Impact of Carrier Stiffness and Microtopology on Two-dimensional Kinetics of P-selectin and P-selectin Glycoprotein Ligand-1 (PSGL-1) Interactions

Abstract

Mechanics and surface microtopology of the molecular carrier influence cell adhesion, but the mechanisms underlying these effects are not well understood. We used a micropipette adhesion frequency assay to quantify how the carrier stiffness and microtopology affected two-dimensional kinetics of interacting adhesion molecules on two apposing surfaces. Interactions of P-selectin with P-selectin glycoprotein ligand-1 (PSGL-1) were used to demonstrate such effects by presenting the molecules on three carrier systems: human red blood cells (RBCs), human promyelocytic leukemia HL-60 cells, and polystyrene beads. Stiffening the carrier alone or in cooperation with roughing the surface lowered the two-dimensional affinity of interacting molecules by reducing the forward rate but not the reverse rate, whereas softening the carrier and roughing the surface had opposing effects in affecting two-dimensional kinetics. In contrast, the soluble antibody bound with similar three-dimensional affinity to surface-anchored P-selectin or PSGL-1 constructs regardless of carrier stiffness and microtopology. These results demonstrate that the carrier stiffness and microtopology of a receptor influences its rate of encountering and binding a surface ligand but does not subsequently affect the stability of binding. This provides new insights into understanding the rolling and tethering mechanism of leukocytes onto endothelium in both physiological and pathological processes.Selectins are important adhesive molecules that mediate the cell adhesions in such biological processes as platelet thrombosis, inflammatory responses, as well as tumor metastasis (1, 2). For example, selectin-ligand interactions mediate the rolling and tethering of leukocytes onto endothelium in blood flow. Three known selectin members, P-, E-, and L-selectin, have a common structure: an N-terminal, calcium-type lectin (Lec) 5 domain, followed by an epidermal growth factor (EGF)-like module, multiple copies of consensus repeat (CR) units characteristic of complement binding proteins, a transmembrane segment, and a short cytoplasmic domain (3, 4). As a biochemically well-characterized selectin ligand, P-selectin glycoprotein ligand-1 (PSGL-1) consists of homodimer cross-linked by disulfate bonds and binds to selectins through its N-terminal sulfoglycopeptide, which includes three tyrosine sulfates and the core-2 O-glycan (5-8).To mediate cell adhesion, receptors and their ligands must be anchored onto two apposed surfaces, which is the so-called two-dimensional interaction. This is different from three-dimensional binding, where at least one of the receptors and ligands is in the fluid phase. Not only is two-dimensional kinetics of receptor-ligand binding determined by their intrinsic structures, but it also depends on their surface presentation as well as the stiffness and microtopology of molecule-bearing carrier. On the one hand, randomizing the selectin construct or rabbit immunoglobulin G (rIgG) or lowering the length of selectin extending outward from the cell s...

Analyses of movement and contact of two nucleated cells using a gas-driven micropipette aspiration technique

“…Briefly, a gas-driven pressure unit was added into a conventional MAT system (Huang et al, 2004;Wu et al, 2007). To manipulate the forward and backward movement of a cell inside the left pipette, a positive pressure was exerted by the pressure unit via controlling gas flux gauged by a pressure regulator while a negative pressure was applied by the conventional suction via adjusting the height of reservoir gauged by a micrometer, resulting in a test cycle of cell approaching to, contacting with, and withdrawing from the counterpart cell held steadily in the right pipette ( Fig.…”

“…While it is well known how the suction pressure affects the contact and retraction of the two apposed cells in a conventional MAT assay (Huang et al, 2004;Wu et al, 2007;Long et al, 2001), the impact of gas-driven impinge pressure remains unclear for cell approach to and contact with the apposed cell, which in turn determines the fate of each contact event. To test its function, the impinge pressure was systematically varied by a gas flux of 150, 175, and 200 ml/min and the approaching velocity, apparent contact area, contact duration, and adhesion probability were compared for a Jurkat cell interacting with a MDA-MB-231 cell.…”

“…Meanwhile, consecutive test cycles are required to determine the time course of adhesion probability, but it is still little known whether and/or how the presence of an adhesion, tether, or non-specific binding event in one test cycle affect the outcome from the next cycle in the sequentially repeated cycles. Specifically, membrane tether is one type of adhesion event but is difficult to distinguish them from the nonspecific events especially when the moving cell interacts with pipette wall (Long et al, 1999;Wu et al, 2007).…”

Surface-bound selectin–ligand binding is regulated by carrier diffusion

“…2D dissociation kinetics (Hammer and Lauffenburger 1987;Alon et al 1995;Chen et al 1997) and forced bond rupture (Florin et al 1994;Dammer et al 1996;Tees et al 2001) as well as their regulating factors (Evans et al 2001;Levin et al 2001;Huang et al 2004;Marshall et al 2005;Wu et al 2007) have been investigated theoretically and experimentally using various approaches or assays, e.g., flow chamber (Kaplanski et al 1993;Alon et al 1995;Finger et al 1996;Yago et al 2007;Paschall et al 2008), biomembrane force probe (BFP) (Evans et al 2001;Evans and Ritchie 1997), atom force microscopy (AFM) (Fritz et al 1998;Merkel et al 1999;Marshall et al 2005;Lü et al 2006), micropipette aspiration (Chesla et al 1998;Long et al 2001;Shao and Xu 2002), optical tweezers (Kulin et al 2002;Rinko et al 2004), fluorescence recovery after photobleaching (FRAP) (Dustin et al 1996;Tolentino et al 2008). Only a few works, however, were focused on quantifying 2D association kinetics mainly due to theoretical and technical limitations.…”

“…For example, association rate (in s -1 ) of homotypic [e.g., cadherin-cadherin (Pierres et al 1998)] or heterotypic [e.g., E-selectin-ligand (Kaplanski et al 1993) or CD2-CD48 (Pierres et al 1997)] molecular pair, defined as the product of 2D forward rate k f (in lm 2 /s) and site density (in lm -2 ), was extracted from the dependence of binding frequency on the distance between apposed surfaces in a flow chamber assay. 2D effective forward rate (in lm 4 /s), the product of 2D forward rate and contact area (in lm 2 ), could also be estimated from the dependence of adhesion probability on contact duration as well as site densities of interacting molecules in an adhesion frequency assay (Chesla et al 1998;Huang et al 2004;Long et al 2001;Wu et al 2007), and from the waiting time distribution of intermittent binding and unbinding events in a biomembrane force probe (BFP) assay (Chen et al 2008). Surface-bound bond formation is determined by 2D forward rate of interacting molecules, k f , the association rate per unit contact area per molecule (lm 2 /s).…”