Torres Alexis J, Vasudevan Lavanya, Holowka David, Baird Barbara A
Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA.
Proc Natl Acad Sci U S A. 2008 Nov 11;105(45):17238-44. doi: 10.1073/pnas.0802138105.
Patterned surfaces that present specific ligands in spatially defined arrays are used to examine structural linkages between clustered IgE receptors (IgE-Fc epsilonRI) and the cytoskeleton in rat basophilic leukemia (RBL) mast cells. We showed with fluorescence microscopy that cytoskeletal F-actin concentrates in the same regions as cell surface IgE-Fc epsilonRI that bind to the micrometer-size patterned ligands. However, the proteins mediating these cytoskeletal connections and their functional relevance were not known. We now show that whereas the adaptor proteins ezrin and moesin do not detectably concentrate with the array of clustered IgE-Fc epsilonRI, focal adhesion proteins vinculin, paxillin, and talin, which are known to link F-actin with integrins, accumulate in these regions on the same time scale as F-actin. Moreover, colocalization of these focal adhesion proteins with clustered IgE-Fc epsilonRI is enhanced after addition of fibronectin-RGD peptides. Significantly, the most prominent rat basophilic leukemia cell integrin (alpha5) avoids the patterned regions occupied by the ligands and associates preferentially with exposed regions of the silicon substrate. Thus, spatial separation provided by the patterned surface reveals that particular focal adhesion proteins, which connect to the actin cytoskeleton, associate with ligand-cross-linked IgE-Fc epsilonRI, independently of integrins. We investigated the functional role of one of these proteins, paxillin, in IgE-Fc epsilonRI-mediated signaling by using small interfering RNA. From these results, we determine that paxillin reduces stimulated phosphorylation of the Fc epsilonRI beta subunit but enhances stimulated Ca(2+) release from intracellular stores. The results suggest that paxillin associated with clustered IgE-Fc epsilonRI has a net positive effect on Fc epsilonRI signaling.
呈现空间定义阵列中特定配体的图案化表面用于研究大鼠嗜碱性白血病（RBL）肥大细胞中聚集的IgE受体（IgE-FcεRI）与细胞骨架之间的结构联系。我们通过荧光显微镜观察到，细胞骨架F-肌动蛋白集中在与结合微米级图案化配体的细胞表面IgE-FcεRI相同的区域。然而，介导这些细胞骨架连接的蛋白质及其功能相关性尚不清楚。我们现在表明，衔接蛋白埃兹蛋白和膜突蛋白不会与聚集的IgE-FcεRI阵列明显聚集，而粘着斑蛋白纽蛋白、桩蛋白和踝蛋白，已知它们将F-肌动蛋白与整合素连接起来，在与F-肌动蛋白相同的时间尺度上聚集在这些区域。此外，添加纤连蛋白-RGD肽后，这些粘着斑蛋白与聚集的IgE-FcεRI的共定位增强。值得注意的是，最突出的大鼠嗜碱性白血病细胞整合素（α5）避开配体占据的图案化区域，优先与硅底物的暴露区域结合。因此，图案化表面提供的空间分离表明，与肌动蛋白细胞骨架相连的特定粘着斑蛋白与配体交联的IgE-FcεRI相关联，与整合素无关。我们使用小干扰RNA研究了其中一种蛋白质桩蛋白在IgE-FcεRI介导的信号传导中的功能作用。从这些结果中，我们确定桩蛋白降低了FcεRIβ亚基的刺激磷酸化，但增强了细胞内储存库中刺激的Ca(2+)释放。结果表明，与聚集的IgE-FcεRI相关的桩蛋白对FcεRI信号传导具有净正向作用。
