Cardiovascular Research Institute and Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, United States.
Elife. 2022 Sep 20;11:e75263. doi: 10.7554/eLife.75263.
T cells use kinetic proofreading to discriminate antigens by converting small changes in antigen-binding lifetime into large differences in cell activation, but where in the signaling cascade this computation is performed is unknown. Previously, we developed a light-gated immune receptor to probe the role of ligand kinetics in T cell antigen signaling. We found significant kinetic proofreading at the level of the signaling lipid diacylglycerol (DAG) but lacked the ability to determine where the multiple signaling steps required for kinetic discrimination originate in the upstream signaling cascade (Tiseher and Weiner, 2019). Here, we uncover where kinetic proofreading is executed by adapting our optogenetic system for robust activation of early signaling events. We find the strength of kinetic proofreading progressively increases from Zap70 recruitment to LAT clustering to downstream DAG generation. Leveraging the ability of our system to rapidly disengage ligand binding, we also measure slower reset rates for downstream signaling events. These data suggest a distributed kinetic proofreading mechanism, with proofreading steps both at the receptor and at slower resetting downstream signaling complexes that could help balance antigen sensitivity and discrimination.
T 细胞通过将抗原结合寿命中的微小变化转化为细胞激活的巨大差异来利用动力学校对来区分抗原,但在信号级联中执行此计算的位置尚不清楚。此前,我们开发了一种光门控免疫受体来探究配体动力学在 T 细胞抗原信号转导中的作用。我们发现信号脂质二酰基甘油 (DAG) 水平存在显著的动力学校对,但缺乏确定多个信号步骤的能力,这些步骤是进行动力学区分所必需的,这些步骤源自上游信号级联(Tiseher 和 Weiner,2019 年)。在这里,我们通过适应我们的光遗传学系统来稳健地激活早期信号事件,从而揭示了动力学校对的执行位置。我们发现,从 Zap70 募集到 LAT 聚集再到下游 DAG 生成,动力学校对的强度逐渐增加。利用我们的系统快速脱离配体结合的能力,我们还测量了下游信号事件的较慢重置率。这些数据表明存在分布式动力学校对机制,校对步骤既在受体上,也在较慢重置的下游信号复合物上,这有助于平衡抗原敏感性和区分能力。
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