Lehrstuhl Zellbiologie, Universitat Konstanz, 78457, Konstanz, Germany.
Konstanz Research School Chemical Biology, Universitat Konstanz, 78457, Konstanz, Germany.
Sci Rep. 2019 Apr 5;9(1):5728. doi: 10.1038/s41598-019-42002-6.
Integrins and integrin-dependent cell-matrix adhesions are essential for a number of physiological processes. Integrin function is tightly regulated via binding of cytoplasmic proteins to integrin intracellular domains. Yet, the complexity of cell-matrix adhesions in mammals, with more than 150 core adhesome proteins, complicates the analysis of integrin-associated protein complexes. Interestingly, the evolutionary origin of integrins dates back before the transition from unicellular life to complex multicellular animals. Though unicellular relatives of metazoa have a less complex adhesome, nothing is known about the initial steps of integrin activation and adhesion complex assembly in protozoa. Therefore, we developed a minimal, microscope-based system using chimeric integrins to investigate receptor-proximal events during focal adhesion assembly. Clustering of the human integrin β1 tail led to recruitment of talin, kindlin, and paxillin and mutation of the known talin binding site abolished recruitment of this protein. Proteins indirectly linked to integrins, such as vinculin, migfilin, p130, or zyxin were not enriched around the integrin β1 tail. With the exception of integrin β4 and integrin β8, the cytoplasmic domains of all human integrin β subunits supported talin binding. Likewise, the cytoplasmic domains of integrin β subunits expressed by the protozoan Capsaspora owczarzaki readily recruited talin and this interaction was based on an evolutionary conserved NPXY/F amino acid motif. The results we present here validate the use of our novel microscopic assay to uncover details of integrin-based protein-protein interactions in a cellular context and suggest that talin binding to integrin β cytoplasmic tails is an ancient feature of integrin regulation.
整合素及其依赖的细胞基质黏附对于许多生理过程都是必不可少的。整合素的功能通过细胞质蛋白与整合素细胞内结构域的结合来紧密调控。然而，哺乳动物细胞基质黏附的复杂性，超过 150 种核心黏着斑蛋白，使得整合素相关蛋白复合物的分析变得复杂。有趣的是，整合素的进化起源可以追溯到从单细胞生命向复杂多细胞动物的过渡之前。尽管后生动物的单细胞亲属的黏着斑结构较为简单，但对于原生动物中整合素的激活和黏附复合物组装的初始步骤还一无所知。因此，我们开发了一种基于显微镜的最小系统，使用嵌合整合素来研究焦点黏附组装过程中受体近端事件。人整合素β1 尾部的聚类导致了 talin、kindlin 和 paxillin 的募集，并且已知的 talin 结合位点的突变则消除了该蛋白的募集。与整合素间接相关的蛋白质，如 vinculin、migfilin、p130 或 zyxin，则不会在整合素β1 尾部周围富集。除了整合素β4 和整合素β8 之外，所有人类整合素β亚基的细胞质结构域都支持 talin 结合。同样，原生动物 Capsaspora owczarzaki 表达的整合素β亚基的细胞质结构域也容易募集 talin，并且这种相互作用基于一个进化保守的 NPXY/F 氨基酸基序。我们在这里呈现的结果验证了我们新型显微镜检测法的使用，该方法可以揭示细胞环境中基于整合素的蛋白-蛋白相互作用的细节，并表明 talin 与整合素β细胞质尾部的结合是整合素调控的一个古老特征。
