Protein-mediated molecular adhesion: AFM studies

描述(申请人提供)：白细胞整合素，淋巴细胞功能相关抗原-1(LFA-1)及其天然配体，细胞间黏附分子-1(ICAM-1)之间的黏附相互作用对免疫系统的正常功能至关重要。它在T细胞活化过程中稳定T淋巴细胞与抗原提呈细胞的相互作用。LFA-1/ICAM-1相互作用的生物物理性质在这一过程中被认为是重要的，包括复合体在高亲和力和低亲和力状态之间的调节能力以及LFA-1/ICAM-1复合体的固有机械强度。直到最近，通过包括动力学测量或简单的细胞粘附性分析的间接方法来检测粘附性。现在，原子力显微镜(AFM)的进步使在单配体-受体对水平上直接测量粘附力成为可能。AFM测量与突变实验相结合，可以用来确定导致LFA-1/ICAM-1复合体解离潜力主要特征的分子决定因素。 在这里，我们建议获得AFM力测量，以研究在初始T-CELI/APC接触过程中以及在随后的T细胞黏附进一步加强时T细胞激活过程中，LFA-1和ICAM-1之间相互作用的机制。前三个目标将测量动态强度并确定LFA-1/ICAM-1相互作用的结构成分，后两个目标将探索T细胞激活后增强结合的机制。这些实验结果将回答以下问题：1)LFA-1/ICAM-1复合体是如何解离的？2)LFA-1/ICAM-1复合体的结合强度是如何随着LFA-1构象的变化而变化的？3)使LFA-1/ICAM-1复合体抵抗大拉力的分子决定因素是什么？4)由于受体聚集，细胞激活后淋巴细胞黏附增强？5)ICAM-1的二聚体结构是否增强了其与LFA-1的相互作用？最终，这些实验将帮助我们更好地理解决定配体-受体结合强度的生物物理机制，并有助于开发免疫系统相关疾病的治疗方法。

DESCRIPTION (provided by applicant): The adhesive interactions between the leukocyte integrin, lymphocyte function associated antigen-1 (LFA-1) and its native ligand, intercellular adhesion molecule-1 (ICAM-1) are crucial for normal function of the immune system. It stabilizes the interactions of T lymphocytes and antigen-presenting cells during the process of T cell activation. The biophysical properties of the LFA-1/ICAM-1 interaction that are deemed important in this process include the ability of the complex to modulate between high and low affinity states and the intrinsic mechanical strength of the LFA-1/ICAM-1 complex. Until recently adhesion was examined through indirect methods that involved kinetic measurements or simple cell adhesion assays. Now, advances in atomic force microscopy (AFM) have enabled direct measurements of adhesive forces at the level of single ligand-receptor pairs. The AFM measurements, when combined with mutagensis experiments, can be used to identify the molecular determinants that are responsible for the major features in the dissociation potential of the LFA-1/ICAM-1 complex. Here, we propose to acquire AFM force measurements to investigate the mechanisms that contribute to the interaction between LFA-1 and ICAM-1 during initial T-celI/APC contact and during subsequent T-cell activation when adhesion is further strengthened. The first three objectives will measure the dynamic strength and identify the structural components of the LFA-1/ICAM-1 interaction and the last two objectives will explore mechanisms for the enhanced binding following T-cell activation. Results from these experiments will answer the following questions: 1) How does the LFA-1/ICAM-1 complex unbind? 2) How does the bond strength of the LFA-1/ICAM-1 complex change with the conformation of LFA-1? 3) What are the molecular determinants that permit the LFA-1/ICAM-1 complex to resist large pulling forces? 4) Is enhanced lymphocyte adhesion following cell activation due to receptor clustering? and 5) Does the dimeric structure of ICAM-1 strengthen its interaction with LFA-1? Ultimately, these experiments will help us achieve a better understanding of the biophysical mechanisms that determine ligand-receptor binding strength and could aid in the development of treatments for immune system related disorders.