Dynamics of Subsynaptic Vesicles and Surface Microclusters at the Immunological Synapse
Dynamics of Subsynaptic Vesicles and Surface Microclusters at the Immunological Synapse
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DOI：
10.1126/scisignal.2000645
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发表时间：
2010-05-11
影响因子：
7.3
通讯作者：
Davis, Daniel M.
中科院分区：
文献类型：
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作者：
Purbhoo, Marco A.;Liu, Hebin;Davis, Daniel M.
成像研究已经确定了在T细胞和抗原提呈细胞(APC)之间的接触点上充当信号中心的蛋白激酶和接头蛋白簇。在这里，我们报告了激酶ZAP-70和接头蛋白LAT和SLP-76聚集在T细胞和覆盖有CD3刺激性抗体的盖片之间的不同的簇中，CD3是T细胞抗原受体复合体的一个组成部分。在反复移动到SLP-76表面微簇和适配器蛋白GADS(Shc下游的生长因子受体结合蛋白相关适配器)的活动小泡中检测到一小部分LAT，在那里它们表现出运动性降低。后来，残基酪氨酸171和酪氨酸191(LAT与GADS结合所需)突变为苯丙氨酸的分子没有居住在SLP-76的簇上。在免疫突触，含有LAT的囊泡也与SLP-76的微簇共定位，这是在使用激光镊子垂直定位T细胞-APC结合物以进行高分辨率成像的实验中检测到的。当囊泡状LAT与SLP-76共定位时，LAT的磷酸化最为显著。事实上，SLP-76微簇中磷酸化LAT的丰度在那些最近与含有LAT的囊泡有较多相互作用的簇中最多。最后，抑制受体ILT2的负信号破坏了含有SLP-76的微簇的组装。综上所述，这些数据表明，含有LAT的囊泡的运动与蛋白质微簇的组织有关，并表明囊泡LAT在SLP-76信号小体中发挥着重要作用。
Imaging studies have identified clusters of kinases and adaptor proteins that serve as centers of signaling at the contact points between T cells and antigen-presenting cells (APCs). Here, we report that the kinase ZAP-70 and the adaptor proteins LAT and SLP-76 accumulated in separate clusters at the interface between T cells and coverslips coated with a stimulatory antibody against CD3, a component of the T cell antigen receptor complex. A fraction of LAT was detected in motile vesicles that repeatedly moved to surface microclusters of SLP-76 and the adaptor protein GADS (growth factor receptor-bound protein-related adaptor downstream of Shc), where they exhibited decreased motility. LAT molecules in which the residues tyrosine 171 and tyrosine 191 (which are required for the binding of LAT to GADS) were mutated to phenylalanine did not dwell at clusters of SLP-76. At immunological synapses, LAT-containing vesicles also colocalized with microclusters of SLP-76, as detected in experiments in which laser tweezers were used to position T cell-APC conjugates vertically for high-resolution imaging. Phosphorylation of LAT was most prominent when vesicular LAT colocalized with SLP-76. Indeed, the abundance of phosphorylated LAT within a microcluster of SLP-76 was greatest in those clusters that had more recent interactions with LAT-containing vesicles. Finally, negative signals by the inhibitory receptor ILT2 disrupted the assembly of SLP-76-containing microclusters. Together, these data show that the movement of LAT-containing vesicles is linked to the organization of protein microclusters and suggest an important role for vesicular LAT in the SLP-76 signalosome.