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.

整合素是一类普遍表达的细胞粘附受体。为了与配体紧密结合，它们需要一个激活步骤，然后组装成复杂的粘附结构。这两个过程都需要 talin 和 kindlin 与整合素的细胞质结构域结合。虽然talin诱导整合素激活的机制似乎已被了解，但kindlins的分子作用仍然很大程度上未知。特别是talin和kindlin如何合作调节整合素活性和功能仍然是整合素研究领域最大的谜团之一。我们最近提供了通过衔接蛋白桩蛋白间接相互作用的证据，桩蛋白充当两种蛋白质之间的桥梁。我们假设 talin/paxillin/kindlin 复合物将活化的整合素编排成微簇，使其能够立即与其多价配体紧密结合。 虽然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，但现在重要的是在更多的生理条件和细胞培养系统下研究talin-paxillin和paxillin-kindlin相互作用的重要性，并为其提供明确的证据。体内相关性。我们的研究将集中于该复合物在白细胞整合素调节中的作用，因为这些细胞特别依赖于稳定粘附的快速形成。在此提出的提案中，我们使用基因工程化的 Hoxb8 祖细胞系，能够在体外分化为功能类似于其原代细胞的中性粒细胞样细胞和巨噬细胞。使用 CRISPR/Cas9/12 介导的基因编辑，我们生成了在内源性 talin1 和 kindlin3 蛋白的桩蛋白结合位点上携带突变的 Hoxb8 细胞克隆。我们的初步研究表明，在静态条件和流动条件下，踝蛋白-桩蛋白相互作用的破坏显着降低了整合素介导的中性粒细胞粘附。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合素中的重要性。特别是，我们的目标是了解它在白细胞粘附和信号转导、整合素激活和微簇形成以及复杂粘附结构组装中的作用，以及它对细胞迁移的影响。此外，我们的目标是利用生化方法和质谱分析来深入了解白细胞粘附级联过程中talin/paxillin/kindlin3复合物的启动、形成和调节。此外，我们已经成功地产生了talin1小鼠突变体，其talin1-rod结构域中的桩蛋白结合位点发生了突变。使用这些动物，我们将研究稳态和炎症条件下离体和体内白细胞粘附级联的不同步骤。