Force, Conformation & Affinity in VLA-4 and LFA-1 Adhesion

描述(由申请人提供)：白细胞整合素在宿主防御、炎症、止血和转移的血管细胞黏附中发挥关键作用。在过去的五年里，我们开发了一套新的工具，利用流式细胞术来分析细胞黏附相互作用。我们使用流式细胞术：1)实时分析配体与VLA-4的结合和解离：2)测量在无剪切条件下将细胞结合在一起的键数；3)测量机械转导(剪切诱导信号和剪切诱导的结合加强)；4)通过荧光能量转移实时测量活细胞上VLA-4和LFA-1的分子构象(黏附分子长度)。结果提示了一种新的粘附键强化机制，其中施加的力有助于VLA-4的延伸，增加亲和力和亲和力，使VLA-4有助于低亲和力的白细胞滚动和高亲和力的牢固粘附性。我们的总体目标是定义构象、亲和力、作用力和粘合功能之间的关系。首先，我们将填补我们在没有剪切力的情况下对VLA-4构象调控的理解的关键空白。我们将区分由配基结合事件引起的构象变化和由其他细胞激活方式引起的构象变化，如内向外信号。其次，我们将比较VLA-4(一种可变捕获受体)和LFA-1(一种牢固的依附受体)，后者与VLA-4协同工作。这种比较的一个组成部分是对含有整合素的I结构域和非L结构域之间的结构活性差异的理解。第三，我们将评估亲和力和构象对细胞黏附的意义。我们已经证明，由PKC和PLC启动的不完全信号传递区分了亲和力和构象变化。我们将测试构象调节细胞黏附效率，而亲和力调节细胞黏附持续时间的想法。第四，我们将使用一种集成了试管粘度计和流式细胞仪的新设备来区分力(从外到内)对整合素黏附持续时间的影响。通过将流式细胞术中的黏附持续时间与Evans的Bioforce探针的单分子结合行为相关联，我们将确定整合素构象在黏附过程中的作用。综上所述，这些结果将从分子构象、亲和力和应用剪切的角度，以单细胞-单分子分析模式和纳米级分辨率来解释黏附分子的行为。

DESCRIPTION (provided by applicant): Leukocyte integrins play key roles in vascular cell adhesion in host defense, inflammation, hemostasis, and metastasis. In the last five years we have developed a set of novel tools that exploit flow cytometry to analyze cell adhesive interactions. We used flow cytometry: 1) for the real-time analysis of ligand association and dissociation with VLA-4: 2) to measure the number of bonds holding cells together under no shear conditions; 3) to measure mechanotransduction (shear-induced signaling and shear-induced bond strengthening); 4) to measure molecular conformation (adhesion molecule length) of both VLA-4 and LFA-1 in real-time on living cells by fluorescence energy transfer. The results suggest a novel mechanism of adhesion bond strengthening in which the applied force contributes to VLA-4 extension, increasing affinity and avidity, allowing VLA-4 to contribute both to low avidity leukocyte rolling and high avidity firm adhesion. Our overall goal is to define relationships among conformation, affinity, applied force and adhesive function. First, we will fill critical gaps in our understanding of VLA-4 conformational regulation in the absence of shear. We will distinguish conformational changes induced by a ligand binding event from those induced by other means of cell activation such as inside-out signaling. Second, we will compare VLA-4 (a variable capture receptor) to LFA-1 (a firm attachment receptor) which works in concert with VLA-4. Integral to this comparison is an understanding of structure-activity differences between I domain and non-l domain containing integrins. Third, we will evaluate the significance of affinity and conformation to cell adhesion. We have shown that incomplete signaling initiated by PKC and PLC differentiates between the affinity and conformational changes. We will test the idea that conformation regulates the efficiency of cell adhesion, whereas affinity regulates it duration. Fourth, we will distinguish the contributions of force (outside-in) to the duration of integrin adhesion, using a new device integrating a cuvette viscometer and a flow cytometer. By relating adhesive duration in flow cytometry to single molecule bond behavior with Evans' bioforce probe we will determine the role of integrin conformation in the adhesive process. Taken together, the results will interpret adhesion molecule behavior in terms of molecular conformation, affinity, and applied shear, in a single-cell single-molecule analysis mode and with nanoscale resolution.