We used a combination of laminar flow chamber and reflection interference microscopy to study the formation and rupture of single bonds formed between Fc-ICAM-1 attached to a substrate and ...anti-ICAM-1 carried by micrometric beads in the presence of a repulsive hyaluronan (HA) layer adsorbed onto the substrate. The absolute distance between the colloids and the surface was measured under flow with an accuracy of a few nanometers. We could verify the long-term prediction of classical lubrication theory for the movement of a sphere near a wall in a shear flow. The HA polymer layer exerted long-range repulsive steric force on the beads and the hydrodynamics at the boundary remained more or less unchanged. By incubating HA at various concentrations, the thickness of the layer, as estimated by beads most probable height, was tuned in the range 20–200nm. Frequency of bond formation was decreased by more than one order of magnitude by increasing the thickness of the repulsive layer, while the lifetime of individual bonds was not affected. This study opens the way for further quantitative studies of the effect of molecular environment and separation distance on ligand-receptor association and dissociation.
The efficiency of many cell-surface receptors is dependent on the rate of binding soluble or surface-attached ligands. Much effort was exerted to measure association rates between soluble molecules ...(three-dimensional k(on)) and, more recently, between surface-attached molecules (two-dimensional 2D k(on)). According to a generally accepted assumption, the probability of bond formation between receptors and ligands is proportional to the first power of encounter duration. Here we provide new experimental evidence and review published data demonstrating that this simple assumption is not always warranted. Using as a model system the (2D) interaction between ICAM-1-coated surfaces and flowing microspheres coated with specific anti-ICAM-1 antibodies, we show that the probability of bond formation may scale as a power of encounter duration that is significantly higher than 1. Further, we show that experimental data may be accounted for by modeling ligand-receptor interaction as a displacement along a single path of a rough energy landscape. Under a wide range of conditions, the probability that an encounter of duration t resulted in bond formation varied as erfc(t(0)/t)(1/2), where t(0) was on the order of 10 ms. We conclude that the minimum contact time for bond formation may be a useful parameter to describe a ligand-receptor interaction, in addition to conventional association rates.
The rupture forces and adhesion frequencies of single recognition complexes between an affinityselected peptide/MHC complex and a TCR at a murine hybridoma surface were measured usingAtomic Force ...Microscopy. When the CD8 coreceptor is absent, the adhesion frequency depends onthe nature of the peptide but the rupture force does not. When CD8 is present, no effect of the natureof the peptide is observed. CD8 is proposed to act as a time and distance lock, enabling the shorterTCR molecule to bridge the pMHC and have time to finely read the peptide. Ultimately, suchexperiments could help the dissection of the sequential steps by which the TCR reads thepeptide/MHC complex in order to control T cell activation.
Transparent nanopatterned plastic substrates are prepared by molding a photopolymer inside nanoporous ordered anodic alumina and subsequently etching away the mold. The array of glass‐supported, ...index‐matched nanopillars is ordered over a large area. They can serve as a platform for studying the interaction of living cells with nanotopography (see image) using quantitative optical microscopy.
We report the collective and single-filament dynamics of long semiflexible actin filaments flowing in an evaporating droplet adhering on glass and accumulating along the physical barrier constituted ...by the droplet triple line. The observation of fluorescent reporter filaments embedded in the entangled network enables us to relate the final collective organization of the accumulated filaments to the individual filament dynamics. Three areas corresponding to distinct filament organizations are observed in the region of the initial triple line pinning, after complete evaporation of the droplet. A nematic liquid-crystal-like alignment of the filaments is observed at the edge of the droplet because of the dynamic filament alignment, whereas a less-ordered packing is generated because of the bending and folding of most of the filaments. The latter unconventional dynamics is analyzed in terms of the amplification of undulation modes typical of semiflexible polymers. The receding regime of the droplet triple line leads finally to a remaining film of actin filaments showing random organization.
Bioanalogue models of composite cell envelopes were designed by electrostatically driven self-assembly of actin shells inside giant vesicles. Viscoelastic relaxation moduli were measured between 0.03 ...and 20 s as a function of actin density by magnetic bead microrheometry. The shear relaxation spectra exhibited by the composite shells compare well with those of natural cell envelopes and bulk entangled actin networks. Absolute value of the shear modulus was measured for the first time by deformation field mapping. Shear and bending moduli agree well with values obtained by bead fluctuations analysis.
Functional microdomains of glycolipids were designed by mixing neoglycolipids with partially fluorinated alkyl (F-alkyl) chains and matrix lipids with alkyl chains. Fluorescence images of the mixed ...lipid monolayers at the air-water interface demonstrated that it is possible to control both size and distribution of the microdomains by means of the strong demixing of alkyl and F-alkyl membrane anchors, while the carbohydrate head groups seemed to play a rather minor role. These microdomains in monolayers could be transferred onto hydrophobized substrates and subjected to experiments in a dynamic flow chamber. The results obtained here clearly indicated that the dynamic adhesion of Chinese hamster ovarial cells expressing E-selectin (CHO-E cells) on a lipid monolayer containing microdomains of sialyl Lewisx (sLex) can be both enhanced and reduced by controlled demixing of ligands and matrices. Moreover, the same clusters of sLex could also be formed in giant lipid vesicles, which can be used as a model cell that locally expresses biospecific functions.
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