Cai Ning, Wong Chee C, Tan Samuel C W, Chan Vincent, Liao Kin
School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore 639798.
Langmuir. 2009 Sep 15;25(18):10939-47. doi: 10.1021/la901527x.
Cell adhesion is generally concomitant to the formation of focal adhesion. Although it is well-known that focal adhesion plays an important role in the functional regulations of anchorage dependent cells, previous experimental studies have not provided quantitative description of the relation between focal adhesion and biophysical responses of cells. Furthermore, there is lack of knowledge on the importance of the beta1 integrin subunit to the dynamic responses of cells during initial cell seeding. In this study, we attempt to bridge the quantitative connection between focal adhesion density and cell-substrate interactions and evaluate the influence on functional blocking of beta1 integrin on adhesion strength. Total internal reflection fluorescence microscopy (TIRFM), fluorescence microscopy, and phase contrast microscopy was employed to study the time-dependent evolvement of vinculin pattern, distribution of actin filament, and morphological change, respectively, during 4 h of culture for porcine esophageal fibroblasts (non-blocked and beta1-blocked) on a fibronectin-coated surface. Micropipet aspiration technique was used to study the change of mechanotransduction through the determination of adhesion force and strength. It is shown in our experimental results that spread area, adhesion force, and adhesion strength increases over time on the two types of cells. Throughout the culture period, the two key mechanotransduction parameters of non-blocked cells is higher than those of beta1-blocked cells. Interestingly, adhesion strength initially ascends, then begins to diminish at a critical time point, and finally resumes increasing linearly against the increase of focal adhesion density. This variation as mentioned above can be explained by peeling and fracture models based on the dissimilar vinculin pattern of cells after being cultured for different time periods. Moreover, the averaged focal adhesion strength and non-focal adhesion strength of beta1-blocked cells are significantly less than those of non-blocked of cells. The weaker adhesion strength on beta(1)-blocked cells is directly caused by lower focal and non-focal adhesion strength, as well as by smaller focal adhesion density.
细胞黏附通常与黏着斑的形成相伴发生。尽管众所周知黏着斑在锚定依赖性细胞的功能调节中起着重要作用，但先前的实验研究尚未对黏着斑与细胞生物物理反应之间的关系进行定量描述。此外，对于β1整合素亚基在细胞初始接种过程中对细胞动态反应的重要性缺乏了解。在本研究中，我们试图建立黏着斑密度与细胞-基质相互作用之间的定量联系，并评估β1整合素功能阻断对黏附强度的影响。采用全内反射荧光显微镜（TIRFM）、荧光显微镜和相差显微镜，分别研究猪食管成纤维细胞（未阻断和β1阻断）在纤连蛋白包被表面培养4小时期间纽蛋白模式的时间依赖性演变、肌动蛋白丝的分布和形态变化。使用微吸管抽吸技术通过测定黏附力和强度来研究机械转导的变化。我们的实验结果表明，两种类型的细胞上的铺展面积、黏附力和黏附强度均随时间增加。在整个培养期间，未阻断细胞的两个关键机械转导参数高于β1阻断细胞。有趣的是，黏附强度最初上升，然后在一个关键时间点开始下降，最后随着黏着斑密度的增加又恢复线性增加。上述变化可以用基于细胞在不同培养时间段后不同纽蛋白模式的剥离和断裂模型来解释。此外，β1阻断细胞的平均黏着斑强度和非黏着斑强度明显低于未阻断细胞。β1阻断细胞上较弱的黏附强度直接由较低的黏着斑和非黏着斑强度以及较小的黏着斑密度引起。
