Deciphering the role of the talin1/paxillin/kindlin3 complex in regulating integrin activity and function in hematopoietic cells and mice

破译talin1/paxillin/kindlin3复合物在调节造血细胞和小鼠整合素活性和功能中的作用

• 批准号: 520308008
• 负责人: Privatdozent Dr. Markus Moser
• 金额: --
• 依托单位: Institute for Experimental Hematology
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/520308008?language=en
• 关键词: Deciphering; role; talin1; paxillin; kindlin3

Integrins are a family of ubiquitously expressed cell adhesion receptors. To bind tightly to their ligands, they require an activation step followed by their assembly into complex adhesion structures. Both processes require the binding of talin and kindlin to the cytoplasmic domain of integrins. While the mechanism of talin-induced activation of integrins appears to be understood, the molecular role of kindlins is still largely unknown. In particular, how talin and kindlin cooperate to regulate integrin activity and function remains one of the greatest mysteries in the field of integrin research. We have recently provided evidence for an indirect interaction via the adapter protein paxillin, which serves as a bridge between the two proteins. We hypothesize that the talin/paxillin/kindlin complex orchestrates activated integrins into micro-clusters that allow immediate tight binding to their multivalent ligands. While our recent in vitro studies provided unequivocal evidence for the existence of this trimeric complex, it is now important to investigate the significance of the talin-paxillin and paxillin-kindlin interactions under more physiological conditions and cell culture systems and to provide unequivocal evidence for its relevance in vivo. Our study will focus on the role of this complex in the regulation of leukocyte integrins, as these cells are particularly dependent on the rapid formation of stable adhesions. In the proposal presented here, we use genetically engineered Hoxb8 progenitor cell lines capable of differentiating in vitro into neutrophil-like cells and macrophages that functionally resemble their primary counterparts. Using CRISPR/Cas9/12-mediated gene editing, we have generated Hoxb8 cell clones carrying mutations at the paxillin-binding sites of the endogenous talin1 and kindlin3 proteins. Our preliminary studies show that disruption of the talin-paxillin interaction significantly reduces integrin-mediated adhesion of neutrophils under static conditions and under flow. In comprehensive cell biological and biochemical studies using these and other novel Hoxb8 cell lines, we will study the importance of the talin/paxillin/kindlin3 complex in regulating leukocyte integrins. In particular, we aim to understand its role in leukocyte adhesion and signal transduction, integrin activation and micro-cluster formation followed by the assembly of complex adhesion structures, as well as its effects on cell migration. In addition, we aim to use biochemical methods and mass spectrometry to gain mechanistic insights into the initiation, formation, and regulation of the talin/paxillin/kindlin3 complex during the leukocyte adhesion cascade. In addition, we have already succeeded in generating talin1 mouse mutants with a mutated paxillin binding site in the talin1-rod domain. Using these animals, we will study the different steps of the leukocyte adhesion cascade ex vivo and in vivo under homeostatic and inflammatory conditions.

整联蛋白是一个普遍表达的细胞粘附受体家族。要与配体紧密结合，它们需要一个激活步骤，然后将其组装成复杂的粘附结构。这两个过程都需要塔林和Kindlin与整联蛋白的细胞质结构域结合。虽然塔林诱导的整联蛋白的激活的机制似乎是可以理解的，但Kindlins的分子作用仍然在很大程度上未知。特别是，塔林（Talin）和金林（Kindlin）如何合作调节整联蛋白的活动和功能仍然是整合素研究领域中最大的奥秘之一。我们最近通过衔接蛋白帕西林（Paxillin）提供了间接相互作用的证据，后者是两种蛋白质之间的桥梁。我们假设Talin/Paxillin/Kindlin复合物将活化的整联蛋白协调到微群体中，从而可以立即与其多价配体紧密结合。 尽管我们最近的体外研究为这种三聚体复合物的存在提供了明确的证据，但现在重要的是要研究塔林 - 帕西林和帕西林 - 金德林在更生理条件和细胞培养系统下的重要性，并为其在VIVO中的相关性提供明确的证据。我们的研究将重点关注该复合物在白细胞整合蛋白调节中的作用，因为这些细胞特别取决于稳定粘附的快速形成。在此处提出的提案中，我们使用了基因设计的HOXB8祖细胞系，能够将体外区分为中性粒细胞样细胞和巨噬细胞，这些细胞和巨噬细胞在功能上类似于其主要对应物。使用CRISPR/CAS9/12介导的基因编辑，我们在内源性talin1和Kindlin3蛋白的Paxillin结合位点携带突变的HOXB8细胞克隆。我们的初步研究表明，在静态条件下和流动范围内，塔林 - 帕西林相互作用的破坏显着降低了整联蛋白介导的中性粒细胞的粘附。在使用这些和其他新型HOXB8细胞系的综合细胞生物学和生化研究中，我们将研究Talin/Paxillin/Kindlin3复合物在调节白细胞整联蛋白中的重要性。特别是，我们旨在了解其在白细胞粘附和信号转导，整合素激活和微簇形成中的作用，然后是组装复杂的粘附结构及其对细胞迁移的影响。此外，我们旨在使用生化方法和质谱法来获得对白细胞粘附级联过程中塔林/帕克西林/Kindlin3复合物的起始，形成和调节的机械见解。此外，我们已经成功地生成了talin1-Rod结构域中具有突变的帕西林结合位点的Talin1小鼠突变体。使用这些动物，我们将研究白细胞粘附级联反体的不同步骤在体内和体内在体内和炎症条件下。