Nano- to microscale dynamics of P-selectin detachment from leukocyte interfaces. I. Membrane separation from the cytoskeleton

We have used a biomembrane force probe decorated with P-selectin to form point attachments with PSGL-1 receptors on a human neutrophil (PMN) in a calcium-containing medium and then to quantify the forces experienced by the attachment during retraction of the PMN at fixed speed. From first touch to final detachment, the typical force history exhibited the following sequence of events: i), an initial linear-elastic displacement of the PMN surface, ii), an abrupt crossover to viscoplastic flow that signaled membrane separation from the interior cytoskeleton and the beginning of a membrane tether, and iii), the final detachment from the probe tip by usually one precipitous step of P-selectin:PSGL-1 dissociation. In this first article I, we focus on the initial elastic response and its termination by membrane separation from the cytoskeleton, initiating tether formation. Quantifying membrane unbinding forces for rates of loading (force/time) in the elastic regime from 240 pN/s to 38,000 pN/s, we discovered that the force distributions agreed well with the theory for kinetically limited failure of a weak bond. The kinetic rate for membrane unbinding was found to increase as an exponential function of the pulling force, characterized by an e-fold scale in force of approximately 17 pN and a preexponential factor, or apparent unstressed off rate, of approximately 1/s. The rheological properties of tether growth subsequent to the membrane unbinding events are presented in a companion article II.     

Direct observation of membrane tethers formed during neutrophil attachment to platelets or P-selectin under physiological flow

Adhesion and subsequent aggregation between neutrophils and platelets is dependent upon the initial binding of P-selectin on activated platelets to P-selectin glycoprotein ligand 1 (PSGL-1) on the microvilli of neutrophils. High speed, high resolution videomicroscopy of flowing neutrophils interacting with spread platelets demonstrated that thin membrane tethers were pulled from neutrophils in 32 +/- 4% of the interactions. After capture by spread platelets, neutrophil membrane tethers (length of 5.9 +/- 4.1 microm, n = 63) were pulled at an average rate of 6-40 microm/s as the wall shear rate was increased from 100-250 s(-1). The average tether lifetime decreased significantly (P < 0.001) from 630 to 133 ms as the shear rate was increased from 100 s(-1) (F(bond) = 86 pN) to 250 s(-1) (F(bond) = 172 pN), which is consistent with P-selectin/PSGL-1 bond dynamics under stress. Tether formation was blocked by antibodies against P-selectin or PSGL-1, but not by anti-CD18 antibodies. During neutrophil rolling on P-selectin at 150 s(-1), thin membrane tethers were also pulled from the neutrophils. The characteristic jerking motion of the neutrophil coexisted with tether growth (8.9 +/- 8.8 microm long), whereas tether breakage (average lifetime of 3.79 +/- 3.32 s) caused an acute jump in the rolling velocity, proving multiple bonding in the cell surface and the tether surface contact area. Extremely long membrane tethers (>40 microm) were sometimes pulled, which detached in a flow-dependent mechanism of microparticle formation. Membrane tethers were also formed when neutrophils were perfused over platelet monolayers. These results are the first visualization of the often hypothesized tethers that shield the P-selectin/PSGL-1 bond from force loading to regulate neutrophil rolling during inflammation and thrombosis.     

Membrane tether formation from outer hair cells with optical tweezers

Optical tweezers were used to characterize the mechanical properties of the outer hair cell (OHC) plasma membrane by pulling tethers with 4.5-microm polystyrene beads. Tether formation force and tether force were measured in static and dynamic conditions. A greater force was required for tether formations from OHC lateral wall (499 +/- 152 pN) than from OHC basal end (142 +/- 49 pN). The difference in the force required to pull tethers is consistent with an extensive cytoskeletal framework associated with the lateral wall known as the cortical lattice. The apparent plasma membrane stiffness, estimated under the static conditions by measuring tether force at different tether length, was 3.71 pN/microm for OHC lateral wall and 4.57 pN/microm for OHC basal end. The effective membrane viscosity was measured by pulling tethers at different rates while continuously recording the tether force, and estimated in the range of 2.39 to 5.25 pN x s/microm. The viscous force most likely results from the viscous interactions between plasma membrane lipids and the OHC cortical lattice and/or integral membrane proteins. The information these studies provide on the mechanical properties of the OHC lateral wall is important for understanding the mechanism of OHC electromotility.     

Nano-to-micro scale dynamics of P-selectin detachment from leukocyte interfaces. III. Numerical simulation of tethering under flow

Transient capture of cells or model microspheres from flow over substrates sparsely coated with adhesive ligands has provided significant insight into the unbinding kinetics of leukocyte:endothelium adhesion complexes under external force. Whenever a cell is stopped by a point attachment, the full hydrodynamic load is applied to the adhesion site within an exceptionally short time-less than the reciprocal of the hydrodynamic shear rate (e.g., typically <0.01 s). The decay in numbers of cells or beads that remain attached to a surface has been used as a measure of the kinetics of molecular bond dissociation under constant force, revealing a modest increase in detachment rate at growing applied shear stresses. On the other hand, when detached under steady ramps of force with mechanical probes (e.g., the atomic force microscope and biomembrane force probe), P-selectin:PSGL-1 adhesion bonds break at rates that increase enormously under rising force, yielding 100-fold faster off rates at force levels comparable to high shear. The comparatively weak effect of force on tether survival in flow chamber experiments could be explained by a possible partition of the load amongst several bonds. However, a comprehensive understanding of the difference in kinetic behavior requires us to also inspect other factors affecting the dynamics of attachment-force buildup, such as the interfacial compliance of all linkages supporting the adhesion complex. Here, combining the mechanical properties of the leukocyte interface measured in probe tests with single-bond kinetics and the kinetics of cytoskeletal dissociation, we show that for the leukocyte adhesion complex P-selectin:PSGL-1, a detailed adhesive dynamics simulation accurately reproduces the tethering behavior of cells observed in flow chambers. Surprisingly, a mixture of 10% single bonds and 90% dimeric bonds is sufficient to fully match the data of the P-selectin:PSGL-1 experiments, with the calculated decay in fraction of attached cells still appearing exponential.     

Comparison of PSGL-1 microbead and neutrophil rolling: microvillus elongation stabilizes P-selectin bond clusters

A cell-scaled microbead system was used to analyze the force-dependent kinetics of P-selectin adhesive bonds independent of micromechanical properties of the neutrophil's surface microvilli, an elastic structure on which P-selectin ligand glycoprotein-1 (PSGL-1) is localized. Microvillus extension has been hypothesized in contributing to the dynamic range of leukocyte rolling observed in vivo during inflammatory processes. To evaluate PSGL-1/P-selectin bond kinetics of microbeads and neutrophils, rolling and tethering on P-selectin-coated substrates were compared in a parallel-plate flow chamber. The dissociation rates for PSGL-1 microbeads on P-selectin were briefer than those of neutrophils for any wall shear stress, and increased more rapidly with increasing flow. The microvillus length necessary to reconcile dissociation constants of PSGL-1 microbeads and neutrophils on P-selectin was 0.21 microm at 0.4 dyn/cm2, and increased to 1.58 microm at 2 dyn/cm2. The apparent elastic spring constant of the microvillus ranged from 1340 to 152 pN/microm at 0.4 and 2.0 dyn/cm2 wall shear stress. Scanning electron micrographs of neutrophils rolling on P-selectin confirmed the existence of micrometer-scaled tethers. Fixation of neutrophils to abrogate microvillus elasticity resulted in rolling behavior similar to PSGL-1 microbeads. Our results suggest that microvillus extension during transient PSGL-1/P-selectin bonding may enhance the robustness of neutrophil rolling interactions.     

Ethanol enhances neutrophil membrane tether growth and slows rolling on P-selectin but reduces capture from flow and firm arrest on IL-1-treated endothelium

The effects of ethanol at physiological concentrations on neutrophil membrane tether pulling, adhesion lifetime, rolling, and firm arrest behavior were studied in parallel-plate flow chamber assays with adherent 1-microm-diameter P-selectin-coated beads, P-selectin-coated surfaces, or IL-1-stimulated human endothelium. Ethanol (0.3% by volume) had no effect on P-selectin glycoprotein ligand-1 (PSGL-1), L-selectin, or CD11b levels but caused PSGL-1 redistribution. Also, ethanol prevented fMLP-induced CD11b up-regulation. During neutrophil collisions with P-selectin-coated beads at venous wall shear rates of 25-100 s(-1), ethanol increased membrane tether length and membrane growth rate by 2- to 3-fold but reduced the adhesion efficiency (detectable bonding per total collisions) by 2- to 3-fold, compared with untreated neutrophils. Without ethanol treatment, adhesion efficiency and adhesion lifetime declined as wall shear rate was increased, whereas ethanol caused the adhesion lifetime over all events to increase from 0.1 s to 0.5 s as wall shear rate was increased, an example of pharmacologically induced hydrodynamic thresholding. Consistent with this increased membrane fluidity and reduced capture, ethanol reduced rolling velocity by 37% and rolling flux by 55% on P-selectin surfaces at 100 s(-1), compared with untreated neutrophils. On IL-1-stimulated endothelium, rolling velocity was unchanged by ethanol treatment, but the fraction of cells converting to firm arrest was reduced from 35% to 24% with ethanol. Overall, ethanol caused competing biophysical and biochemical effects that: 1) reduced capture due to PSGL-1 redistribution, 2) reduced rolling velocity due to increased membrane tether growth, and 3) reduced conversion to firm arrest.     

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

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/μm at 0.2 dyn/cm² to 0.014 ± 0.003 bonds/μm at 1.0 dyn/cm². In contrast, L-selectin tether bond formation increased from 0.017 ± 0.005 bonds/μm at 0.2 dyn/cm² to 0.031 ± 0.005 bonds/μm at 1.0 dyn/cm². 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.     

Neutrophil-bead collision assay: pharmacologically induced changes in membrane mechanics regulate the PSGL-1/P-selectin adhesion lifetime

Visualization of flowing neutrophils colliding with adherent 1-mum-diameter beads presenting P-selectin allowed the simultaneous measurement of collision efficiency (epsilon), membrane tethering fraction (f), membrane tether growth dynamics, and PSGL-1/P-selectin binding lifetime. For 1391 collisions analyzed over venous wall shear rates from 25 to 200 s(-1), epsilon decreased from 0.17 to 0.004, whereas f increased from 0.15 to 0.70, and the average projected membrane tether length, L(tether)(m), increased from 0.35 mum to approximately 2.0 mum over this shear range. At all shear rates tested, adhesive collisions lacking membrane tethers had average bond lifetimes less than those observed for collisions with tethers. For adhesive collisions that failed to form membrane tethers, the regressed Bell parameters (consistent with single bond Monte Carlo simulation) were zero-stress off-rate, k(off)(0) = 0.56 s(-1) and reactive compliance, r = 0.10 nm, similar to published atomic force microscopy (AFM) measurements. For all adhesion events (+/- tethers), the bond lifetime distributions were more similar to those obtained by rolling assay and best simulated by Monte Carlo with the above Bell parameters and an average of 1.48 bonds (n = 1 bond (67%), n = 2 (22%), and n = 3-5 (11%)). For collisions at 100 s(-1), pretreatment of neutrophils with actin depolymerizing agents, latrunculin or cytochalasin D, had no effect on epsilon, but increased L(tether)(m) by 1.74- or 2.65-fold and prolonged the average tether lifetime by 1.41- or 1.65-fold, respectively. Jasplakinolide, an actin polymerizing agent known to cause blebbing, yielded results similar to the depolymerizing agents. Conversely, cholesterol-depletion with methyl-beta-cyclodextrin or formaldehyde fixation had no effect on epsilon, but reduced L(tether)(m) by 66% or 97% and reduced the average tether lifetime by 30% or 42%, respectively. The neutrophil-bead collision assay combines advantages of atomic force microscopy (small contact zone), aggregometry (discrete interactions), micropipette manipulation (tether visualization), and rolling assays (physiologic flow loading). Membrane tether growth can be enhanced or reduced pharmacologically with consequent effects on PSGL-1/P-selectin lifetimes.     

Single molecule characterization of P-selectin/ligand binding

P-selectin expressed on activated platelets and vascular endothelium mediates adhesive interactions to polymorphonuclear leukocytes (PMNs) and colon carcinomas critical to the processes of inflammation and blood-borne metastasis, respectively. How the overall adhesiveness (i.e. the avidity) of receptor/ligand interactions is controlled by the affinity of the individual receptors to single ligands is not well understood. Using single molecule force spectroscopy, we probed in situ both the tensile strength and off-rate of single P-selectin molecules binding to single ligands on intact human PMNs and metastatic colon carcinomas and compared them to the overall avidity of these cells for P-selectin substrates. The use of intact cells rather than purified proteins ensures the proper orientation and preserves post-translational modifications of the P-selectin ligands. The P-selectin/PSGL-1 interaction on PMNs was able to withstand forces up to 175 pN and had an unstressed off-rate of 0.20 s(-1). The tensile strength of P-selectin binding to a novel O-linked, sialylated protease-sensitive ligand on LS174T colon carcinomas approached 125 pN, whereas the unstressed off-rate was 2.78 s(-1). Monte Carlo simulations of receptor/ligand bond rupture under constant loading rate for both P-selectin/PSGL-1 and P-selectin/LS174T ligand binding give distributions and mean rupture forces that are in accord with experimental data. The pronounced differences in the affinity for P-selectin/ligand binding provide a mechanistic basis for the differential abilities of PMNs and carcinomas to roll on P-selectin substrates under blood flow conditions and underline the requirement for single molecule affinity measurements.     

Membrane tether extraction from human umbilical vein endothelial cells and its implication in leukocyte rolling

During the rolling of human neutrophils on the endothelium, tethers (cylindrical membrane tubes) are likely extracted from the neutrophil. Tether extraction reduces the force imposed on the adhesive bond between the neutrophil and endothelium, thereby facilitating the rolling. However, whether tethers can be extracted from the endothelium is still unknown. Here, with the micropipette-aspiration technique, we show that tethers can be extracted from either suspended or attached human umbilical vein endothelial cells. We also show that a linear relationship between the pulling force and tether growth velocity exists and this relationship does not depend on the receptor type (used to impose point forces), tumor necrosis factor-alpha stimulation, or cell attachment state. With linear regression, we determined that the threshold force was 50 pN and the effective viscosity was 0.50 pN.s/microm. Therefore, tethers might be simultaneously extracted from the neutrophil and endothelial cell during the rolling and, more importantly, the endothelial cell might contribute much more to the total composite tether length than the neutrophil. Compared with tether extraction from the neutrophil alone, simultaneous tether extraction results in a larger increase in the lifetime of the adhesive bond, and thus further stabilizes the rolling of neutrophils under high physiological shear stresses.     

Cell protrusions and tethers: a unified approach

Low pulling forces applied locally to cell surface membranes produce viscoelastic cell surface protrusions. As the force increases, the membrane can locally separate from the cytoskeleton and a tether forms. Tethers can grow to great lengths exceeding the cell diameter. The protrusion-to-tether transition is known as the crossover. Here we propose a unified approach to protrusions and tethers providing, to our knowledge, new insights into their biomechanics. We derive a necessary and sufficient condition for a crossover to occur, a formula for predicting the crossover time, conditions for a tether to establish a dynamic equilibrium (characterized by constant nonzero pulling force and tether extension rate), a general formula for the tether material after crossover, and a general modeling method for tether pulling experiments. We introduce two general protrusion parameters, the spring constant and effective viscosity, valid before and after crossover. Their first estimates for neutrophils are 50 pN μm⁻¹ and 9 pN s μm⁻¹, respectively. The tether elongation after crossover is described as elongation of a viscoelastic-like material with a nonlinearly decaying spring (NLDs-viscoelastic material). Our model correctly describes the results of the published protrusion and tether pulling experiments, suggesting that it is universally applicable to such experiments.     

Multiple membrane tethers probed by atomic force microscopy

Using the atomic force microscope to locally probe the cell membrane, we observed the formation of multiple tethers (thin nanotubes, each requiring a similar pulling force) as reproducible features within force profiles recorded on individual cells. Forces obtained with Chinese hamster ovary cells, a malignant human brain tumor cell line, and human endothelial cells (EA hy926) were found to be 28 +/- 10 pN, 29 +/- 9 pN, and 29 +/- 10 pN, respectively, independent of the nature of attachment to the cantilever. The rather large variation of the tether pulling forces measured at several locations on individual cells points to the existence of heterogeneity in the membrane properties of a morphologically homogeneous cell. Measurement of the summary lengths of the simultaneously extracted tethers provides a measure of the size of the available membrane reservoir through which co-existing tethers are associated. As expected, partial disruption of the actin cytoskeleton and removal of the hyaluronan backbone of the glycocalyx were observed to result in a marked decrease (30-50%) in the magnitude and a significant sharpening of the force distribution indicating reduced heterogeneity of membrane properties. Taken together, our results demonstrate the ability of the plasma membrane to locally produce multiple interdependent tethers-a process that could play an important role in the mechanical association of cells with their environment.     

Neutrophils, monocytes, and dendritic cells express the same specialized form of PSGL-1 as do skin-homing memory T cells: cutaneous lymphocyte antigen.

Memory T cells in inflamed skin express the cutaneous lymphocyte-associated antigen (CLA), a glycosylated epitope defined by the mAb HECA-452. We previously reported that on T cells, CLA occurs almost exclusively on the protein backbone of P-selectin glycoprotein ligand-1 (PSGL-1). T cells exhibiting the CLA isoform of PSGL-1 can tether and roll on both E- and P-selectin, while T cells expressing PSGL-1 without the CLA epitope do not bind E-selectin, though they may bind P-selectin. We show here that circulating neutrophils and monocytes, and cultured blood dendritic cells, also express CLA almost entirely as an isoform of PSGL-1. These cells all tether and roll on both E- and P-selectin. A chimeric fusion protein incorporating the 19 N-terminal amino acids of mature PSGL-1 exhibited HECA-452 immunoreactivity and supported rolling of CHO cells expressing either E- or P-selectin. These findings indicate a site for the CLA modification within the distal tip of PSGL-1, previously shown to be critical for P-selectin binding and to mediate some, but not all, of the E-selectin binding of PSGL-1. We hypothesize that the types of circulating leukocytes discussed above all use CLA/PSGL-1 to tether and roll on E- and P-selectin along the vascular endothelium.     

重组人P-选凝素及P-选凝素糖蛋白配基-1的表达与鉴定

P-选凝素表达于血管内皮细胞及血小板膜上,它可以与白细胞膜表面的P-选凝素糖蛋白配基-1(P-selectin glyco-protein ligand-1,PSGL-1)相互作用,在炎症过程中介导白细胞的滚动并启动随后的白细胞迁移级联过程.我们构建了重组人野生型可溶性P-选凝素及其钙离子结合位点突变体,同时构建了重组PSGL-1免疫球蛋白融合分子(PSGL-1-Rg),并应用昆虫杆状病毒表达系统在Sf9细胞中表达这些重组蛋白,最后用镍金属螯和柱或Protein A亲和柱予以纯化.结果显示,用该系统表达的P-选凝素或PSGL-1是有活性的,但是P-选凝素的4个钙离子结合位点突变体却没有活性.该研究证明了P-选凝素钙离子结合位点在其与配基相互作用中的重要性.     

Single membrane tether extraction from adult and neonatal dermal microvascular endothelial cells

Membrane tethers were found to be extracted from leukocytes and macrovascular endothelial cells (e.g., human umbilical vein endothelial cells or HUVECs) when a point pulling force was exerted. These tethers stabilize leukocyte rolling on the endothelium during the inflammatory response. However, little is known about tether extraction from other vascular cells like microvascular endothelial cells (MECs). In this study, we extracted tethers from both adult and neonatal dermal MECs with the micropipette aspiration technique. We found a linear relationship between the pulling force and tether growth velocity for both cell lines. This constitutive relationship is mainly determined by the membrane mechanical property and the underlying actin-based cytoskeleton for both attached and suspended endothelial cells. It is independent of cell surface receptor type, attachment state, cytokine stimulation, or cell lineage. For both types of MECs, the threshold forces are 50 pN and the effective viscosities are around 0.5 pN·s/µm. These results, which are close to what was obtained from HUVECs, indicate that homogeneity is preserved in terms of tether extraction among different types of endothelial cells, and simultaneous tethers are likely extracted when leukocytes roll on either microvascular or macrovascular surfaces. leukocyte rolling; cell mechanics; micropipette; cytoskeleton     

Characteristics of a membrane reservoir buffering membrane tension.

When membrane-attached beads are pulled vertically by a laser tweezers, a membrane tube of constant diameter (tether) is formed. We found that the force on the bead (tether force) did not depend on tether length over a wide range of tether lengths, which indicates that a previously unidentified reservoir of membrane and not stretch of the plasma membrane provides the tether membrane. Plots of tether force vs. tether length have an initial phase, an elongation phase, and an exponential phase. During the major elongation phase, tether force is constant, buffered by the "membrane reservoir." Finally, there is an abrupt exponential rise in force that brings the tether out of the trap, indicating depletion of the membrane reservoir. In chick embryo fibroblasts and 3T3 fibroblasts, the maximum tether lengths that can be pulled at a velocity of 4 microm/s are 5.1 +/- 0. 3 and 5.0 +/- 0.2 microm, respectively. To examine the importance of the actin cytoskeleton, we treated cells with cytochalasin B or D and found that the tether lengths increased dramatically to 13.8 +/- 0.8 and 12.0 +/- 0.7 microm, respectively. Similarly, treatment of the cells with colchicine and nocodazole results in more than a twofold increase in tether length. We found that elevation of membrane tension (through osmotic pressure, a long-term elevation of tether force, or a number of transitory increases) increased reservoir size over the whole cell. Using a tracking system to hold tether force on the bead constant near its maximal length in the exponential phase, the rate of elongation of the tethers was measured as a function of tether force (membrane tension). The rate of elongation of tethers was linearly dependent on the tether force and reflected an increase in size of the reservoir. Increases in the reservoir caused by tension increases on one side of the cell caused increases in reservoir size on the other side of the cell. Thus, we suggest that cells maintain a plasma membrane reservoir to buffer against changes in membrane tension and that the reservoir is increased with membrane tension or disruption of the cytoskeleton.     

Functional analysis of the combined role of the O-linked branching enzyme core 2 beta1-6-N-glucosaminyltransferase and dimerization of P-selectin glycoprotein ligand-1 in rolling on P-selectin

Leukocyte P-selectin glycoprotein ligand-1 (PSGL-1) is expressed as a homodimer and mediates leukocyte rolling through interactions with endothelial P-selectin. Previous studies have shown that PSGL-1 must be properly modified by specific glycosyltransferases including alpha1,3-fucosyltransferase-VII, core 2 beta1-6-N-glucosaminyltransferase (C2GlcNAcT-I), one or more alpha2,3-sialytransferases, and a tyrosulfotransferase. In addition, dimerization of PSGL-1 through its sole extracellular cysteine (Cys(320)) is essential for rolling on P-selectin under shear conditions. In this report, we measured the contributions of both C2GlcNAcT-I glycosylation and dimerization of PSGL-1 to adhesive bonds formed during tethering and rolling of transfected cell lines on purified P-selectin. Tethering to P-selectin under flow increased with dimerization compared with cells expressing monomeric PSGL-1 (referred to as C320A). The rolling defects (decreased cellular accumulation, PSGL-1/P-selectin bond strengths and tethering rates, and increased velocities and skip distance) demonstrated by transfectants expressing monomeric PSGL-1 could be overcome by increasing the substrate P-selectin site density and by overexpressing C2GlcNAcT-I in C320A transfectants. Two molecular weight variants of PSGL-1 were isolated from cell lines transfected with PSGL-1, C320A, and/or C2GlcNAcT-I cDNAs, and these differences in electrophoretic mobility appeared to correlate with C2GlcNAcT-I expression. C320A transfectants expressing low molecular weight PSGL-1 had lower C2GlcNAcT-I levels (measured by reactivity to core 2 specific linkage antibody, CHO-131) and compromised rolling on P-selectin (regardless of site density) compared with C320A cells with high levels of C2GlcNAcT-I and high molecular weight PSGL-1. Both C2GlcNAcT-I glycosylation and PSGL-1 dimerization increased the rate of tethering to P-selectin under flow, whereas C2GlcNAcT-I levels primarily influenced tether bond strength.     

Platelets enhance neutrophil transendothelial migration via P-selectin glycoprotein ligand-1

Platelets are increasingly recognized as important for inflammation in addition to thrombosis. Platelets promote the adhesion of neutrophils [polymorphonuclear neutrophils (PMNs)] to the endothelium; P-selectin and P-selectin glycoprotein ligand (PSGL)-1 have been suggested to participate in these interactions. Whether platelets also promote PMN transmigration across the endothelium is less clear. We tested the hypothesis that platelets enhance PMN transmigration across the inflamed endothelium and that PSGL-1 is involved. We studied the effects of platelets on PMN transmigration in vivo and in vitro using a well-characterized corneal injury model in C57BL/6 mice and IL-1β-stimulated human umbilical vein endothelial cells (HUVECs) under static and dynamic conditions. In vivo, platelet depletion altered PMN emigration from limbal microvessels after injury, with decreased emigration 6 and 12 h after injury. Both PSGL-1-/- and P-selectin-/- mice, but not Mac-1-/- mice, also had reduced PMN emigration at 12 h after injury relative to wild-type control mice. In the in vitro HUVEC model, platelets enhanced PMN transendothelial migration under static and dynamic conditions independent of firm adhesion. Anti-PSGL-1 antibodies markedly inhibited platelet-PMN aggregates, as assessed by flow cytometry, and attenuated the effect of platelets on PMN transmigration under static conditions without affecting firm adhesion. These data support the notion that platelets enhance neutrophil transmigration across the inflamed endothelium both in vivo and in vitro, via a PSGL-1-dependent mechanism.     

Deformation and flow of membrane into tethers extracted from neuronal growth cones.

Membrane tethers are extracted at constant velocity from neuronal growth cones using a force generated by a laser tweezers trap. A thermodynamic analysis shows that as the tether is extended, energy is stored in the tether as bending and adhesion energies and in the cell body as "nonlocal" bending. It is postulated that energy is dissipated by three viscous mechanisms including membrane flow, slip between the two monolayers that form the bilayer, and slip between membrane and cytoskeleton. The analysis predicts and the experiments show a linear relation between tether force and tether velocity. Calculations based on the analytical results and the experimental measurements of a tether radius of approximately 0.2 micron and a tether force at zero velocity of approximately 8 pN give a bending modulus for the tether of 2.7 x 10(-19) N.m and an extraordinarily small "apparent surface tension" in the growth cone of 0.003 mN/m, where the apparent surface tension is the sum of the far-field, in-plane tension and the energy of adhesion. Treatments with cytochalasin B and D, ethanol, and nocodazole affect the apparent surface tension but not bending. ATP depletion affects neither, whereas large concentrations of DMSO affect both. Under conditions of flow, data are presented to show that the dominant viscous mechanism comes from the slip that occurs when the membrane flows over the cytoskeleton. ATP depletion and the treatment with DMSO cause a dramatic drop in the effective viscosity. If it is postulated that the slip between membrane and cytoskeleton occurs in a film of water, then this water film has a mean thickness of only approximately 10 A.