Applications of AFM for the analysis of biomolecular interaction
Biomolecular Interaction Technology Center
Basic biomedical research in the post-genomic era is confronted with the difficult question of how protein interactions give rise to cellular functions. To aid in addressing this question, there have been significant advances in high throughput screening methods (e.g., protein microarrays) and single molecule measurements in recent years. Our research uses the AFM to investigate the biophysical mechanisms of leukocyte adhesion. Cell adhesion is mediated by interactions of complementary adhesion molecules from the two apposing cell surfaces. Recent advances in atomic force microscopy have made it possible to measure the unbinding force of individual adhesion complexes. These single molecule measurements demonstrated that a single rupture force cannot adequately describe the bond strength of a ligand-receptor complex. Instead, the dynamic strength of a ligand-receptor complex is characterized by a force spectrum that relates the unbinding force of the complex to the loading rate of the unbinding process. Such characterization of a ligand-receptor pair is referred to as dynamic force spectroscopy (DFS) and has been used to deduce the dissociation potentials of ligand-receptor complexes. The overall goal of this proposal is to make this technology available to the members of the Biomolecular Interaction Technology Center (BITC). More information...
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NIH Grant
National Institutes of Health
 Cell-cell adhesion plays a crucial role in the function of the immune system. The formation of a stable conjugate between a T lymphocyte and an antigen-presenting cell (APC) is a pre-requisite for T cell activation and is mediated by the interaction between lymphocyte function-associated antigen 1 (LFA 1) and intercellular adhesion molecule 1 (ICAM 1). Although it has been shown that T lymphocytes can modulate the LFA 1/ICAM 1 interaction, it is not known how changes in the affinity state of LFA-1 translate to changes in the mechanical strength of the LFA 1/ICAM 1 bond. Moreover, it is not known how the lateral redistribution of LFA 1 following cell activation modifies the strength of cell-cell adhesion. This gap in knowledge limits the extent to which an effective strategy can be developed to regulate the immune response through the modulation of cell-cell adhesion. Our long-range goal is to understand the mechanisms that regulate leukocyte adhesion during immune and inflammatory processes. The main objective of the proposed research is to determine the biophysical parameters that enable the LFA 1/ICAM 1 interaction to facilitate firm adhesion between T lymphocytes and APCs. The central hypothesis of this application is that cell adhesion is governed by the intrinsic properties of the LFA 1/ICAM 1 complex and by the ability of the complex to modulate between different adhesive states. We have formulated this hypothesis based on preliminary findings that (i) the LFA 1/ICAM 1 complex is especially well-suited to resist large pulling forces and (ii) the avidity modulation of LFA-1 plays an important role in promoting enhanced cell adhesion. More information...
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American Heart Association Grant
American Heart Association
Summary. Atherosclerosis is caused by the recruitment of monocytes into the artery walls, followed by their transformation into foam cells and subsequently the formation of fatty streaks. The selective recruitment of monocytes by vascular endothelium during atherogenesis involves multiple types of cell adhesion molecules. The initial attachment of circulating monocytes to the endothelium is mediated by the interaction of P-selectin and P-selectin Glycoprotein Ligand-1 (PSGL-1). Subsequent intercellular interaction by a second pair of cell adhesion molecules, Very Late Antigen-4 (VLA-4) and Vascular Cell Adhesion Moleule-1 (VCAM-1), permits the monocytes to roll along and eventually adhere firmly to the endothelium. Although much is known about the adhesion molecules involved in the recruitment of monocytes, the underlying mechanisms that permit a cell to resist the shear force of the blood stream are poorly understood. Our research in cell adhesion focuses on understanding how cell adhesion molecules contribute to the selective recruitment of monocytes into the arteries. It is based on the application of advanced biophysical approaches such as the Atomic Force Microscope (AFM) to probe the intrinsic properties of the individual adhesion complexes and the interplay between the adhesion complexes. In this application, we propose to elaborate on the mechanisms of cell adhesion by: 1) investigating the temporal and spatial dependences of the mechanical force generated by the interaction between monocyte and endothelial cells and 2) evaluating potential mechanisms for avidity modulation of integrin-mediated adhesion. The proposed research will provide an improved quantitative understanding of the mechanisms of leukocyte adhesion at the molecular level that would be of great relevance to atherosclerosis and in the design of inhibitors that modulate the trafficking of cells. More information... |
Funding for the Moy Lab Web site has been provided by a grant awarded to Dr. Vincent T. Moy by the Biomolecular Interaction Technologies Center (BITC).