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

整合素是一类广泛表达的细胞黏附受体家族。为了与配体紧密结合，它们需要一个活化步骤，然后组装成复杂的黏附结构。这两个过程都需要Talin和kindlin与整合素的细胞质区域结合。虽然Talin诱导整合素激活的机制似乎已被了解，但kindlins的分子作用在很大程度上仍不清楚。特别是，talin和kindlin如何协同调节整合素的活性和功能仍然是整合素研究领域最大的谜团之一。我们最近提供的证据表明，通过连接两种蛋白质的连接蛋白--帕克西林，可以间接相互作用。我们假设talin/paxlin/kindlin复合体将激活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体的存在提供了明确的证据，但现在重要的是研究在更多的生理条件和细胞培养系统中他林-帕西林和帕西林-Kindlin相互作用的意义，并为其在体内的相关性提供明确的证据。我们的研究将集中在这种复合体在调节白细胞整合素中的作用，因为这些细胞特别依赖于稳定的黏附的快速形成。在这里提出的建议中，我们使用基因工程的Hoxb8祖细胞株，能够在体外分化为功能类似于其初级同种细胞的中性粒细胞样细胞和巨噬细胞。利用CRISPR/Cas9/12介导的基因编辑，我们已经产生了在内源性talin1和kindlin3蛋白的paxlin结合位点上携带突变的Hoxb8细胞克隆。我们的初步研究表明，在静态和流动条件下，阻断他林-帕西林相互作用显著降低整合素介导的中性粒细胞黏附。在使用这些和其他新的Hoxb8细胞株进行的全面的细胞生物学和生化研究中，我们将研究talin/paxlin/kindlin3复合体在调节白细胞整合素方面的重要性。特别是，我们的目标是了解它在白细胞黏附和信号转导、整合素激活和微簇形成以及随后组装复杂的黏附结构中的作用，以及它对细胞迁移的影响。此外，我们的目标是使用生化方法和质谱学来获得关于Talin/paxlin/kindlin3复合体在白细胞粘附级联过程中的启动、形成和调节的机械性见解。此外，我们已经成功地获得了在talin1-rod结构域上带有突变的巴西林结合位点的talin1小鼠突变体。利用这些动物，我们将研究体内和体外在动态平衡和炎症条件下白细胞黏附级联的不同步骤。

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.