Department of Chemistry, The Children's Hospital of Philadelphia and Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States of America.
PLoS One. 2012;7(2):e32398. doi: 10.1371/journal.pone.0032398. Epub 2012 Feb 22.
T cell receptor (TCR) engagement induces clustering and recruitment to the plasma membrane of many signaling molecules, including the protein tyrosine kinase zeta-chain associated protein of 70 kDa (ZAP70) and the adaptor SH2 domain-containing leukocyte protein of 76 kDa (SLP76). This molecular rearrangement results in formation of the immunological synapse (IS), a dynamic protein array that modulates T cell activation. The current study investigates the effects of apparent long-range ligand mobility on T cell signaling activity and IS formation. We formed stimulatory lipid bilayers on glass surfaces from binary lipid mixtures with varied composition, and characterized these surfaces with respect to diffusion coefficient and fluid connectivity. Stimulatory ligands coupled to these surfaces with similar density and orientation showed differences in their ability to activate T cells. On less mobile membranes, central supramolecular activation cluster (cSMAC) formation was delayed and the overall accumulation of CD3ζ at the IS was reduced. Analysis of signaling microcluster (MC) dynamics showed that ZAP70 MCs exhibited faster track velocity and longer trajectories as a function of increased ligand mobility, whereas movement of SLP76 MCs was relatively insensitive to this parameter. Actin retrograde flow was observed on all surfaces, but cell spreading and subsequent cytoskeletal contraction were more pronounced on mobile membranes. Finally, increased tyrosine phosphorylation and persistent elevation of intracellular Ca(2+) were observed in cells stimulated on fluid membranes. These results point to ligand mobility as an important parameter in modulating T cell responses.
T 细胞受体 (TCR) 结合诱导许多信号分子的聚集和募集到质膜，包括蛋白酪氨酸激酶 ζ 链相关蛋白 70 kDa（ZAP70）和衔接蛋白 SH2 结构域含有白细胞蛋白 76 kDa（SLP76）。这种分子重排导致免疫突触（IS）的形成，这是一种调节 T 细胞活化的动态蛋白质阵列。本研究探讨了明显的长程配体流动性对 T 细胞信号转导活性和 IS 形成的影响。我们从具有不同组成的二元脂质混合物在玻璃表面上形成刺激脂质双层，并对这些表面的扩散系数和流体连通性进行了表征。与这些表面结合具有相似密度和取向的刺激配体显示出激活 T 细胞能力的差异。在流动性较低的膜上，中央超分子激活簇（cSMAC）的形成延迟，并且 CD3ζ 在 IS 上的整体积累减少。对信号微簇（MC）动力学的分析表明，ZAP70 MCs 的轨迹速度更快，轨迹更长，随着配体流动性的增加，而 SLP76 MCs 的运动对这个参数相对不敏感。在所有表面上都观察到肌动蛋白逆行流动，但在流动膜上细胞铺展和随后的细胞骨架收缩更为明显。最后，在流动膜上刺激的细胞中观察到酪氨酸磷酸化增加和细胞内 Ca(2+)持续升高。这些结果表明配体流动性是调节 T 细胞反应的一个重要参数。
