Dataset Information

Enhancement of L-selectin, but not P-selectin, bond formation frequency by convective flow.

ABSTRACT: L-selectin-mediated leukocyte rolling has been proposed to require a high rate of bond formation compared to that of P-selectin to compensate for its much higher off-rate. To test this hypothesis, a microbead system was utilized to measure relative L-selectin and P-selectin bond formation rates on their common ligand P-selectin glycoprotein ligand-1 (PSGL-1) under shear flow. Using video microscopy, we tracked selectin-coated microbeads to detect the formation frequency of adhesive tether bonds. From velocity distributions of noninteracting and interacting microbeads, we observed that tether bond formation rates for P-selectin on PSGL-1 decreased with increasing wall shear stress, from 0.14 +/- 0.04 bonds/microm at 0.2 dyn/cm(2) to 0.014 +/- 0.003 bonds/microm at 1.0 dyn/cm(2). In contrast, L-selectin tether bond formation increased from 0.017 +/- 0.005 bonds/microm at 0.2 dyn/cm(2) to 0.031 +/- 0.005 bonds/microm at 1.0 dyn/cm(2). L-selectin tether bond formation rates appeared to be enhanced by convective transport, whereas P-selectin rates were inhibited. The transition force for the L-selectin catch-slip transition of 44 pN/bond agreed well with theoretical models (Pereverzev et al. 2005. Biophys. J. 89:1446-1454). Despite catch bond behavior, hydrodymanic shear thresholding was not detected with L-selectin beads rolling on PSGL-1. We speculate that shear flow generated compressive forces may enhance L-selectin bond formation relative to that of P-selectin and that L-selectin bonds with PSGL-1 may be tuned for the compressive forces characteristic of leukocyte-leukocyte collisions during secondary capture on the blood vessel wall. This is the first report, to our knowledge, comparing L-selectin and P-selectin bond formation frequencies in shear flow.

SUBMITTER: Paschall CD

PROVIDER: S-EPMC2186251 | biostudies-literature | 2008 Feb

REPOSITORIES: biostudies-literature

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Enhancement of L-selectin, but not P-selectin, bond formation frequency by convective flow.

Paschall Christopher D CD Guilford William H WH Lawrence Michael B MB

Biophysical journal 20070921 3

L-selectin-mediated leukocyte rolling has been proposed to require a high rate of bond formation compared to that of P-selectin to compensate for its much higher off-rate. To test this hypothesis, a microbead system was utilized to measure relative L-selectin and P-selectin bond formation rates on their common ligand P-selectin glycoprotein ligand-1 (PSGL-1) under shear flow. Using video microscopy, we tracked selectin-coated microbeads to detect the formation frequency of adhesive tether bonds. ...[more]

PMID: 17890384

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Project description:The interaction of proteins at cellular interfaces is critical for many biological processes, from intercellular signaling to cell adhesion. For example, the selectin family of adhesion receptors plays a critical role in trafficking during inflammation and immunosurveillance. Quantitative measurements of binding rates between surface-constrained proteins elicit insight into how molecular structural details and post-translational modifications contribute to function. However, nano-scale transport effects can obfuscate measurements in experimental assays. We constructed a biophysical simulation of the motion of a rigid microsphere coated with biomolecular adhesion receptors in shearing flow undergoing thermal motion. The simulation enabled in silico investigation of the effects of kinetic force dependence, molecular deformation, grouping adhesion receptors into clusters, surface-constrained bond formation, and nano-scale vertical transport on outputs that directly map to observable motions. Simulations recreated the jerky, discrete stop-and-go motions observed in P-selectin/PSGL-1 microbead assays with physiologic ligand densities. Motion statistics tied detailed simulated motion data to experimentally reported quantities. New deductions about biomolecular function for P-selectin/PSGL-1 interactions were made. Distributing adhesive forces among P-selectin/PSGL-1 molecules closely grouped in clusters was necessary to achieve bond lifetimes observed in microbead assays. Initial, capturing bond formation effectively occurred across the entire molecular contour length. However, subsequent rebinding events were enhanced by the reduced separation distance following the initial capture. The result demonstrates that vertical transport can contribute to an enhancement in the apparent bond formation rate. A detailed analysis of in silico motions prompted the proposition of wobble autocorrelation as an indicator of two-dimensional function. Insight into two-dimensional bond formation gained from flow cell assays might therefore be important to understand processes involving extended cellular interactions, such as immunological synapse formation. A biologically informative in silico system was created with minimal, high-confidence inputs. Incorporating random effects in surface separation through thermal motion enabled new deductions of the effects of surface-constrained biomolecular function. Important molecular information is embedded in the patterns and statistics of motion.

Project description:Selectin-ligand interactions mediate tethering and rolling of circulating leukocytes on the vessel wall during inflammation. Extensive study has been devoted to elucidating the kinetic and mechanical constraints of receptor-ligand-interaction-mediated leukocyte adhesion, yet many questions remain unanswered. Here, we describe our design of an inverted flow chamber to compare adhesions of HL-60 cells to E-selectin in the upright and inverted orientations. This new, to our knowledge, design allowed us to evaluate the effect of gravity and to investigate the mechanisms of flow-enhanced adhesion. Cell rolling in the two orientations was qualitatively similar, and the quantitative differences can be explained by the effect of gravity, which promotes free-flowing cells to tether and detached cells to reattach to the surface in the upright orientation but prevents such attachment from happening in the inverted orientation. We characterized rolling stability by the lifetime of rolling adhesion and detachment of rolling cells, which could be easily measured in the inverted orientation, but not in the upright orientation because of the reattachment of transiently detached cells. Unlike the transient tether lifetime of E-selectin-ligand interaction, which exhibited triphasic slip-catch-slip bonds, the lifetime of rolling adhesion displayed a biphasic trend that first increased with the wall shear stress, reached a maximum at 0.4 dyn/cm(2), and then decreased gradually. We have developed a minimal mathematical model for the probability of rolling adhesion. Comparison of the theoretical predictions to data has provided model validation and allowed evaluation of the effective two-dimensional association on-rate, kon, and the binding affinity, Ka, of the E-selectin-ligand interaction. kon increased with the wall shear stress from 0.1 to 0.7 dyn/cm(2). Ka first increased with the wall shear stress, reached a maximum at 0.4 dyn/cm(2), and then decreased gradually. Our results provide insights into how the interplay between flow-dependent on-rate and off-rate of E-selectin-ligand bonds determine flow-enhanced cell rolling stability.

Dataset Information

Enhancement of L-selectin, but not P-selectin, bond formation frequency by convective flow.

Publications

Enhancement of L-selectin, but not P-selectin, bond formation frequency by convective flow.

Similar Datasets

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