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

整合素是一类普遍表达的细胞粘附受体。为了与它们的配体紧密结合，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结合到整合素的细胞质区域。虽然talin诱导整合素激活的机制似乎已经被理解，但kindlins的分子作用仍然很大程度上是未知的。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。我们最近提供了间接相互作用的证据，通过适配器蛋白paxillin，作为两个蛋白之间的桥梁。我们假设talin/paxillin/kindlin复合物将活化的整合素编排成微团簇，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，但现在重要的是要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。我们的研究将集中于该复合物在白细胞整合素调控中的作用，因为这些细胞特别依赖于稳定粘附的快速形成。在这里提出的建议中，我们使用基因工程的Hoxb8祖细胞系，能够在体外分化为中性粒细胞样细胞和巨噬细胞，其功能类似于它们的主要对生细胞。利用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，在静态条件和流动条件下，破坏talin-paxillin相互作用可显著降低整合素介导的中性粒细胞粘附。在利用这些和其他新型Hoxb8细胞系进行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，我们旨在了解其在白细胞粘附和信号转导，整合素激活和微团簇形成以及复杂粘附结构组装中的作用，以及其对细胞迁移的影响。此外，我们的目标是利用生化方法和质谱分析来获得在白细胞粘附级联过程中talin/paxillin/kindlin3复合物的启动、形成和调控的机制。此外，我们已经成功地产生了talin1小鼠突变体，talin1-rod结构域的paxillin结合位点发生了突变。利用这些动物，我们将研究白细胞粘附级联在体内和体外在稳态和炎症条件下的不同步骤。

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.