
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Correspondence to Yves F Dufrêne.

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Francius, G., Alsteens, D., Dupres, V. et al. Stretching polysaccharides on live cells using single molecule force spectroscopy. Nat Protoc 4, 939–946 (2009). https://doi.org/10.1038/nprot.2009.65

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