Integrin activation by the intracellular adaptor proteins talin1 and kindlin-3 is essential for lymphocyte adhesion. These adaptors cooperatively control integrin activation through bidirectional (inside-out and outside-in) activation signals. Using single-molecule measurements, we revealed the distinct dynamics of talin1 and kindlin-3 interactions with the integrin LFA1 (αLβ2) and their functions in LFA1 activation and LFA1-mediated adhesion. The kinetics of talin1 binding to the tail of the β2 subunit corresponded to those of LFA1 binding to its ligand ICAM1. ICAM1 binding induced transient interactions between the membrane-proximal cytoplasmic region of the β2 subunit with an N-terminal domain of kindlin-3, leading to disruption of the association between the integrin subunits (the α/β clasp) and unbending of the ectodomains of the α/β heterodimer. These conformational changes promoted high-affinity talin1 binding to the β2 tail that required the talin rod domain and the actomyosin cytoskeleton. Inside-out signaling induced by the GTPase Rap1 did not markedly stabilize the binding of talin1 and kindlin-3 to LFA1. In contrast, ligand-induced outside-in signaling, the stabilization of open LFA1 conformers, or shear force substantially altered the dynamics of talin1 and kindlin-3 association with LFA1 and enhanced both Rap1 and LFA1 activation. In migrating lymphocytes, asymmetrical distribution of talin1 and kindlin-3 correlated with the maturation of LFA1 from a low-affinity conformation at the leading edge to a high-affinity conformation in the adherent mid-body. Our results suggest that kindlin-3 spatiotemporally mediates a positive feedback circuit of LFA1 activation to control dynamic adhesion and migration of lymphocytes.

细胞内衔接蛋白 talin1 和 kindlin-3 激活整合素对淋巴细胞粘附至关重要。这些适配器通过双向（由内向外和由外向内）激活信号协同控制整合素激活。使用单分子测量，我们揭示了 talin1 和 kindlin-3 与整合素 LFA1 (αLβ2) 相互作用的独特动力学及其在 LFA1 激活和 LFA1 介导的粘附中的功能。talin1 与 β2 亚基尾部结合的动力学对应于 LFA1 与其配体 ICAM1 结合的动力学。ICAM1结合诱导β2亚基的近膜细胞质区域与kindlin-3的N端结构域之间的瞬时相互作用，导致整联蛋白亚基（α/β 扣）之间的关联破坏和 α/β 异二聚体的胞外域不弯曲。这些构象变化促进了高亲和力 talin1 与需要 talin 杆结构域和肌动球蛋白细胞骨架的 β2 尾部结合。由 GTPase Rap1 诱导的内向外信号传导并未显着稳定 talin1 和 kindlin-3 与 LFA1 的结合。相比之下，配体诱导的外向内信号传导、开放 LFA1 构象异构体的稳定化或剪切力显着改变了 talin1 和 kindlin-3 与 LFA1 结合的动力学，并增强了 Rap1 和 LFA1 的激活。在迁移的淋巴细胞中，talin1 和 kindlin-3 的不对称分布与 LFA1 从前缘的低亲和力构象到粘附中间体的高亲和力构象的成熟相关。我们的研究结果表明，kindlin-3 在时空上介导 LFA1 激活的正反馈回路，以控制淋巴细胞的动态粘附和迁移。