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.W.H. (hwh@biochem.duke.edu). .............................................................. Direct observation of catch bonds involving cell-adhesion molecules Bryan T. Marshall*, Mian Long*†, James W. Piper*†, Tadayuki Yago‡, Rodger P. McEver‡§ & Cheng Zhu*k * Woodruff School of Mechanical Engineering and k Coulter School of Biomedical Engineering, Georgia Institute of Technology, Atlanta, Georgia 30332, USA ‡ Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, and § Department of Biochemistry and Molecular Biology and Oklahoma Center for Medical Glycobiology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA ............................................................................................................................................................................. 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. Tran- sitions between catch and slip bonds might explain why leuko- cyte 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-selec- tin 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 reliable resolution of mean forces as low as a few piconewtons, and allowed the detected differences in mean forces to achieve high statistical significance (Fig. 1f). The binding frequency was kept low (12–20%) to ensure that most (about 90%) adhesions dissociated as a single step (Fig. 1c, lower tracing). Only single-step dissociations were analysed. Binding was highly specific. Rendering the cantilever tip func- tional with (s)PSGL-1 increased adhesion frequencies 3–10-fold (Fig. 2a and b) and also increased bond lifetimes (Fig. 3a and b). The inclusion of blocking mAbs against P-selectin (G1) or against PSGL-1 (PL1 20 ) or the divalent-cation chelator EDTA in the chamber solution decreased adhesion to nonspecific levels. G1-coated cantilever tips had significantly higher adhesion frequen- cies and longer bond lifetimes than control PL2-coated tips (Figs 2c and 3c). Remarkably, both the P-selectin–sPSGL-1 interaction (Fig. 3a) and the P-selectin–PSGL-1 interaction (Fig. 3b) exhibited a biphasic relationship between lifetime and force. The bond lifetimes initially increased with force, indicating the presence of catch bonds. After reaching a maximum, the lifetimes decreased with force, indicating slip bonds. This biphasic pattern was detected in individual experi- ments with a single cantilever tip on a single bilayer, which included as few as about five lifetime measurements to calculate a mean and a standard deviation at each of four force levels to cover the biphasic region. This pattern remained unchanged as more data were accumulated (about 400 lifetime measurements for each form of PSGL-1), which allowed us to examine the distributions of lifetimes. As with published flow-chamber data 7,9,11–13 , lifetimes at a given force seemed to follow an exponential distribution, which was made linear by plotting ln(number of events with a lifetime of t or more) Figure 1 AFM system. a, Schematic diagram. b, Making the AFM functional. The cantilever tip depicted represents a composite of all molecules adsorbed or captured. sPSGL-1 and PSGL-1 are depicted as monomer and dimer, respectively. c, Force-scan curves illustrating the cantilever bending (insets) when a compressive or tensile force was applied to the tip. The upper curves illustrate a contact cycle without binding, where the retraction curve (from left to right) retraced the approach curve (from right to left). The lower curves illustrate a contact cycle with binding and lifetime measurement. d, Plot of instantaneous force against time before (red) and after (blue) an unbinding event. e, Running means (thick curves) ^ s.e.m. (paired thin curves) against number of data points. f, P value of the Student’s t-test comparing the difference of the two running means against numbers of point pairs. † Present addresses: National Microgravity Laboratory, Institute of Mechanics, Chinese Academy of Sciences, Beijing 100080, China (M.L.), and Immucor, Inc., Norcross, Georgia 30091,USA (J.W.P.). letters to nature NATURE | VOL 423 | 8 MAY 2003 | www.nature.com/nature 190 © 2003 Nature Publishing Group