Acknowledgements We thank M. Inouye for the gift of the RU1012 strain, L. Loew for the gift of styryl dyes, S. Conrad and G. Shirman for assistance with mutagenesis and protein chemistry, and M. G. Prisant for construction of the computer cluster. This work was supported by grants from the Office of Naval Research, the Defense Advanced Research Project Agency and the National Institutes of Health.Competing interests statement The authors declare that they have no competing financial interests.Correspondence and requests for material should be addressed to H. Bonds between adhesion molecules are often mechanically stressed. A striking example is the tensile force applied to selectin-ligand bonds, which mediate the tethering and rolling of flowing leukocytes on vascular surfaces 1-3 . It has been suggested that force could either shorten bond lifetimes, because work done by the force could lower the energy barrier between the bound and free states 4 ('slip'), or prolong bond lifetimes by deforming the molecules such that they lock more tightly 5,6 ('catch'). Whereas slip bonds have been widely observed 7-14 , catch bonds have not been demonstrated experimentally. Here, using atomic force microscopy and flow-chamber experiments, we show that increasing force first prolonged and then shortened the lifetimes of P-selectin complexes with P-selectin glycoprotein ligand-1, revealing both catch and slip bond behaviour. Transitions between catch and slip bonds might explain why leukocyte rolling on selectins first increases and then decreases as wall shear stress increases 9,15,16 . This dual response to force provides a mechanism for regulating cell adhesion under conditions of variable mechanical stress.Using atomic force microscopy (AFM) (Fig. 1a), we measured the force dependence of bond lifetimes of P-selectin with two forms of P-selectin glycoprotein ligand-1 (PSGL-1) or with G1, a blocking monoclonal antibody (mAb) against P-selectin 17 (see Methods). P-selectin is an extended C-type lectin expressed on activated endothelial cells and platelets. PSGL-1 is a mucin expressed on leukocytes. Ca 2þ -dependent interactions of P-selectin with PSGL-1 mediate the tethering and rolling of flowing leukocytes on vascular surfaces in response to infection or tissue injury 1-3 .We captured dimeric PSGL-1 purified from human neutrophils 18 or monomeric recombinant soluble PSGL-1 (sPSGL-1) 19 with PL2, a non-blocking anti-PSGL-1 mAb 20 adsorbed on the cantilever tip (Fig. 1b). Cantilever tips bearing (s)PSGL-1 or G1 were repeatedly brought into contact with lipid bilayers reconstituted with P-selectin purified from human platelets 21 to allow bond formation. The cantilever was then retracted a prescribed distance to apply a constant tensile force to the bond or bonds (if any resulted from the contact), and the duration or lifetime of the adhesion at that force was recorded (Fig. 1c). To measure lifetime at forces lower than the level of their fluctuations, many instantaneous forces were averaged (Fig. 1d, e). This enabled the reli...

Receptor-ligand bonds that mediate cell adhesion are often subjected to forces that regulate their dissociation via modulating off-rates. Off-rates control how long receptor-ligand bonds last and how much force they withstand. One should therefore be able to determine off-rates from either bond lifetime or unbinding force measurements. However, substantial discrepancies exist between the force dependence of off-rates derived from the two types of measurements even for the same interactions, e.g., selectins dissociating from their ligands, which mediate the tethering and rolling of leukocytes on vascular surfaces during inflammation and immune surveillance. We used atomic force microscopy to measure survival times of P-selectin dissociating from P-selectin glycoprotein ligand 1 or from an antibody in both bond lifetime and unbinding force experiments. By a new method of data analysis, we showed that the discrepancies resulted from the assumption that off-rates were functions of force only. The off-rates derived from forced dissociation data depended not only on force but also on the history of force application. This finding provides a new paradigm for understanding how force regulates receptor-ligand interactions.

In single-molecule mechanics experiments the molecular elasticity is usually measured from the deformation in response to a controlled applied force, e.g., via an atomic force microscope cantilever. We have tested the validity of an alternative method based on a recently developed theory. The concept is to measure the change in thermal fluctuations of the cantilever tip with and without its coupling to a rigid surface via the molecule. The new method was demonstrated by its application to the elasticity measurements of L- and P-selectin complexed with P-selectin glycoprotein ligand-1 or their respective antibodies, which showed values comparable to those measured from the slope of the force-extension curve. L- and P-selectin were found to behave as nearly linear springs capable of sustaining large forces and strains without sudden unfolding. The measured spring constants of approximately 4 and approximately 1 pN/nm for L- and P-selectin, respectively, suggest that a physiological force of approximately 100 pN would result in an approximately 200% strain for the respective selectins.

During inflammation, selectin-ligand interactions provide forces for circulating leukocytes to adhere to vascular surfaces, which stretch the interacting molecules, suggesting that mechanical properties may be pertinent to their biological function. From mechanical measurements with atomic force microscopy, we analyzed the molecular characteristics of selectins complexed with ligands and antibodies. Respective stiffness of L-, E-, and P-selectins (4.2, 1.4, and 0.85 piconewton/nm) correlated inversely with the number (2, 6, and 9) of consensus repeats in the selectin structures that acted as springs in series to dominate their compliance. After reconstitution into a lipid bilayer, purified membrane P-selectin remained a dimer, capable of forming dimeric bonds with P-selectin glycoprotein ligand (PSGL)-1, endoglycan-Ig, and a dimeric form of a glycosulfopeptide modeled after the N terminus of PSGL-1. By comparison, purified membrane L-and E-selectin formed only monomeric bonds under identical conditions. Ligands and antibodies were much less stretchable than selectins. The length of endoglycan-Ig was found to be 51 ؎ 12 nm. These results provide a comprehensive characterization of the molecular stiffness of selectins and illustrate how mechanical measurements can be utilized for molecular analysis, e.g. evaluating the multimericity of selectins and determining the molecular length of endoglycan.The physical properties of biomolecules can be exploited as tools for their analysis. For instance, electrophoresis that separates proteins according to mass and charge can be exploited to analyze molecular identity and abundance. Physical properties of many biomolecules also have critical roles in their functions. As an example, bending rigidities of actin and microtubule enable these cytoskeletal proteins to provide mechanical support to the cell. Adhesion molecules are also subjected to forces because they anchor cells to other cells or to the extracellular matrix. Mechanical properties and their relevance to biological function of DNA and muscle proteins (e.g. titin and ubiquitin) have been extensively documented (1-8). However, limited studies exist on the mechanical characterization of adhesion molecules and on the utilization of such mechanical measurements for molecular analysis. Here, we employ atomic force microscopy (AFM) 4 to measure mechanical properties of molecular complexes of selectins and their ligands or monoclonal antibodies (mAbs) and use mechanical measurements to address biological issues involving these molecules.Each of the three selectins consists of an N-terminal lectin domain, an epidermal growth factor (EGF)-like domain, 2, 6, or 9 (for L-, E-or P-selectin, respectively) short consensus repeats (CRs), a transmembrane domain, and a short cytoplasmic tail (Fig. 1A). During inflammation, selectin-ligand interactions mediate tethering and rolling of circulating leukocytes on vascular surfaces under a mechanically stressful milieu (9). These molecular complexes are subjected to physical forces that ...

No abstract