Focal adhesion proteins connect IgE receptors to the cytoskeleton as revealed by micropatterned ligand arrays
Focal adhesion proteins connect IgE receptors to the cytoskeleton as revealed by micropatterned ligand arrays
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DOI：
10.1073/pnas.0802138105
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发表时间：
2008-11-11
影响因子：
11.1
通讯作者：
Baird, Barbara A.
中科院分区：
文献类型：
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作者：
Torres, Alexis J.;Vasudevan, Lavanya;Baird, Barbara A.
在空间定义的阵列中呈现特定配体的图案化表面被用来检查聚集的IgE受体(IgE-Fc epsilon RI)和大鼠嗜碱性白血病(RBL)肥大细胞的细胞骨架之间的结构联系。我们用荧光显微镜显示，细胞骨架F-肌动蛋白集中在与微米级图案配体结合的细胞表面IgE-Fc epsilon RI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。我们现在证明，虽然接头蛋白Ezrin和moesin不能与聚集的IgE-Fc epsilon RI阵列一起检测到浓集，但已知将F-肌动蛋白与整合素联系在一起的焦点黏附蛋白vinculin、paxlin和talin在这些区域积累的时间尺度与F-肌动蛋白相同。此外，加入纤维连接蛋白-RGD多肽后，这些焦点黏附蛋白与聚集的IgE-Fc epsilon RI的共局性增强。值得注意的是，最突出的大鼠嗜碱性白血病细胞整合素(α5)避免了配体占据的图案化区域，并优先与硅衬底的暴露区域相关联。因此，图案化表面提供的空间分离揭示了连接到肌动蛋白细胞骨架的特定焦点黏附蛋白与配体交联的IgE-Fc epsilon RI相关联，而不依赖于整合素。我们利用小干扰RNA研究了其中一种蛋白质paxlin在IgE-Fc epsilon RI介导的信号转导中的功能作用。根据这些结果，我们确定帕西林减少了Fc epsilon RIβ亚基的受刺激的磷酸化，但增加了细胞内储存的受刺激的钙释放。这些结果表明，与聚集型IgE-Fc epsilon RI结合的巴西林对Fc epsilon RI信号转导具有净正向作用。
Patterned surfaces that present specific ligands in spatially defined arrays are used to examine structural linkages between clustered IgE receptors (IgE-Fc epsilon RI) 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 epsilon RI 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 epsilon RI, 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 epsilon RI is enhanced after addition of fibronectin-RGD peptides. Significantly, the most prominent rat basophilic leukemia cell integrin (alpha 5) 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 epsilon RI, independently of integrins. We investigated the functional role of one of these proteins, paxillin, in IgE-Fc epsilon RI-mediated signaling by using small interfering RNA. From these results, we determine that paxillin reduces stimulated phosphorylation of the Fc epsilon RI beta subunit but enhances stimulated Ca2+ release from intracellular stores. The results suggest that paxillin associated with clustered IgE-Fc epsilon RI has a net positive effect on Fc epsilon RI signaling.