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.

利用超分辨率荧光显微镜技术，本研究探讨了整合素β6在无牵引力RGD膜上的时空调控机制。实时定量PCR检测显示，CHO-B2细胞中talin1、kindlin1和kindlin2含量丰富。然而，仅kindlin2在RGD配体与整合素β6之间的粘附簇形成中发挥关键作用。当整合素β6的胞质尾与整合素β1的胞质尾互换后，通过像素强度图像分析观察到更密集的RGD簇集。为此，我们进行了生化实验（免疫沉淀和蛋白质印迹），最终证实整合素β1相较于整合素β6，对kindlin2的招募具有偏好性。引入kindlin2也无疑增强了整合素β6的激活，并强化了在RGD膜上的粘附形成。值得注意的是，额外的kindlin2被发现能够支持细胞在柔软基质上的迁移，从而克服了整合素β6的刚性依赖性迁移。