Anne Pierres

Granulocyte-endothelium initial adhesion. Analysis of transient binding events mediated by E-selectin in a laminar shear flow.

The adhesion of moving cells to receptor-bearing surfaces is a key step to many important biological processes. Attachment was subjected to extensive modeling. However, the numerical values of kinetic bonding parameters relevant to realistic models of cell adhesion remain poorly known. In this report, we describe the motion of human granulocytes to interleukin-1-activated endothelial cells in presence of a low hydrodynamic drag (a few piconewtons) estimated to be much weaker than a standard ligand-receptor bond. It was thus expected to visualize the formation and rupture of individual bonds. We observed multiple short-time cell arrests with a median duration of 2.43 s. Stop frequency, not duration, was significantly inhibited by anti-E-selectin antibodies. Binding efficiency exhibited an almost linear relationship with the inverse of cell velocity. The distribution of arrest duration was determined: results were consistent with the view that these arrests reflected the formation/dissociation of single ligand-receptor bonds with a spontaneous dissociation rate of 0.5 s-1. The rate of bond formation was on the order of 0.04 s-1 when cells were freely rolling (mean velocity: 19 microns/s) and it exhibited an approximately 10-fold increase after the formation of a first adhesion.

How Cells Tiptoe on Adhesive Surfaces before Sticking

Cell membranes are studded with protrusions that were thoroughly analyzed with electron microscopy. However, the nanometer-scale three-dimensional motions generated by cell membranes to fit the topography of foreign surfaces and initiate adhesion remain poorly understood. Here, we describe the dynamics of surface deformations displayed by monocytic cells bumping against fibronectin-coated surfaces. We observed membrane undulations with typically 5 nm amplitude and 5-10 s lifetime. Cell membranes behaved as independent units of micrometer size. Cells detected the presence of foreign surfaces at 50 nm separation, resulting in time-dependent amplification of membrane undulations. Molecular contact then ensued with apparent cell-membrane separation of 30-40 nm, and this distance steadily decreased during the following tens of seconds. Contact maturation was associated with in-plane egress of bulky molecules and robust membrane fluctuations. Thus, membrane undulations may be the major determinant of cell sensitivity to substrate topography, outcome of interaction, and initial kinetics of contact extension.

Fast dissociation kinetics between individual E-cadherin fragments revealed by flow chamber analysis

E‐cadherin is the predominant adhesion molecule of epithelia. The interaction between extracellular segments of E‐cadherin in the membrane of opposing cells is homophilic and calcium dependent. Whereas it is widely accepted that the specificity of the adhesive interaction is localized to the N‐terminal domain, the kinetics of the recognition process are unknown. We report the first quantitative data describing the dissociation kinetics of individual E‐cadherin interactions. Aggregation assays indicate that the two outermost domains of E‐cadherin (E/EC1–2) retain biological activity when chemically immobilized on glass beads. Cadherin fragment trans‐interaction was analysed using a flow chamber technique. Transient tethers had first‐order kinetics, suggesting a unimolecular interaction. The unstressed lifetime of individual E‐cadherin interactions was as brief as 2 s. A fast off rate and the low tensile strength of the E‐cadherin bond may be necessary to support the high selectivity and plasticity of epithelial cell interactions.

Dissecting streptavidin-biotin interaction with a laminar flow chamber.

A laminar flow chamber was used to study single molecule interactions between biotinylated surfaces and streptavidin-coated spheres subjected to a hydrodynamic drag lower than a piconewton. Spheres were tracked with 20 ms and 40 nm resolution. They displayed multiple arrests lasting between a few tens of milliseconds and several minutes or more. Analysis of about 500,000 positions revealed that streptavidin-biotin interaction was multiphasic: transient bound states displayed a rupture frequency of 5.3 s(-1) and a rate of transition toward a more stable configuration of 1.3 s(-1). These parameters did not display any significant change when the force exerted on bonds varied between 3.5 and 11 pN. However, the apparent rate of streptavidin-biotin association exhibited about 10-fold decrease when the wall shear rate was increased from 7 to 22 s(-1), which supports the existence of an energy barrier opposing the formation of the transient binding state. It is concluded that a laminar flow chamber can yield new and useful information on the formation of molecular bonds, and especially on the structure of the external part of the energy landscape of ligand-receptor complexes.

Diffusion of microspheres in shear flow near a wall: use to measure binding rates between attached molecules.

The rate and distance-dependence of association between surface-attached molecules may be determined by monitoring the motion of receptor-bearing spheres along ligand-coated surfaces in a flow chamber (Pierres et al., Proc. Natl. Acad. Sci. U.S.A. 95:9256-9261, 1998). Particle arrests reveal bond formation, and the particle-to-surface distance may be estimated from the ratio between the velocity and the wall shear rate. However, several problems are raised. First, data interpretation requires extensive computer simulations. Second, the relevance of standard results from fluid mechanics to micrometer-size particles separated from surfaces by nanometer distances is not fully demonstrated. Third, the wall shear rate must be known with high accuracy. Here we present a simple derivation of an algorithm permitting one to simulate the motion of spheres near a plane in shear flow. We check that theoretical predictions are consistent with the experimental dependence of motion on medium viscosity or particle size, and the requirement for equilibrium particle height distribution to follow Boltzmans law. The determination of the statistical relationship between particle velocity and acceleration allows one to derive the wall shear rate with 1-s(-1) accuracy and the Hamaker constant of interaction between the particle and the wall with a sensitivity better than 10(-21) J. It is demonstrated that the correlation between particle height and mean velocity during a time interval Deltat is maximal when Deltat is about 0.1-0.2 s for a particle of 1.4-microm radius. When the particle-to-surface distance ranges between 10 and 40 nm, the particle height distribution may be obtained with a standard deviation ranging between 8 and 25 nm, provided the average velocity during a 160-ms period of time is determined with 10% accuracy. It is concluded that the flow chamber allows one to detect the formation of individual bonds with a minimal lifetime of 40 ms in presence of a disruptive force of approximately 5 pN and to assess the distance dependence within the tens of nanometer range.

Measuring the Lifetime of Bonds Made between Surface-linked Molecules

It is not well known how the kinetic constants of association between soluble receptors and ligands may be used to predict the behavior of these molecules when they are bound to cell surfaces. Spherical beads were coated with varying densities of anti-rabbit immunoglobulin monoclonal antibodies and driven along glass surfaces derivatized with rabbit anti-dinitrophenol. Particle motion was analyzed. The velocity, attachment frequency, and duration of binding events were determined on individual particles. It was found that i) beads exhibited frequent arrests lasting between a few tenths of a second and more than one minute; ii) when antibodies were diluted, the median arrest duration remained fairly constant (≈1 s) whereas binding frequency varied as the first power of the antibody concentration, suggesting that most particle arrests were due to the formation of a single bond; iii) when the shear rate was increased 7-fold, the duration of transient binding events remained constant. The disruptive force exerted on attachment points was estimated to range between about 6 and 37 piconewtons; and iv) the distribution of arrest durations suggested that binding was not a monophasic reaction but involved at least one intermediate step. Therefore, transient binding events reflected the formation of unstable associations that are not detected with standard techniques.

Cell Membrane Alignment along Adhesive Surfaces: Contribution of Active and Passive Cell Processes

Cell adhesion requires nanometer scale membrane alignment to allow contact between adhesion receptors. Little quantitative information is presently available on this important biological process. Here we present an interference reflection microscopic study of the initial interaction between monocytic THP-1 cells and adhesive surfaces, with concomitant determination of cell deformability, using micropipette aspiration, and adhesiveness, using a laminar flow assay. We report that 1), during the first few minutes after contact, cells form irregular-shaped interaction zones reaching approximately 100 micro m(2) with a margin extension velocity of 0.01-0.02 micro m/s. This happens before the overall cell deformations usually defined as spreading. 2), These interference reflection microscopic-detected zones represent bona fide adhesion inasmuch as cells are not released by hydrodynamic forces. 3), Alignment is markedly decreased but not abolished by microfilament blockade with cytochalasin or even cell fixation with paraformaldehyde. 4), In contrast, exposing cells to hypotonic medium increased the rate of contact extension. 5), Contacts formed in presence of cytochalasin, after paraformaldehyde fixation or in hypotonic medium, were much more regular-shaped than controls and their extension matched cell deformability. 6), None of the aforementioned treatments altered adhesiveness to the surface. It is concluded that adhesive forces and passive membrane deformations are sufficient to generate initial cell alignment to adhesive surfaces, and this process is accelerated by spontaneous cytoskeletally-driven membrane motion.

Measuring bonds between surface-associated molecules

Adhesive interactions play an essential role in immune function. Much information on these phenomena was recently obtained by applying sophisticated methods such as the surface forces apparatus, atomic force microscopy, lipid vesicle-based technology or flow chambers. In the present review it is shown that the use of hydrodynamic flow allows quantitative study of the formation and dissociation of individual molecular bonds between receptor-bearing cells or particles and ligand-derivatized surfaces. In addition, it should be possible to determine particle-surface interaction forces with subpiconewton sensitivity and nanometer resolution. Data analysis shows that the classical concepts of bond strength, or association and dissociation rates must be reexamined in order to achieve a correct understanding of the behavior of individual molecules.

Motion of cells sedimenting on a solid surface in a laminar shear flow

Cell adhesion often occurs under dynamic conditions, as in flowing blood. A quantitative understanding of this process requires accurate knowledge of the topographical relationships between the cell membrane and potentially adhesive surfaces. This report describes an experimental study made on both the translational and rotational velocities of leukocytes sedimenting of a flat surface under laminar shear flow. The main conclusions are as follows: (a) Cells move close to the wall with constant velocity for several tens of seconds. (b) The numerical values of translational and rotational velocities are inconsistent with Goldmans model of a neutrally buoyant sphere in a laminar shear flow, unless a drag force corresponding to contact friction between cells and the chamber floor is added. The phenomenological friction coefficient was 7.4 millinewton.s/m. (c) Using a modified Goldmans theory, the width of the gap separating cells (6 microns radius) from the chamber floor was estimated at 1.4 micron. (d) It is shown that a high value of the cell-to-substrate gap may be accounted for by the presence of cell surface protrusions of a few micrometer length, in accordance with electron microscope observations performed on the same cell population. (e) In association with previously reported data (Tissot, O., C. Foa, C. Capo, H. Brailly, M. Delaage, and P. Bongrand. 1991. Biocolloids and Biosurfaces. In press), these results are consistent with the possibility that cell-substrate attachment be initiated by the formation of a single molecular bond, which might be considered as the rate limiting step.

A Novel Leukocyte Adhesion Deficiency III Variant: Kindlin-3 Deficiency Results in Integrin- and Nonintegrin-Related Defects in Different Steps of Leukocyte Adhesion

Leukocyte adhesion deficiency type III is a recently described condition involving a Glanzmann-type bleeding syndrome and leukocyte adhesion deficiency. This was ascribed to a defect of the FERMT3 gene resulting in abnormal expression of kindlin-3, a protein expressed in hematopoietic cells with a major role in the regulation of integrin activation. In this article, we describe a patient with a new mutation of FERMT3 and lack of kindlin-3 expression in platelets and leukocytes. We assayed quantitatively the first steps of kindlin-3–defective leukocyte adhesion, namely, initial bond formation, bond strengthening, and early spreading. Initial bond formation was readily stimulated with neutrophils stimulated by fMLF, and neutrophils and lymphocytes stimulated by a phorbol ester or Mn2+. In contrast, attachment strengthening was defective in the patient’s lymphocytes treated with PMA or Mn2+, or fMLF-stimulated neutrophils. However, attachment strengthening was normal in patient’s neutrophils treated with phorbol ester or Mn2+. In addition, the patient’s T lymphocytes displayed defective integrin-mediated spreading and a moderate but significant decrease of spreading on anti-CD3–coated surfaces. Patient’s neutrophils displayed a drastic alteration of integrin-mediated spreading after fMLF or PMA stimulation, whereas signaling-independent Mn2+ allowed significant spreading. In conclusion, the consequences of kindlin-3 deficiency on β2 integrin function depend on both cell type and the stimulus used for integrin activation. Our results suggest looking for a possible kindlin-3 involvement in membrane dynamical event independent of integrin-mediated adhesion.