Protein-mediated molecular adhesion: AFM studies

描述（由申请人提供）：白细胞整合素，淋巴细胞功能相关抗原-1 （LFA-1）与其天然配体，细胞间粘附分子-1 （ICAM-1）之间的粘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淋巴细胞和抗原提呈细胞的相互作用。在这一过程中，LFA-1/ICAM-1相互作用的生物物理性质被认为是重要的，包括复合物在高亲和力和低亲和力状态之间调节的能力，以及LFA-1/ICAM-1复合物的内在机械强度。直到最近，粘连都是通过间接的方法来检测的，包括动力学测量或简单的细胞粘附测定。现在，原子力显微镜（AFM）的进步已经能够在单个配体-受体对的水平上直接测量粘附力。AFM测量与诱变实验相结合，可用于确定LFA-1/ICAM-1复合物解离电位主要特征的分子决定因素。

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.