QUANTIFYING RUPTURE FORCE AND BOND LIFETIME OF P-SELECTIN-PSGL-I INTERACTIONS AT LOWER FORCES

摘要

Cell adhesions mediated by receptor-ligand interactions are crucial to many biological processes such as inflammatory cascade,tumor metastasis,and thrombosis formation.Rupture force of receptor-ligand bond is enhanced with logarithm of loading rate (=spring constant × retracting velocity).Bond lifetime is found to be prolonged (so-called catch bond) or shortened (so-called slip bond) with applied forces and presents catch-slip bond transition,when atomic force microscopy and flow chamber assays are used to monitor bond rupture at relatively higher force rang (＞ 10 pN).The mechanism how bond rupture is regulated at lower forces (＜ 10 pN),however,has been poorly understood.Here we developed an optical trap assay to quantify the forced dissociation of binding of P-selectin to P-selectin glycoprotein ligand 1 (PSGL-1). To conduct the measurements,an optical trap assay with as low spring constant as 10-3 pNm was used to trap two silicon beads coupled by P-selectin and PSGL-1 molecules via their respective capturing antibodies (Fig.I).Bond rupture forces and lifetimes were measured at systematically-varied trap spring constants and retracting velocities.Most probable rupture forces were enhanced when higher spring constants were used at a given retracting velocity,but remained same when higher retracting velocities were applied at a pre-set spring constant (except at the highest constant of 0.047 pNm),suggesting that the spring constant of optical trap plays an more important role in regulating forced bond dissociation (Fig.2).Bond lifetime exhibited a slip-catch bond transition when applied forces varied from 0.5 to 15 pN,validating the hypothesis that another slip-catch bond transition phase appears when lower external forces are applied.It was also tbund that,at a given applied force,bond lifetime varied when high spring constant with low retracting velocity or low spring constant with high retracting velocity,was used.These findings further the understanding of forced dissociation of receptor-ligand bonds at lower applied forces,and provide a new insight into manipulating the cell adhesions in physiological flow.