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International audience; The binding properties of biomolecules play a crucial role in many biological phenomena, specially cell adhesion. While the attachment kinetics of soluble proteins is considered as well known, complex behavior arises when protein molecules are bound to the cell membrane. We probe the hidden kinetics of ligand-receptor bond formation using single molecule flow chamber assays and brownian dynamics simulations. We show that, consistent with our recently proposed hypothesis, association requires a minimum duration of contact between the reactive species. In our experiments, ICAM-1 anchored on a flat substrate bind to anti-ICAM-1 coated on flowing microbeads. The interaction potential between bead and substrate is measured by micro-interferometry and is used as an ingredient to simulate bead movement. Our simulation calculates the duration of ligand-receptor contacts imposed by the bead movement. We quantitatively predict the reduction of adhesion probability measured for shorter tether length of the ligand or if a repulsive hyaluronan layer is added on the surface. To account for our results, we propose that bond formation may occur in our system by crossing of a diffusive plateau in the energy landscape, on the timescale of 5 ms and an energy barrier of 5 kBT, before reaching the first detectable bound state. Our results show how to relate cell scale behavior to the combined information of molecular reactivity and biomolecules submicron scale environment.

The aim of the Conference Series on Cell Biophysics is to gather scientist whose vocation is to understand the workings of living cells in a quantitative manner. As such, this conference series provides a unique opportunity for the cross-fertilisation of ideas emerging in the physics, chemistry biology and medical laboratories in Europe and around the world. It also informs scientists about the state of the art research in this rapidly expanding field allowing the transfer of information necessary for tackling processes governing cellular structure and function.

Structural properties of Na-DNA dilute and semidilute aqueous solutions are quantified using dielectric spectroscopy in the frequency range 100 Hz – 100 MHz[1, 2, 3]. Two relaxation modes are revealed that can be attributed to diffusive motion of DNA counterions. The overall study in the wide concentration, added salt and chain length range demonstrates that the motion detected at lower frequencies probes single-chain structural properties, whereas the one at higher frequencies characterizes the collectives properties of the DNA solution composed of many chains. The characteristic length scales found in the measurements compare well with the fundamental length scales predicted by the theory, as well as respective roles in ionic screening of intrinsic DNA counterions and added salt ions [4]. Surprisingly, results obtained on 146 bp double-stranded DNA indicate that these short DNA fragments exhibit much higher flexibility than expected from the conventional worm-like chain model. Our results are in accord with those obtained in similar studies using small-angle X-ray scattering and fluorescence resonance energy transfer [5]. At the end, open issues and prospects for further research are designated. References [1] S.Tomic, T.Vuletic, S.Dolanski Babic, S.Krca, D.Ivankovic, L.Griparic and R.Podgornik, Phys.Rev.Lett. 97, 098303 (2006). [2] S.Tomic, S.Dolanski Babic, T.Vuletic, S.Krca, D.Ivankovic, L.Griparic and R.Podgornik, Phys.Rev.E 75, 021905 (2007). [3] S.Tomic, S.Dolanski Babic, T.Ivek, T. Vuletic, S. Krca, F.Livolant and R. Podgornik, Europhys.Letters 81, 68003 (2008). [4] A.V.Dobrynin and M.Rubinstein, Prog.Polym.Sci. 30, 1049 (2005) ; A.V.Dobrynin, R.H.Colby and M.Rubinstein, Macromolecules 28, 1859 (1995). [5] C.Yuan, H.Chen, X.W.Lou and L.A.Archer, Phys.Rev.Lett.100, 018102 (2008).

International audience; 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 to 200 nm. 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.

Surface microtopography and nanotopography have been shown to influence cell adhesion and function, including proliferation and differentiation, leading both to fundamental questions and practical applications in the field of biomaterials and nanomedicine. However, the mechanisms of how cells sense topography remain obscure. In this study, we measured directly the effect of nanotopography on the kinetics of association and dissociation of ligand–receptor bonds, which are critically involved in the first steps of cell adhesion. We designed models of biological functionalised surfaces with controlled roughness varying from 2 to 400 nm of root mean square, and controlled ligand density. Tests of transient adhesion of receptor–coated microspheres on these surfaces were performed, using a laminar flow chamber assay. We probed Intercellular Adhesion Molecule ICAM–1–anti–ICAM–1 bond adhesion kinetics in the single molecule limit on smooth and rough substrates. Frequency of adhesion did not exhibit any noticeable dependence on roughness parameter, except at high bead velocity. Detachment rate was also independent of roughness. Finally, leucocyte transient adhesion tests were performed on similar substrates, using variable activating incubating media. Here also, no strong effect of roughness was observed in these conditions. Results are rationalised in terms of the role of local geometry on the access of ligands to receptors.

The adhesion of giant unilamellar phospholipid vesicles to planar substrates coated with extracellular matrix mimetic cushions of hyaluronan is studied using quantitative reflection interference contrast microscopy. The absolute height of the vesicle membrane at the vicinity of the substrate is measured by considering, for the first time, the refractive indices of the reflecting media. The thickness of the cushion is varied in the range of approximately 50-100 nm, by designing various coupling strategies. On bare protein-coated substrates, the vesicles spread fast (0.5 s) and form a uniform adhesion disk, with the average membrane height approximately 4 nm. On thick hyaluronan cushions (80 nm), the membrane height is approximately the same as the thickness of the cushion, implying that the vesicle lies on top of the cushion. On a thin and inhomogeneous hyaluronan cushion, the adhesion is modified but not prevented. The spreading is slow ( approximately 20 s) compared to the no-cushion case. The average membrane height is approximately 10 nm and the adhesion disk is studded with blisterlike structures. Observations with fluorescent hyaluronan indicate that the polymer is compressed under, rather than expelled from, the adhesion disk. The adhesion energy density is approximately threefold higher in the no-cushion case (1.2 microJ/m(2)) as compared to the thin-cushion case (0.54 microJ/m(2)). In the thin-cushion case, the presence of short ( approximately 4 nm) glyco-polymers on the vesicles results in a hitherto unreported stable partial adhesion state--the membrane height ranges from zero to approximately 250 nm. The minimal model system presented here mimics in vitro the hyaluronan-modulated early stages of cell adhesion, and demonstrates that the presence of a polymer cushion influences both the final equilibrium adhesion-state and the spreading kinetics.