L-selectin Dimerization Enhances Tether Formation to Properly Spaced Ligand

Abstract

Selectin counterreceptors are glycoprotein scaffolds bearing multiple carbohydrate ligands with exceptional ability to tether flowing cells under disruptive shear forces. Bond clusters may facilitate formation and stabilization of selectin tethers. L-selectin ligation has been shown to enhance L-selectin rolling on endothelial surfaces. We now report that monoclonal antibodies-induced L-selectin dimerization enhances L-selectin leukocyte tethering to purified physiological L-selectin ligands and glycopeptides. Microkinetic analysis of individual tethers suggests that leukocyte rolling is enhanced through the dimerization-induced increase in tether formation, rather than by tether stabilization. Notably, L-selectin dimerization failed to augment Lselectin-mediated adhesion below a threshold ligand density, suggesting that L-selectin dimerization enhanced adhesiveness only to properly clustered ligand. In contrast, an epidermal growth factor domain substitution of L-selectin enhanced tether formation to L-selectin ligands irrespective of ligand density, suggesting that this domain controls intrinsic ligand binding properties of L-selectin without inducing L-selectin dimerization. Strikingly, at low ligand densities, where L-selectin tethering was not responsive to dimerization, elevated shear stress restored sensitivity of tethering to selectin dimerization. This is the first indication that shear stress augments effective selectin ligand density at local contact sites by promoting L-selectin encounter of immobilized ligand.Selectins are specialized C-type lectins that mediate the reversible capture (tethering) of circulating cell subsets to specific vessel walls and its subsequent rolling tethers in the direction of flow (1, 2). The biophysical basis for the exceptional ability of selectins to mediate cell capture (tethering) and rolling adhesions under highly disruptive forces is still obscure (3-6). This ability has been primarily attributed to fast formation rates of selectin bonds and to the ability of selectin tethers to tolerate rupture by elevated shear forces (7-10). A major unresolved question regarding selectin function is the high effective formation rate of selectin tethers to their physiological counterreceptors. Efficient tether formation, in particular by L-selectin, contrasts with the fairly low k on of intrinsic Lselectin bonds (11). This k on falls in a range shared by other molecular pairs with poor tethering capacity under shear flow (12, 13). It has thus become increasingly evident that selectin tethering to ligand is controlled by mechanical properties in addition to the biochemical properties measured under shearfree conditions (14,15).In addition to the fast effective kinetics of bond formation and intrinsically high mechanical stability of individual bonds (5), three other mechanisms have been proposed to account for the exceptionally high capacity of selectins to form tethers under physiological flow. Association of L-selectin with the actin cytoskeleton through its cytoplasmic domain wa...

Selectin catch–slip kinetics encode shear threshold adhesive behavior of rolling leukocytes

“…Within these limits, cell velocity varied inversely with substrate density and consistently exhibited a minimum near a shear stress of 0.8 dynes/cm 2 , in good agreement with experimental measurements of the shear threshold for neutrophils adhering to PSGL-1. In a similar manner, we confirmed that dimerization of adhesion receptors promoted more robust rolling while leaving the magnitude of the shear threshold unaltered (34). Much like a doubling in substrate density, dimerizing L-selectin in our simulations resulted in slower cell rolling but an identical shear threshold at 0.8 dynes/cm 2 (Fig.…”

Membranal Cholesterol Is Not Required for L-Selectin Adhesiveness in Primary Lymphocytes but Controls a Chemokine-Induced Destabilization of L-Selectin Rolling Adhesions

“…Notably, although L-selectin ligation was reported to drive the selectin into raft microdomains and induce shedding (15), the chemokine-mediated suppressive signals studied in our system are not associated with L-selectin shedding, because chemokine-mediated suppression of L-selectin rolling cannot be rescued by inhibition of L-selectin shedding (20). Ab-driven dimerization of the ectodomain of L-selectin known to augment L-selectin adhesiveness under shear flow (46) was also unable to rescue chemokine-mediated destabilization of rolling (20). Our data are therefore consistent with the possibility that chemokinedriven GPCR assemblies do not interfere with the ability of Lselectin to dimerize, but rather inhibit key cytoplasmic associations of L-selectin necessary for adhesion stabilization under external forces (47).…”

Cytoskeletal interactions regulate inducible L-selectin clustering

“…Considering that all physiological ligands of L-selectin present multiple low-affinity oligosaccharide binding sites, inducible L-selectin clustering would likely provide a highly efficient means of regulating its binding activity. Indeed, L-selectin when clustered greatly enhances leukocyte tethering (12,48). This process also results in signaling and the induction of various postadhesion events, including oxidative burst, degranulation, cytokine expression, actin polymerization, and CD18 integrin activation (9,21,22,30,43,64,67,71,76,80,81).…”