Supporting data for 'Kindlin-driven inside-out activation governs force-dependent adhesion assembly of integrin beta6'

Super-resolution fluorescence microscopy was utilized to examine the spatiotemporal regulation of integrin beta6 on RGD-membrane devoid of traction force. RT-qPCR has revealed a high abundance of talin1, kindlin1 and kindlin2 in CHO-B2 cells. However, solely kindlin2 critically reinforced the adhesion clustering between RGD ligands and integrin beta6. When integrin beta6 cytoplasmic tail was swapped with that of integrin beta1, denser RGD clustering was observed via the pixel-intensity image analysis. To that end, we conducted biochemistry assay (immunoprecipitation and western blot), and ultimately demonstrated a preferential recruitment of kindlin2 by integrin beta1 comparing to integrin beta6. Kindlin2 introduction also certainly enhanced integrin beta6 activation and strengthened the adhesion formation on RGD-membrane. Remarkably, additional kindlin2 was found to support cell locomotion even on the soft substrate, thereby overcame the rigidity-dependent migration of integrin beta6.本研究借助超分辨率荧光显微镜（super-resolution fluorescence microscopy），对缺失牵引力的RGD膜（RGD-membrane）表面整合素β6（integrin beta6）的时空调控机制展开了考察。实时定量聚合酶链反应（RT-qPCR）结果显示，CHO-B2细胞中踝蛋白1（talin1）、kindlin1及kindlin2均呈现高丰度表达。但仅kindlin2能够显著强化RGD配体与整合素β6之间的黏附聚集。将整合素β6的胞质尾区（cytoplasmic tail）替换为整合素β1（integrin beta1）的胞质尾区后，通过像素强度图像分析可观察到更为致密的RGD聚集现象。为此，本研究开展了生化检测（免疫共沉淀（immunoprecipitation）与蛋白质印迹（western blot）实验），最终证实相较于整合素β6，整合素β1对kindlin2具有更显著的招募偏好性。引入kindlin2同样可有效增强整合素β6的活化水平，并强化其在RGD膜上的黏附形成。值得注意的是，即便在软性基底（soft substrate）上，额外引入kindlin2仍可支持细胞运动，从而逆转了整合素β6的刚度依赖性迁移特性。