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ABSTRACT: Single-molecule measurements of the interaction of leukocyte function-associated antigen-1 (LFA-1), expressed on Jurkat T cells, with intercellular adhesion molecules-1 and -2 (ICAM-1 and ICAM-2) were conducted using atomic force microscopy (AFM). The force spectra (i.e., unbinding force versus loading rate) of both the LFA-1/ICAM-1 and LFA-1/ICAM-2 interactions were acquired at a loading rate range covering 3 orders of magnitude (50-60,000 pN/s) and revealed a fast loading regime and a slow loading regime. This indicates that the dissociation of both complexes involves overcoming a steep inner and a wide outer activation barrier. LFA-1 binding to ICAM-1 and ICAM-2 was strengthened in the slow loading regime by the addition of Mg(2+). Differences in the dynamic strength of the LFA-1/ICAM-1 and LFA-1/ICAM-2 interactions can be attributed to the presence of wider barriers in the ICAM-2 complex, making it more responsive to a pulling force than the ICAM-1 complex.
Biomacromolecules 12/2006; 7(11):3188-95. · 5.48 Impact Factor
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ABSTRACT: The recruitment of T lymphocytes to lymphoid organs or sites of inflammation is a crucial step in adaptive immunity. These processes require endothelial activation and expression of adhesion molecules, including E- and P-selectins, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1). However, the complete characterization of the adhesion strength and dynamics between lymphocytes and endothelial cells has been hampered by the lack of sensitive quantitative techniques. Here we report on the application of atomic force microscopy to characterize the interaction between individual pairs of living T lymphocytes (i.e., Jurkat cells) and human umbilical vein endothelial cells (HUVECs). The detachment of individual cell-cell conjugates was a complex process involving several step-like rupture events and the viscoelastic deformation of cells on the scale of several microns. Adhesion between Jurkat cells and activated endothelial cells increased with compression force and contact time, with the most dramatic changes occurring within the first half second of contact. After 0.25 sec of contact, E-selectin, ICAM-1, and VCAM-1 contributed to 18%, 39%, and 41% of total adhesion strength, respectively, suggesting that ICAM-1 and VCAM-1 contributed more than the selectins in supporting cell attachment.
Experimental Biology and Medicine 10/2006; 231(8):1306-12. · 2.64 Impact Factor
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ABSTRACT: Intercellular adhesion mediated by integrin alpha4beta1 and vascular cell adhesion molecule-1 (VCAM-1) plays a crucial role in both the rolling and firm attachment of leukocytes onto the vascular endothelium. Essential to the alpha4beta1/VCAM-1 interaction is its mechanical strength that allows the complex to resist the large shear forces imposed by the bloodstream. Herein we employed single-molecule dynamic force spectroscopy to investigate the dynamic strength of the alpha4beta1/VCAM-1 complex. Our force measurements revealed that the dissociation of the alpha4beta1/VCAM-1 complex involves overcoming at least two activation potential barriers: a steep inner barrier and a more elevated outer barrier. The inner barrier grants the complex the tensile strength to withstand large pulling forces (>50 pN) and was attributed to the ionic interaction between the chelated Mg2+ ion at the N-terminal A-domain of the beta1 subunit of alpha4beta1 and the carboxyl group of Asp-40 of VCAM-1 through the use of site-directed mutations. In general, additional mutations within the C-D loop of domain 1 of VCAM-1 suppressed both inner and outer barriers of the alpha4beta1/VCAM-1 complex, while a mutation at Asp-143 of domain 2 of VCAM-1 resulted in the suppression of the outer barrier, but not the inner barrier. In contrast, the outer barrier of alpha4beta1/VCAM-1 complex was stabilized by integrin activation. Together, these findings provide a molecular explanation for the functionally relevant kinetic properties of the alpha4beta1/VCAM-1 interaction.
Biophysical Journal 11/2004; 87(5):3470-8. · 3.65 Impact Factor
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ABSTRACT: The selectins are Ca(2+)-dependent cell adhesion molecules that facilitate the initial attachment of leukocytes to the vascular endothelium by binding to a carbohydrate moiety as exemplified by the tetrasaccharide, sialyl Lewis X (sLeX). An important property of the selectin-sLeX interaction is its ability to withstand the hydrodynamic force of the blood flow. Herein, we used single-molecule dynamic force spectroscopy (DFS) to identify the molecular determinants within sLeX that give rise to the dynamic properties of the selectin/sLeX interaction. Our atomic force microscopy (AFM) measurements revealed that the unbinding of the selectin/sLeX complexes involves overcoming at least two activation barriers. The inner barrier, which determines the dynamic response of the complex at high forces, is governed by the interaction between the Fuc residue of sLeX and a Ca2+ ion chelated to the lectin domain of the selectin molecule, whereas the outer activation barrier can be attributed to interactions involving the sialic acid residue of sLeX. Due to their steep inner activation barriers, the selectin-sLeX complexes are less sensitive to high pulling forces. Hence, besides its contribution to the bond energy, the Ca2+ ion also grants the selectin-sLeX complexes a tensile strength that is crucial for the selectin-mediated rolling of leukocytes.
ChemPhysChem 03/2004; 5(2):175-82. · 3.41 Impact Factor
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ABSTRACT: We describe the use of atomic force microscopy (AFM) in studies of cell adhesion and cell compliance. Our studies use the interaction between leukocyte function associated antigen-1 (LFA-1)/intercellular adhesion molecule-1 (ICAM-1) as a model system. The forces required to unbind a single LFA-1/ICAM-1 bond were measured at different loading rates. This data was used to determine the dynamic strength of the LFA-1/ICAM-1 complex and characterize the activation potential that this complex overcomes during its breakage. Force measurements acquired at the multiple- bond level provided insight about the mechanism of cell adhesion. In addition, the AFM was used as a microindenter to determine the mechanical properties of cells. The applications of these methods are described using data from a previous study.
Biological Procedures Online 02/2004; 6:1-9. · 1.29 Impact Factor
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ABSTRACT: Leukocyte adhesion to vascular endothelium is a key initiating step in the pathogenesis of many inflammatory diseases. In this study, we present real-time force measurements of the interaction between monocytic human promyelocytic leukemia cells (HL-60) cells and a monolayer of human umbilical vein endothelial cells (HUVECs) by using atomic force microscopy (AFM). The detachment of HL-60-HUVEC conjugates involved a series of rupture events with force transitions of 40-100 pN. The integrated force of these rupture events provided a quantitative measure of the adhesion strength on a whole cell level. The AFM measurements revealed that HL-60 adhesion is heightened in the borders formed by adjacent HUVECs. The average force and mechanical work required to detach a single HL-60 from the borders of a tumor necrosis factor-alpha-activated HUVEC layer were twice as high as those of the HUVEC bodies. HL-60 adhesion to the monolayer was significantly reduced by a monoclonal antibody against beta1-integrins and partially inhibited by antibodies against selectins ICAM-1 and VCAM-1 but was not affected by anti-alphaVbeta3. Interestingly, adhesion was also inhibited in a dose-dependent manner (IC50 approximately 100 nM) by a cyclic arginine-glycine-aspartic acid (cRGD) peptide. This effect was mediated via interfering with the VLA-4-VCAM-1 binding. In parallel measurements, transmigration of HL-60 cells across a confluent HUVEC monolayer was inhibited by the cRGD peptide and by both anti-beta1 and anti-alphaVbeta3 antibodies. In conclusion, these data demonstrate the role played by beta1-integrins in leukocyte-endothelial adhesion and transmigration and the role played by alphaVbeta3 in transmigration, thus underscoring the high efficacy of cRGD peptide in blocking both the adhesion and transmigration of monocytes.
AJP Heart and Circulatory Physiology 02/2004; 286(1):H359-67. · 3.71 Impact Factor
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ABSTRACT: The interaction of leukocyte function-associated antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) is central to the regulation of adhesion in leukocytes. In this report, we investigated the mechanisms by which phorbol myristate acetate (PMA) promotes LFA-1-dependent cell adhesion. The adhesion of PMA-stimulated cells to immobilized ICAM-1 was quantified in direct force measurements acquired by atomic force microscopy (AFM). Enhanced adhesion of PMA-stimulated cells to immobilized ICAM-1 stemmed from an increase in the number of LFA-1-ICAM-1 complexes formed between the two apposing surfaces on contact, rather than by affinity modulation of LFA-1. Single molecule force measurements revealed that the force spectrum of the LFA-1-ICAM-1 complex formed by PMA-stimulated cells is identical to the force spectrum of the complex formed by resting cells. Thus, PMA stimulation does not modify the mechanical strength of the individual LFA-1-ICAM-1 interaction. Instead, the enhanced cell adhesion of PMA-stimulated cells appears to be a complex process that correlates with changes in the mechanical properties of the cell. We estimate that changes in the elasticity of the cell gave rise to a more than 10-fold increase in cell adhesion.
Journal of Cell Science 07/2003; 116(Pt 12):2531-9. · 6.11 Impact Factor
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ABSTRACT: Cooperative (simultaneous) breakage of multiple adhesive bonds has been proposed as a mechanism for enhanced binding strength between adhesion molecules on apposing cell surfaces. In this report, we used the atomic force microscopy (AFM) to study how changes in binding affinity and separation rate of force-induced ligand-receptor dissociation affect binding cooperativity. The AFM force measurements were carried out using (strept)avidin-functionalized cantilever tips and biotinylated agarose beads under conditions where multiple (strept)avidin-biotin linkages were formed following surface contact. At slow surface separation of the AFM cantilever from the bead's surface, the (strept)avidin-biotin linkages appeared to rupture sequentially. Increasing the separation rate from 210 to 1950 nm/s led to a linear increase in the average rupture force. Moreover, force histograms revealed a quantized force distribution that shifted toward higher values with increasing separation rate. In measurements of streptavidin-iminobiotin adhesion, the force distribution also shifted toward higher values when the buffer was adjusted to a higher pH to raise the binding affinity. Together, these results demonstrate that the cooperativity of ligand-receptor bonds is significantly enhanced by increases in surface separation rate and/or binding affinity.
Biophysical Chemistry 06/2003; 104(1):271-8. · 2.20 Impact Factor
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ABSTRACT: Interactions between leukocyte function-associated antigen-1 (LFA-1) with its cognate ligand, intercellular adhesion molecule-1 (ICAM-1) play a crucial role in leukocyte adhesion. Because the cell and its adhesive components are subject to external perturbation from the surrounding flow of blood, it is important to understand the binding properties of the LFA-1/ICAM-1 interaction in both steady state and in the presence of an external pulling force. Here we report on atomic force microscopy (AFM) measurements of the unbinding of LFA-1 from ICAM-1. The single molecule measurements revealed the energy landscape corresponding to the dissociation of the LFA-1/ICAM-1 complex and provided the basis for defining the energetic determinants of the complex at equilibrium and under the influence of an external force. The AFM force measurements were performed in an experimental system consisting of an LFA-1-expressing T cell hybridoma, 3A9, attached to the end of the AFM cantilever and an apposing surface expressing ICAM-1. In measurements covering three orders of magnitude change in force loading rate, the LFA-1/ICAM-1 force spectrum (i.e., unbinding force versus loading rate) revealed a fast and a slow loading regime that characterized a steep inner activation barrier and a wide outer activation barrier, respectively. The addition of Mg(2+), a cofactor that stabilizes the LFA-1/ICAM-1 interaction, elevated the unbinding force of the complex in the slow loading regime. In contrast, the presence of EDTA suppressed the inner barrier of the LFA-1/ICAM-1 complex. These results suggest that the equilibrium dissociation constant of the LFA-1/ICAM-1 interaction is regulated by the energetics of the outer activation barrier of the complex, while the ability of the complex to resist a pulling force is determined by the divalent cation-dependent inner activation barrier.
Biophysical Journal 11/2002; 83(4):2270-9. · 3.65 Impact Factor
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ABSTRACT: Since its invention in 1986, the atomic force microscope (AFM) has emerged as a flexible and powerful tool for exploring a
variety of biological processes, including cell adhesion, protein folding, and protein–protein interactions. This review focuses
on the application of the AFM to studies of protein–protein interactions. It describes the commonly used methodologies and
reviews the theoretical framework used to analyze single-molecule protein–protein unbinding measurements. Finally, the chapter
summarizes recent progress in the field and shows that the AFM provides an excellent tool for probing interactions on the
cell surface and for understanding the energy landscapes that govern the dynamics of protein interactions.
01/1970: pages 555-570;
