Gonnermann Carina, Huang Chaolie, Becker Sarah F, Stamov Dimitar R, Wedlich Doris, Kashef Jubin, Franz Clemens M
Karlsruhe Institute of Technology (KIT), Center for Functional Nanostructures, Wolfgang-Gaede-Strasse 1a, 76131 Karlsruhe, Germany.
Integr Biol (Camb). 2015 Mar;7(3):356-63. doi: 10.1039/c4ib00282b. Epub 2015 Feb 24.
AFM-based force spectroscopy in combination with optical microscopy is a powerful tool for investigating cell mechanics and adhesion on the single cell level. However, standard setups featuring an AFM mounted on an inverted light microscope only provide a bottom view of cell and AFM cantilever but cannot visualize vertical cell shape changes, for instance occurring during motile membrane blebbing. Here, we have integrated a mirror-based sideview system to monitor cell shape changes resulting from motile bleb behavior of Xenopus cranial neural crest (CNC) cells during AFM elasticity and adhesion measurements. Using the sideview setup, we quantitatively investigate mechanical changes associated with bleb formation and compared cell elasticity values recorded during membrane bleb and non-bleb events. Bleb protrusions displayed significantly lower stiffness compared to the non-blebbing membrane in the same cell. Bleb stiffness values were comparable to values obtained from blebbistatin-treated cells, consistent with the absence of a functional actomyosin network in bleb protrusions. Furthermore, we show that membrane blebs forming within the cell-cell contact zone have a detrimental effect on cell-cell adhesion forces, suggesting that mechanical changes associated with bleb protrusions promote cell-cell detachment or prevent adhesion reinforcement. Incorporating a sideview setup into an AFM platform therefore provides a new tool to correlate changes in cell morphology with results from force spectroscopy experiments.
基于原子力显微镜（AFM）的力谱技术与光学显微镜相结合，是在单细胞水平上研究细胞力学和粘附的有力工具。然而，将AFM安装在倒置光学显微镜上的标准装置仅能提供细胞和AFM悬臂的底部视图，无法可视化垂直方向上的细胞形状变化，例如在运动性膜泡形成过程中发生的变化。在此，我们集成了一个基于镜子的侧视图系统，以监测非洲爪蟾颅神经嵴（CNC）细胞在AFM弹性和粘附测量过程中因运动性膜泡行为而导致的细胞形状变化。使用侧视图装置，我们定量研究了与膜泡形成相关的力学变化，并比较了在膜泡和非膜泡事件期间记录的细胞弹性值。与同一细胞中的非膜泡化膜相比，膜泡突起显示出明显更低的硬度。膜泡硬度值与用blebbistatin处理的细胞所获得的值相当，这与膜泡突起中缺乏功能性的肌动蛋白-肌球蛋白网络一致。此外，我们表明在细胞-细胞接触区内形成的膜泡对细胞-细胞粘附力有不利影响，这表明与膜泡突起相关的力学变化促进了细胞-细胞分离或阻止了粘附增强。因此，将侧视图装置整合到AFM平台中提供了一种新工具，可将细胞形态变化与力谱实验结果相关联。
