Direct observation of catch bonds involving cell-adhesion molecules

Abstract

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...

Single-molecule binding of CD44 to fibrin versus P-selectin predicts their distinct shear-dependent interactions in cancer

“…The P-selectin-PSGL-1 bond exhibits 'catch-slip' dissociation under force, in which bond lifetime is initially prolonged (catch) and then diminishes (slip) with increasing applied force (Evans et al, 2004;Marshall et al, 2003). The unstressed off-rate of P-selectin-PSGL-1 along the single pathway is 0.37±0.07 second -1 , whereas its reactive compliance is 0.23±0.01 nm, and displays a tensile strength of 70-150 pN for loading rates in the range of 300 to 30,000 pN/second, respectively (Evans et al, 2004).…”

AFM as a tool to probe and manipulate cellular processes

“…For example, Benoit et al described the interaction of cells with diverse artificially tailored surfaces with the goal of guiding the rational design of medical implants [17]. Similar experiments have revealed molecular mechanisms that might explain the divergences observed in the rolling behaviours of leukocytes over different selectin-decorated surfaces [9,22]. AFM experiments have also characterised specific cell signalling pathways affecting fibronectin-mediated adhesion in the gastrulation process in the early development of zebrafish embryos [26].…”

“…The application of atomic force microscopy (AFM) to biology has provided unique opportunities to visualise [1][2][3][4][5] and manipulate [5][6][7][8][9][10][11][12] functional biological structures with nanometre resolution in physiological conditions and has enabled researchers to address biological questions that could not otherwise be investigated. Force measurement with the AFM has become a tool of choice to probe and manipulate a wide variety of cellular events.…”

Liaisons « accrocheuses » et mécanique moléculaire

“…Anne Pierres, Anne-Marie Benoliel, Pierre Bongrand > Dans un récent article [1], Cheng Zhu et al décrivent pour la première fois la propriété des molécules de P-sélectine de former avec leur ligand des «liaisons accrocheuses» (catch bonds), c'est-à-dire des liaisons dont la durée de vie augmente lorsqu'elles sont soumises à des contraintes mécaniques. Ce travail démontre la réalité d'un concept imaginé par un théoricien, Micah Dembo, quinze ans auparavant [2].…”

“…La durée de vie des liaisons formées était alors de l'ordre de la seconde. Ces estimations ont été confirmées par l'équipe de T. Springer [17]: la mesure de la constante de dissociation (k off ) pour plusieurs valeurs de la force hydrodynamique a conduit à une vérification de la loi de a pu être obtenue par une utilisation habile de la microscopie de force atomique et des chambres à flux laminaire en conditions de faible flux hydrodynamique [1]. Ce travail montre une fois de plus combien la réalisation de progrès nécessite l'association d'une idée originale, d'une bonne maîtrise technologique et d'un acharnement suffisant.…”