Biomedical subjects

Arkadiusz G Klopocki

Publications and source records attributed to Arkadiusz G Klopocki.

5 recordsLinked to original sources

PubMed2006

Transport governs flow-enhanced cell tethering through L-selectin at threshold shear.

Flow-enhanced cell adhesion is a counterintuitive phenomenon that has been observed in several biological systems. Flow augments L-selectin-dependent adhesion by increasing the initial tethering of leukocytes to vascular surfaces and by strengthening their subsequent rolling interactions. Tethering or rolling might be influenced by physical factors that affect the formation or dissociation of selectin-ligand bonds. We recently demonstrated that flow enhanced rolling of L-selectin-bearing microspheres or neutrophils on P-selectin glycoprotein ligand-1 by force decreased bond dissociation. Here, we show that flow augmented tethering of these microspheres or cells to P-selectin glycoprotein ligand-1 by three transport mechanisms that increased bond formation: sliding of the sphere bottom on the surface, Brownian motion, and molecular diffusion. These results elucidate the mechanisms for flow-enhanced tethering through L-selectin.

Biological Transport↗

PubMed2006

Flow-enhanced adhesion regulated by a selectin interdomain hinge.

L-selectin requires a threshold shear to enable leukocytes to tether to and roll on vascular surfaces. Transport mechanisms govern flow-enhanced tethering, whereas force governs flow-enhanced rolling by prolonging the lifetimes of L-selectin-ligand complexes (catch bonds). Using selectin crystal structures, molecular dynamics simulations, site-directed mutagenesis, single-molecule force and kinetics experiments, Monte Carlo modeling, and flow chamber adhesion studies, we show that eliminating a hydrogen bond to increase the flexibility of an interdomain hinge in L-selectin reduced the shear threshold for adhesion via two mechanisms. One affects the on-rate by increasing tethering through greater rotational diffusion. The other affects the off-rate by strengthening rolling through augmented catch bonds with longer lifetimes at smaller forces. By forcing open the hinge angle, ligand may slide across its interface with L-selectin to promote rebinding, thereby providing a mechanism for catch bonds. Thus, allosteric changes remote from the ligand-binding interface regulate both bond formation and dissociation.

Cell Adhesion↗

PubMed2004

Catch bonds govern adhesion through L-selectin at threshold shear.

Flow-enhanced cell adhesion is an unexplained phenomenon that might result from a transport-dependent increase in on-rates or a force-dependent decrease in off-rates of adhesive bonds. L-selectin requires a threshold shear to support leukocyte rolling on P-selectin glycoprotein ligand-1 (PSGL-1) and other vascular ligands. Low forces decrease L-selectin-PSGL-1 off-rates (catch bonds), whereas higher forces increase off-rates (slip bonds). We determined that a force-dependent decrease in off-rates dictated flow-enhanced rolling of L-selectin-bearing microspheres or neutrophils on PSGL-1. Catch bonds enabled increasing force to convert short-lived tethers into longer-lived tethers, which decreased rolling velocities and increased the regularity of rolling steps as shear rose from the threshold to an optimal value. As shear increased above the optimum, transitions to slip bonds shortened tether lifetimes, which increased rolling velocities and decreased rolling regularity. Thus, force-dependent alterations of bond lifetimes govern L-selectin-dependent cell adhesion below and above the shear optimum. These findings establish the first biological function for catch bonds as a mechanism for flow-enhanced cell adhesion.

Antibodies, Monoclonal↗

PubMed2003

Low force decelerates L-selectin dissociation from P-selectin glycoprotein ligand-1 and endoglycan.

Selectin-ligand interactions mediate the tethering and rolling of circulating leukocytes on vascular surfaces during inflammation and immune surveillance. To support rolling, these interactions are thought to have rapid off-rates that increase slowly as wall shear stress increases. However, the increase of off-rate with force, an intuitive characteristic named slip bonds, is at odds with a shear threshold requirement for selectin-mediated cell rolling. As shear drops below the threshold, fewer cells roll and those that do roll less stably and with higher velocity. We recently demonstrated a low force regime where the off-rate of P-selectin interacting with P-selectin glycoprotein ligand-1 (PSGL-1) decreased with increasing force. This counter-intuitive characteristic, named catch bonds, might partially explain the shear threshold phenomenon. Because L-selectin-mediated cell rolling exhibits a much more pronounced shear threshold, we used atomic force microscopy and flow chamber experiments to determine off-rates of L-selectin interacting with their physiological ligands and with an antibody. Catch bonds were observed at low forces for L-selectin-PSGL-1 interactions coinciding with the shear threshold range, whereas slip bonds were observed at higher forces. These catch-slip transitional bonds were also observed for L-selectin interacting with endoglycan, a newly identified PSGL-1-like ligand. By contrast, only slip bonds were observed for L-selectin-antibody interactions. These findings suggest that catch bonds contribute to the shear threshold for rolling and are a common characteristic of selectin-ligand interactions.

Animals↗

PubMed2002

Adhesive properties of carcinoembryonic antigen glycoforms expressed in glycosylation-deficient Chinese hamster ovary cell lines.

Carcinoembryonic antigen (CEA) is an oncofoetal cell surface glycoprotein that serves as an important tumour marker for colorectal and some other carcinomas. Its immunoglobulin-like structure places CEA within the immunoglobulin superfamily. CEA functions in several biological roles including homotypic and heterotypic (with other CEA family members) cell adhesion. Cell-cell interaction can be modulated by different factors, e.g., post-translational modifications such as glycosylation. The purpose of this study was to examine whether changes in carbohydrate composition of CEA oligosaccharides can influence homotypic (CEA-CEA) interactions. In order to modulate glycosylation of CEA we used two different glycosylation mutants of Chinese hamster ovary (CHO) cells, Lec2 and Lec8. Lec2 cells should produce CEA with nonsialylated N-glycans, while Lec8 cells should yield more truncated sugar structures than Lec2. Parental CHO (Pro5) cells and the glycosylation deficient mutants were stably transfected with CEA cDNA. All three CEA glycoforms, tested in a solid-phase cell adhesion assay, showed an ability to mediate CEA-dependent cell adhesion, and no qualitative differences in the adhesion between the glycoforms were observed. Thus, it may be assumed that carbohydrates do not play a role in homotypic adhesion, and the interactions between CEA molecules depend solely on the polypeptide structure.

Animals↗