A High-Throughput Technique Reveals the Load- and Site Density-Dependent Kinetics of E-Selectin.

The kinetics of bond rupture between receptors and ligand are critically dependent on applied mechanical force. Force spectroscopy of single receptor-ligand pairs to measure kinetics is a laborious and time-consuming process that is generally performed using individual force probes and making one measurement at a time when typically hundreds of measurements are needed. A high-throughput approach is thus desirable. We report here a magnetic bond puller that provides high-throughput measurements of single receptor-ligand bond kinetics. Electromagnets are used to apply pN tensile and compressive forces to receptor-coated magnetic microspheres while monitoring their contact with a ligand-coated surface. Bond lifetimes and the probability of forming a bond are measured via videomicroscopy, and the data are used to determine the load dependent rates of bond rupture and bond formation. The approach is simple, customizable, relatively inexpensive, and can make dozens of kinetic measurements simultaneously. We used the device to investigate how compressive and tensile forces affect the rates of formation and rupture, respectively, of bonds between E-selectin and sialyl Lewisa (sLea), a sugar on P-selectin glycoprotein ligand-1 to which selectins bind. We confirmed earlier findings of a load-dependent rate of bond formation between these two molecules, and that they form a catch-slip bond like other selectin family members. We also make the novel observation of an "ideal" bond in a highly multivalent system of this receptor-ligand pair.

The Effects of Load on E-Selectin Bond Rupture and Bond Formation.

Molecular dissociation rates have long been known to be sensitive to applied force. We use a laser trap to provide evidence that rates of association may also be force-dependent. We use the thermal fluctuation assay to study single bonds between E-selectin and sialyl Lewis(a) (sLe(a)), the sugar on PSGL-1 to which the three selectins bind. Briefly, an E-selectin-coated bead is held in a laser trap and pressed with various compressive loads against the vertical surface of a bead coated with sLe(a). The time it takes for a bond to form is used to calculate a specific two-dimensional on-rate, kono. We observe an increase in kono with increasing compressive force, providing single molecule evidence that on-rate, in addition to off-rate, is influenced by load. By measuring bond lifetimes at known tensile loads, we show that E-selectin, like its family members L- and P-selectin, is capable of forming catch bonds. Our data support a reverse Bell model, in which compressive forces lower the activation energy for binding. Load-dependent on-rates may be a general feature of all intermolecular bonds.

The viscoelastic properties of microvilli are dependent upon the cell-surface molecule.

We studied at nanometer resolution the viscoelastic properties of microvilli and tethers pulled from myelogenous cells via P-selectin glycoprotein ligand 1 (PSGL-1) and found that in contrast to pure membrane tethers, the viscoelastic properties of microvillus deformations are dependent upon the cell-surface molecule through which load is applied. A laser trap and polymer bead coated with anti-PSGL-1 (KPL-1) were used to apply step loads to microvilli. The lengthening of the microvillus in response to the induced step loads was fitted with a viscoelastic model. The quasi-steady state force on the microvillus at any given length was approximately fourfold lower in cells treated with cytochalasin D or when pulled with concanavalin A-coated rather than KPL-1-coated beads. These data suggest that associations between PSGL-1 and the underlying actin cytoskeleton significantly affect the early stages of leukocyte deformation under flow.

Peroxynitrite inhibits myofibrillar protein function in an in vitro assay of motility.

We determined the effects of peroxynitrite (ONOO-) on cardiac myosin, actin, and thin filaments in order to more clearly understand the impact of this reactive compound in ischemia/reperfusion injury and heart failure. Actin filaments, native thin filaments, and alpha-cardiac myosin from rat hearts were exposed to ONOO- in the presence of 2 mM bicarbonate. Filament velocities over myosin, calcium sensitivity, and relative force generated by myosin were assessed in an in vitro motility assay in the absence of reducing agents. ONOO- concentrations > or =10 microM significantly reduced the velocities of thin filaments or bare actin filaments over alpha-cardiac myosin when any of these proteins were exposed individually. These functional deficits were linearly related to the degree of tyrosine nitration, with myosin being the most sensitive. However, at 10 microM ONOO- the calcium sensitivity of thin filaments remained unchanged. Cotreatment of myosin and thin filaments, analogous to the in vivo situation, resulted in a significantly greater functional deficit. The load supported by myosin after ONOO- exposure was estimated using mixtures experiments to be increased threefold. These data suggest that nitration of myofibrillar proteins can contribute to cardiac contractile dysfunction in pathologic states in which ONOO- is liberated.