Franz Clemens M, Taubenberger Anna, Puech Pierre-Henri, Muller Daniel J
BioTechnological Center, University of Technology Dresden, Tatzberg 47-51, 01307 Dresden, Germany.
Sci STKE. 2007 Oct 2;2007(406):pl5. doi: 10.1126/stke.4062007pl5.
The establishment of cell adhesion involves specific recognition events between individual cell-surface receptors and molecules of the cellular environment. However, characterizing single-molecule adhesion events in the context of a living cell presents an experimental challenge. The atomic force microscope (AFM) operated in force spectroscopy mode provides an ultrasensitive method to investigate cell adhesion forces at the level of single receptor-ligand bonds. With a living cell attached to the AFM cantilever, the number of cell-substrate interactions can be controlled and limited to the formation of single receptor-ligand bonds. From force-distance (F-D) curves recorded during cell detachment, the strength of single receptor-ligand bonds can be determined. Furthermore, by varying the rate of force application during bond rupture, a dynamic force spectrum (DFS) can be generated from which additional parameters that describe the energy landscape of the interaction, such as dissociation rate and energy barrier width, can be obtained. Using the example of alpha(2)beta(1) integrin-mediated adhesion to type I collagen, we provide a detailed description of how dynamic AFM single-cell force spectroscopy (SCFS) adhesion measurements can be performed with single-molecule sensitivity, and how specific energy landscape parameters of the integrin-collagen bond can be extracted from the DFS.
细胞黏附的建立涉及单个细胞表面受体与细胞外环境分子之间的特异性识别事件。然而,在活细胞环境中表征单分子黏附事件面临着实验挑战。以力谱模式操作的原子力显微镜(AFM)提供了一种超灵敏的方法,可在单受体-配体键水平上研究细胞黏附力。将活细胞附着于AFM悬臂上,细胞与底物之间的相互作用数量可得到控制,并限制在形成单个受体-配体键的范围内。从细胞脱离过程中记录的力-距离(F-D)曲线,可以确定单个受体-配体键的强度。此外,通过改变键断裂过程中的力施加速率,可以生成动态力谱(DFS),从中可以获得描述相互作用能量态势的其他参数,如解离速率和能垒宽度。以α(2)β(1)整合素介导的与I型胶原的黏附为例,我们详细描述了如何以单分子灵敏度进行动态AFM单细胞力谱(SCFS)黏附测量,以及如何从DFS中提取整合素-胶原键的特定能量态势参数。