Probing the effect of clustering on EphA2 receptor signaling efficiency by subcellular control of ligand-receptor mobility.
Probing the effect of clustering on EphA2 receptor signaling efficiency by subcellular control of ligand-receptor mobility.
复制标题
通过配体-受体迁移率的亚细胞控制来探索聚集对EphA 2受体信号传导效率的影响。
DOI：
10.7554/elife.67379
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发表时间：
2021-08-20
期刊：
影响因子：
7.7
通讯作者：
Groves JT
中科院分区：
文献类型：
--
作者：
Chen Z;Oh D;Biswas KH;Zaidel-Bar R;Groves JT
配体的聚集：细胞膜上的受体复合体被广泛认为对随后的信号转导具有功能影响。然而，在不改变细胞系统其他生化方面的情况下，选择性地操纵受体聚集在实验上是具有挑战性的。在这里，我们开发了一种微制造策略，以生产显示移动和固定配体的底物，它们之间的距离约为1微米，从而体验到相同的细胞质信号状态，从而能够精确比较下游信号反应。应用这一方法表征ePhinA1：EphA2信号系统表明，EphA2簇既增强了受体磷酸化，又增强了下游信号活性。单分子成像清楚地解决了Grb2：SOS和NCK：N-WASP信号模块在EphA2簇上增加的分子结合停留时间。这种类型的细胞内比较使定量分析的程度大大高于必须在不同细胞之间进行比较时的可能程度，并基本上消除了细胞对配体操作的反应的影响。
Clustering of ligand:receptor complexes on the cell membrane is widely presumed to have functional consequences for subsequent signal transduction. However, it is experimentally challenging to selectively manipulate receptor clustering without altering other biochemical aspects of the cellular system. Here, we develop a microfabrication strategy to produce substrates displaying mobile and immobile ligands that are separated by roughly 1 µm, and thus experience an identical cytoplasmic signaling state, enabling precision comparison of downstream signaling reactions. Applying this approach to characterize the ephrinA1:EphA2 signaling system reveals that EphA2 clustering enhances both receptor phosphorylation and downstream signaling activity. Single-molecule imaging clearly resolves increased molecular binding dwell times at EphA2 clusters for both Grb2:SOS and NCK:N-WASP signaling modules. This type of intracellular comparison enables a substantially higher degree of quantitative analysis than is possible when comparisons must be made between different cells and essentially eliminates the effects of cellular response to ligand manipulation.