Department of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853-1301, USA.
J Phys Chem B. 2012 Jun 14;116(23):6923-35. doi: 10.1021/jp300197p. Epub 2012 Apr 2.
Antigen-mediated cross-linking of IgE bound to its receptor, FcεRI, initiates a transmembrane signaling cascade that results in mast cell activation in the allergic response. Using immunogold labeling of intact RBL mast cells and scanning electron microscopy (SEM), we visualize molecular reorganization of IgE-FcεRI and early signaling proteins on both leaflets of the plasma membrane, without the need for ripped off membrane sheets. As quantified by pair correlation analysis, we observe dramatic changes in the nanoscale distribution of IgE-FcεRI after binding of multivalent antigen to stimulate transmembrane signaling, and this is accompanied by similar clustering of Lyn and Syk tyrosine kinases, and adaptor protein LAT. We find that Lyn co-redistributes with IgE-FcεRI into clusters that cross-correlate throughout 20 min of stimulation. Inhibition of tyrosine kinase activity reduces the numbers of both IgE-FcεRI and Lyn in stimulated clusters. Coupling of these proteins is also decreased when membrane cholesterol is reduced either before or after antigen addition. These results provide evidence for involvement of FcεRI phosphorylation and cholesterol-dependent membrane structure in the interactions that accompany IgE-mediated activation of RBL mast cells. More generally, this SEM view of intact cell surfaces provides new insights into the nanoscale organization of receptor-mediated signaling complexes in the plasma membrane.
抗原介导的 IgE 与其受体 FcεRI 的交联，启动了跨膜信号级联反应，导致过敏反应中肥大细胞的激活。通过对完整的 RBL 肥大细胞进行免疫金标记和扫描电子显微镜（SEM）观察，我们在质膜的两个叶面上可视化了 IgE-FcεRI 和早期信号蛋白的分子重排，而无需撕下膜片。通过配对相关分析定量，我们观察到在多价抗原结合刺激跨膜信号后，IgE-FcεRI 的纳米尺度分布发生了剧烈变化，Lyn 和 Syk 酪氨酸激酶以及衔接蛋白 LAT 也发生了类似的聚类。我们发现 Lyn 与 IgE-FcεRI 一起重新分布到刺激后 20 分钟内相互关联的簇中。酪氨酸激酶活性的抑制减少了刺激簇中 IgE-FcεRI 和 Lyn 的数量。在用抗原处理之前或之后降低膜胆固醇时，这些蛋白质的偶联也减少了。这些结果为 FcεRI 磷酸化和胆固醇依赖性膜结构在 IgE 介导的 RBL 肥大细胞激活伴随的相互作用中的参与提供了证据。更一般地说，这种完整细胞表面的 SEM 观察为质膜中受体介导的信号复合物的纳米尺度组织提供了新的见解。
