Protein-mediated molecular adhesion: AFM studies

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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.