T cell receptor phosphorylation by Lck is an essential step in T cell activation. It is known that the conformational states of Lck control enzymatic activity; however, the underlying principles of how Lck finds its substrate over the plasma membrane remain elusive. Here, single-particle tracking is paired with photoactivatable localization microscopy to observe the diffusive modes of Lck in the plasma membrane. Individual Lck molecules switched between free and confined diffusion in both resting and stimulated T cells. Lck mutants locked in the open conformation were more confined than Lck mutants in the closed conformation. Further confinement of kinase-dead versions of Lck suggests that Lck confinement was not caused by phosphorylated substrates. Our data support a model in which confined diffusion of open Lck results in high local phosphorylation rates, and inactive, closed Lck diffuses freely to enable long-range distribution over the plasma membrane.

中文翻译：

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构象状态控制 T 细胞中自由扩散模式和受限扩散模式之间的 Lck 切换


Lck 对 T 细胞受体的磷酸化是 T 细胞激活的重要步骤。众所周知，Lck 的构象状态控制酶活性；然而，Lck 如何在质膜上找到其底物的基本原理仍然难以捉摸。在这里，单粒子跟踪与光激活定位显微镜配对，观察质膜中 Lck 的扩散模式。单个 Lck 分子在静息 T 细胞和受刺激 T 细胞中在自由扩散和受限扩散之间切换。锁定在开放构象中的Lck突变体比锁定在闭合构象中的Lck突变体受到更多限制。 Lck 激酶死亡版本的进一步限制表明 Lck 限制不是由磷酸化底物引起的。我们的数据支持一个模型，其中开放 Lck 的有限扩散导致高局部磷酸化率，而无活性的封闭 Lck 自由扩散以实现质膜上的长距离分布。