Francius Grégory, Alsteens David, Dupres Vincent, Lebeer Sarah, De Keersmaecker Sigrid, Vanderleyden Jos, Gruber Hermann J, Dufrêne Yves F
Unité de Chimie des Interfaces, Université Catholique de Louvain, Louvain-la-Neuve, Belgium.
Nat Protoc. 2009;4(6):939-46. doi: 10.1038/nprot.2009.65. Epub 2009 May 28.
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.
了解细胞表面相关分子的粘附和力学特性背后的分子机制是理解其功能的关键。在这种背景下，单分子力谱（SMFS）最近为探究活细胞上多糖和蛋白质的粘附及力学特性提供了新机会。在此，我们展示一种我们用于分析鼠李糖乳杆菌GG上不同性质多糖链的方案。我们描述了以下步骤：（i）用铜绿假单胞菌-I或伴刀豆球蛋白A凝集素对原子力显微镜（AFM）探针进行功能化；（ii）使用带有凝集素探针的SMFS在活细菌上拉伸特定多糖分子；（iii）绘制单个多糖链的定位、粘附和伸展情况。我们还讨论了数据处理，重点强调如何使用扩展自由连接链模型深入了解拉伸大分子的弹性。尽管所展示的方案是针对鼠李糖乳杆菌的，但它可以很容易地针对其他细胞类型进行修改。对于该领域有专业知识的用户，整个方案大约可以在5天内完成。
