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.

我们以前开发了一种表面辅助测定法，以通过延时原子力显微镜（AFM）对细胞诱导的血浆纤连蛋白（FN）原纤维形成的早期步骤进行成像。出乎意料的是，使用荧光标记的FN（Alexa Fluor 488或568）和活细胞光学显微镜观察FN原纤维形成的互补尝试最初始终失败。进一步的分析表明，在荧光成像过程中照射的焦点区域中，原纤维重塑得到了有效抑制，但在基质上的其他位置正常进行，这表明FN原纤维形成过程的光敏性。一致的是，在AFM间隔扫描过程中，可见光的瞬时照射可以阻止FN的主动细胞驱动性纤维状延伸。可以排除对细胞的光毒作用，因为在细胞接种之前预先照射FN层还可以阻止随后的原纤维形成。在400到640 nm之间改变照明波长范围显示了在高达560 nm的可见光谱范围内的强烈抑制作用，并在更长的波长处降低了抑制作用。光效应也影响未标记的FN，但通过FN的荧光团标记而增强。当去除细胞成像介质中的活性氧（ROS）时，抑制作用可能会降低。基于这些发现，可以在成像介质中使用具有长激发波长（Alexa Fluor 633，在632 nm激发）的标记染料和ROS清除剂（例如加氧酶）成功地对FN纤维原纤维形成进行成像。与未暴露的FN相比，通过AFM扫描对暴露的无细胞FN层进行原纤维重塑需要更高的扫描力，包括照明后FN层的机械硬化。与FN力学的变化一致，在预暴露的FN上散布的细胞显示出降低的迁移速度，改变的粘着斑排列以及机械敏感性信号传导途径的变化，包括FAK（Y397）和paxillin（Y118）磷酸化降低。因此，在细胞接种之前将FN预先暴露于可见光下可提供一种有用的工具，以描绘与FN纤维形成相关的机械敏感性信号传导途径。当使用FN包被的细胞粘附基质时，在细胞培养箱中或在活细胞荧光成像过程中比较未暴露的FN层上获得的实验结果时应格外小心，因为FN的原纤维形成和机械敏感的细胞信号通路可能受到不同的影响。