T cell entry into inflamed tissue involves firm adhesion, spreading, and migration of the T cells across endothelial barriers. These events depend on “outside-in” signals through which engaged integrins direct cytoskeletal reorganization. We investigated the molecular events that mediate this process and found that T cells from mice lacking expression of the adaptor protein Crk exhibited defects in phenotypes induced by the integrin lymphocyte function–associated antigen 1 (LFA-1), namely, actin polymerization, leading edge formation, and two-dimensional cell migration. Crk protein was an essential mediator of LFA-1 signaling–induced phosphorylation of the E3 ubiquitin ligase c-Cbl and its subsequent interaction with the phosphatidylinositol 3-kinase (PI3K) subunit p85, thus promoting PI3K activity and cytoskeletal remodeling. In addition, we found that Crk proteins were required for T cells to respond to changes in substrate stiffness, as measured by alterations in cell spreading and differential phosphorylation of the force-sensitive protein CasL. These findings identify Crk proteins as key intermediates coupling LFA-1 signals to actin remodeling and provide mechanistic insights into how T cells sense and respond to substrate stiffness.

T 细胞进入发炎组织涉及 T 细胞穿过内皮屏障的牢固粘附、扩散和迁移。这些事件取决于“由外而内”的信号，整合素通过这些信号直接细胞骨架重组。我们研究了介导这一过程的分子事件，发现来自缺乏衔接蛋白 Crk 表达的小鼠的 T 细胞表现出由整合素淋巴细胞功能相关抗原 1 (LFA-1) 诱导的表型缺陷，即肌动蛋白聚合、前沿形成和二维细胞迁移。Crk 蛋白是 LFA-1 信号诱导的 E3 泛素连接酶 c-Cbl 磷酸化及其随后与磷脂酰肌醇 3-激酶 (PI3K) 亚基 p85 相互作用的重要介质，从而促进 PI3K 活性和细胞骨架重塑。此外，我们发现 T 细胞需要 Crk 蛋白来响应底物硬度的变化，这是通过细胞扩散的改变和力敏感蛋白 CasL 的差异磷酸化来衡量的。这些发现将 Crk 蛋白鉴定为将 LFA-1 信号耦合到肌动蛋白重塑的关键中间体，并提供了关于 T 细胞如何感知和响应底物硬度的机制见解。