Detecting cell-adhesive sites in extracellular matrix using force spectroscopy mapping
Detecting cell-adhesive sites in extracellular matrix using force spectroscopy mapping
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DOI：
10.1088/0953-8984/22/19/194102
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发表时间：
2010-05-19
影响因子：
2.7
通讯作者：
Engler, Adam J.
中科院分区：
文献类型：
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作者：
Chirasatitsin, Somyot;Engler, Adam J.
细胞微环境由细胞外基质(ECM)组成，细胞外基质包含特定的结合部位，使细胞能够与周围环境黏附。细胞使用焦点黏附蛋白，它必须能够抵抗各种力量与细胞外基质结合。然而，目前用于检测这些粘连的空间排列的技术分辨率有限，并且那些检测粘附力的技术缺乏足够的空间特征或分辨率。使用一种独特的力光谱应用，我们在这里展示了能够确定纤维连接蛋白底物粘附性的局部变化，直到纤维连接蛋白抗体功能化的尖端直径的分辨率，类似于20 nm。为了验证力光谱映射(FSM)的检测能力，通过改变加载速度和温度来改变键动力学和粘附力。为了模拟天然细胞外基质的不连续粘附域，我们还利用微接触印迹技术对荧光素异硫氰酸酯偶联的纤维连接蛋白进行了研究。荧光检测被用来识别模式，而FSM被用来与初始荧光图像配准绘制细胞粘附点。结果表明，有限状态机可用于高分辨率的粘附域检测，并可应用于具有随机分布的细胞粘附区的天然ECM。
The cell microenvironment is composed of extracellular matrix (ECM), which contains specific binding sites that allow the cell to adhere to its surroundings. Cells employ focal adhesion proteins, which must be able to resist a variety of forces to bind to ECM. Current techniques for detecting the spatial arrangement of these adhesions, however, have limited resolution and those that detect adhesive forces lack sufficient spatial characterization or resolution. Using a unique application of force spectroscopy, we demonstrate here the ability to determine local changes in the adhesive property of a fibronectin substrate down to the resolution of the fibronectin antibody-functionalized tip diameter, similar to 20 nm. To verify the detection capabilities of force spectroscopy mapping (FSM), changes in loading rate and temperature were used to alter the bond dynamics and change the adhesion force. Microcontact printing was also used to pattern fluorescein isothiocyanate-conjugated fibronectin in order to mimic the discontinuous adhesion domains of native ECM. Fluorescent detection was used to identify the pattern while FSM was used to map cell adhesion sites in registry with the initial fluorescent image. The results show that FSM can be used to detect the adhesion domains at high resolution and may subsequently be applied to native ECM with randomly distributed cell adhesion sites.