Gudzenko Tetyana, Franz Clemens M
DFG-Center for Functional Nanostructures, Karlsruhe Institute of Technology, Karlsruhe, Germany.
WPI Nano Life Science Institute, Kanazawa University, Kanazawa, Japan.
Front Mol Biosci. 2020 Jul 8;7:149. doi: 10.3389/fmolb.2020.00149. eCollection 2020.
We previously developed a surface-assisted assay to image early steps of cell-induced plasma fibronectin (FN) fibrillogenesis by timelapse atomic force microscopy (AFM). Unexpectedly, complementary attempts to visualize FN fibrillogenesis using fluorescently labeled FN (Alexa Fluor 488 or 568) and live-cell light microscopy initially failed consistently. Further analysis revealed that fibrillar remodeling was inhibited efficiently in the focal area illuminated during fluorescence imaging, but progressed normally elsewhere on the substrate, suggesting photo sensitivity of the FN fibrillogenesis process. In agreement, active cell-driven fibrillar extension of FN could be stopped by transient illumination with visible light during AFM timelapse scanning. Phototoxic effects on the cells could be ruled out, because pre-illuminating the FN layer before cell seeding also blocked subsequent fibrillar formation. Varying the illumination wavelength range between 400 and 640 nm revealed strong inhibition across the visible spectrum up to 560 nm, and a decreasing inhibitory effect at longer wavelengths. The photo effect also affected unlabeled FN, but was enhanced by fluorophore labeling of FN. The inhibitory effect could be reduced when reactive oxygen species (ROS) were removed for the cell imaging medium. Based on these findings, FN fibrillogenesis could be imaged successfully using a labeling dye with a long excitation wavelength (Alexa Fluor 633, excitation at 632 nm) and ROS scavengers, such as oxyrase, in the imaging medium. Fibrillar remodeling of exposed cell-free FN layers by AFM scanning required higher scan forces compared to non-exposed FN, consisting with mechanical stiffing of the FN layer after illumination. In agreement with changes in FN mechanics, cells spreading on pre-exposed FN showed reduced migration speeds, altered focal adhesion arrangement, and changes in mechanosensitive signaling pathways, including reduced FAK (Y397) and paxillin (Y118) phosphorylation. Pre-exposure of FN to visible light prior to cell seeding thus provides a useful tool to delineate mechanosensitive signaling pathway related to FN fibrillogenesis. When using FN-coated cell adhesion substrates, care should be taken when comparing experimental results obtained on non-exposed FN layers in cell culture incubators, or during live-cell fluorescence imaging, as FN fibrillogenesis and mechanosensitive cellular signaling pathways may be affected differently.
我们之前开发了一种表面辅助检测方法，通过延时原子力显微镜（AFM）对细胞诱导的血浆纤连蛋白（FN）纤维形成的早期步骤进行成像。出乎意料的是，最初使用荧光标记的FN（Alexa Fluor 488或568）和活细胞光学显微镜可视化FN纤维形成的互补尝试一直失败。进一步分析表明，在荧光成像过程中被照亮的焦点区域，纤维重塑被有效抑制，但在底物的其他位置正常进行，这表明FN纤维形成过程具有光敏感性。同样，在AFM延时扫描期间，用可见光短暂照射可阻止FN由活跃细胞驱动的纤维延伸。可以排除对细胞的光毒性作用，因为在细胞接种前对FN层进行预照射也会阻止随后的纤维形成。在400至640nm之间改变照射波长范围显示，在高达560nm的可见光谱范围内有强烈抑制作用，而在较长波长时抑制作用减弱。光效应也影响未标记的FN，但通过FN的荧光团标记会增强。当从细胞成像培养基中去除活性氧（ROS）时，抑制作用会降低。基于这些发现，使用长激发波长的标记染料（Alexa Fluor 633，在632nm激发）和成像培养基中的ROS清除剂（如氧化酶），可以成功地对FN纤维形成进行成像。与未暴露的FN相比，通过AFM扫描对暴露的无细胞FN层进行纤维重塑需要更高的扫描力，这与照射后FN层的机械硬化一致。与FN力学变化一致，在预先暴露的FN上铺展的细胞显示迁移速度降低、粘着斑排列改变以及机械敏感信号通路的变化，包括FAK（Y397）和桩蛋白（Y118）磷酸化减少。因此，在细胞接种前将FN预先暴露于可见光下，为描绘与FN纤维形成相关的机械敏感信号通路提供了一种有用的工具。当使用FN包被的细胞粘附底物时，在比较在细胞培养箱中未暴露的FN层上获得的实验结果或在活细胞荧光成像期间，应小心，因为FN纤维形成和机械敏感细胞信号通路可能受到不同影响。
