Dynamic Strengths of Leukocyte Adhesion Bonds

描述(申请人提供)：我们的长期目标是在分子水平上对白细胞黏附进行详细的生物物理理解，将黏附结合强度与化学相互作用联系起来，并确定黏附应力如何通过细胞膜传递到受体尾巴和细胞结构之间的内部连接，在那里它可以影响特定的生化途径。我们目前的研究为白细胞粘附键的机械强度及其在力作用下的动力学提供了重要的见解，(I)首先是唾液粘蛋白配体PSGL-1和启动白细胞附着到血管壁的选择素之间的相互作用，(Ii)第二是超级免疫球蛋白(Ig)家族配体和整合素之间的相互作用，这些整合素发出信号并稳定白细胞粘附，使其能够迁移到组织中。我们已经开发了新的方法来固定超灵敏作用力探针尖端的配体，并测试固定在微球上或表达在细胞上的单个受体的机械强度。利用一种令人兴奋的新方法和在受体-细胞骨架相互作用中具有战略变化的已建立的细胞系，这一应用的主要目标是“在细胞内移动”，首先确定细胞结构蛋白调节黏附复合体的机械强度的程度，然后确定这些联系在白细胞信号传递过程中所起的机械作用，这些联系对炎症和损伤部位的募集至关重要。这些研究旨在检验三个假设。假设：白细胞选择素和整合素的相互作用具有特定功能的机械设计，控制着细胞-表面接触内部低应力条件下的结合形成和释放，从而影响结合的增殖，并决定最初粘连事件的命运。假设：分子黏附复合体的机械强度由整个蛋白质相互作用序列中最弱的一环控制，从外部粘附键到连接受体-尾部结构域和细胞结构的内键。假设：将整合素黏附复合体锚定在细胞结构上的连接是白细胞中“由外而内”和“由内而外”信号传递的关键机械效应器，代表着调节黏附强度的重要反馈过程。

DESCRIPTION (provided by applicant): Our long-term objective is to develop a detailed biophysical understanding of leukocyte adhesion at the molecular level, relating adhesive bond strength to chemical interactions and establishing how adhesive bond stress is transmitted through the cell membrane to the interior connections between receptor tails and the cell cytostructure where it can impact specific biochemical pathways. Our current research has provided significant insight into the mechanical strengths of leukocyte adhesion bonds and their kinetics under force, (i) first for interactions between the sialo mucin ligand PSGL-1 and selectins that initiate leukocyte attachments to vessel walls, (ii) second for interactions between super immunoglobulin (Ig) family ligands and integrins that signal and stabilize leukocyte adhesion enabling emigration into tissues. We have developed novel methods to immobilize ligands on the tip of ultrasensitive force probes and test the mechanical strengths of individual bonds to receptors either immobilized on microspheres or expressed on cells. Exploiting an exciting new approach and established cell lines with strategic alterations in receptor- cytoskeletal interactions, the principal objective of this application is to "move inside the cell", first establishing the extent to which cytostructural proteins regulate the mechanical strength of an adhesion complex, and then determining the mechanical role that these linkages play in the leukocyte signalling processes important for recruitment to sites of inflammation and injury. The studies are designed to test three hypotheses. Hypothesis: leukocyte selectin and integrin interactions have function-specific mechanical designs that govern bond formation and release under conditions of low stress interior to a cell- surface contact, thereby impacting bond proliferation and determining the fate of the initial adhesion event. Hypothesis: mechanical strengths of molecular adhesion complexes are governed by the weakest link in entire sequence of protein interactions from the outside adhesive bond to the inside bonds that connect receptor-tail domains to the cell cytostructure. Hypothesis: the linkages that anchor an integrin adhesion complex to the cell structure are key mechanical effectors of "outside-in" and "inside-out" signaling in leukocytes and represent an important feed-back process to regulate adhesion strength.