Department of Communication, Computer and System Sciences, University of Genova, Via Opera Pia, 13-16145 Genova, Italy.
Ultramicroscopy. 2010 Mar;110(4):330-8. doi: 10.1016/j.ultramic.2010.01.005. Epub 2010 Jan 28.
The transforming growth factor beta1 (TGF-beta1) is a human cytokine which has been demonstrated to modulate cell surface integrin repertoire. In this work integrin expression in response to TGF-beta1 stimulation has been investigated on the surface of human osteoblast-like cells. We used atomic force microscopy (AFM) and confocal laser scanning microscopy to assess integrin expression and to evaluate their distribution over the dorsal side of the plasma membrane. AFM probes have been covalently functionalized with monoclonal antibodies specific to the beta1 integrin subunit. Force curves have been collected in order to obtain maps of the interaction between the immobilized antibody and the respective cell membrane receptors. Adhesion peaks have been automatically detected by means of an ad hoc developed data analysis software. The specificity of the detected interactions has been assessed by adding free antibody in the solution and monitoring the dramatic decrease in the recorded interactions. In addition, the effect of TGF-beta1 treatment on both the fluorescence signal and the adhesion events has been tested. The level of expression of the beta1 integrin subunit was enhanced by TGF-beta1. As a further analysis, the adhesion force of the single living cells to the substrate was measured by laterally pushing the cell with the AFM tip and measuring the force necessary to displace it. The treatment with TGF-beta1 resulted in a decrease of the cell/substrate adhesion force. Results obtained by AFM have been validated by confocal laser scanning microscopy thus demonstrating the high potential of the AFM technique for the investigation of cell surface receptors distribution and trafficking at the nanoscale.
转化生长因子-β1（TGF-β1）是一种人类细胞因子，已被证明可调节细胞表面整合素谱。在这项工作中，研究了 TGF-β1 刺激对人成骨样细胞表面整合素表达的影响。我们使用原子力显微镜（AFM）和共焦激光扫描显微镜来评估整合素表达，并评估它们在质膜背侧的分布。AFM 探针已通过与β1 整合素亚基特异性的单克隆抗体共价功能化。已收集力曲线，以获得固定化抗体与各自细胞膜受体之间相互作用的图谱。通过专门开发的数据分析软件自动检测粘附峰。通过在溶液中添加游离抗体并监测记录的相互作用急剧减少，评估检测到的相互作用的特异性。此外，还测试了 TGF-β1 处理对荧光信号和粘附事件的影响。TGF-β1 增强了β1 整合素亚基的表达水平。作为进一步的分析，通过用 AFM 尖端侧向推动细胞并测量将其移位所需的力来测量单个活细胞与底物的粘附力。TGF-β1 处理导致细胞/底物粘附力降低。通过共焦激光扫描显微镜验证了 AFM 获得的结果，从而证明了 AFM 技术在纳米尺度上研究细胞表面受体分布和运输的巨大潜力。
