Adhesion can be quantified by measuring the distance between the interacting surfaces. Reflection interference contrast microscopy (RICM), with its ability to measure inter-surface distances under ...water with nanometric precision and milliseconds time resolution, is ideally suited to studying the dynamics of adhesion in soft systems. Recent technical developments, which include innovative image analysis and the use of multi-coloured illumination, have led to renewed interest in this technique. Unambiguous quantitative measurements have been achieved for colloidal beads and model membranes, thus revealing new insights and applications. Quantification of data from cells shows exciting prospects. Herein, we review the basic principles and recent developments of RICM applied to studies of dynamical adhesion processes in soft matter and cell biology and provide practical hints to potential users.
We designed a strategy, based on a careful examination of the activation capabilities of proteins and antibodies used as substrates for adhering T cells, coupled to protein microstamping to control ...at the same time the position, shape, spreading, mechanics and activation state of T cells. Once adhered on patterns, we examined the capacities of T cells to be activated with soluble anti CD3, in comparison to T cells adhered to a continuously decorated substrate with the same density of ligands. We show that, in our hand, adhering onto an anti CD45 antibody decorated surface was not affecting T cell calcium fluxes, even adhered on variable size micro-patterns. Aside, we analyzed the T cell mechanics, when spread on pattern or not, using Atomic Force Microscopy indentation. By expressing MEGF10 as a non immune adhesion receptor in T cells we measured the very same spreading area on PLL substrates and Young modulus than non modified cells, immobilized on anti CD45 antibodies, while retaining similar activation capabilities using soluble anti CD3 antibodies or through model APC contacts. We propose that our system is a way to test activation or anergy of T cells with defined adhesion and mechanical characteristics, and may allow to dissect fine details of these mechanisms since it allows to observe homogenized populations in standardized T cell activation assays.
Abstract
The role of force application in immune cell recognition is now well established, the force being transmitted between the actin cytoskeleton to the anchoring ligands through receptors such ...as integrins. In this chain, the mechanics of the cytoskeleton to receptor link, though clearly crucial, remains poorly understood. To probe this link, we combine mechanical extraction of membrane tubes from T cells using optical tweezers, and fitting of the resulting force curves with a viscoelastic model taking into account the cell and relevant molecules. We solicit this link using four different antibodies against various membrane bound receptors: antiCD3 to target the T Cell Receptor (TCR) complex, antiCD45 for the long sugar CD45, and two clones of antiCD11 targeting open or closed conformation of LFA1 integrins. Upon disruption of the cytoskeleton, the stiffness of the link changes for two of the receptors, exposing the existence of a receptor to cytoskeleton link—namely TCR-complex and open LFA1, and does not change for the other two where a weaker link was expected. Our integrated approach allows us to probe, for the first time, the mechanics of the intracellular receptor–cytoskeleton link in immune cells.
We examine experimental and theoretical aspects of nonspecific adhesion of giant vesicles on modified surfaces as model systems for cell spreading. Using dual-wave interference microscopy and new ...analysis, membrane undulations as well as large scale vesicle shape are monitored. Measurements and modelling show that the nucleation of adhesion depends critically on the interfacial polymer and membrane tension. Patch growth is governed by local membrane geometry, adhesion energy, and local viscosity. Finally, spreading stops when tension induced by adhesion unfolds excess membrane area.
Cell adhesion is mediated by numerous membrane receptors. It is desirable to derive the outcome of a cell-surface encounter from the molecular properties of interacting receptors and ligands. ...However, conventional parameters such as affinity or kinetic constants are often insufficient to account for receptor efficiency. Avidity is a qualitative concept frequently used to describe biomolecule interactions: this includes incompletely defined properties such as the capacity to form multivalent attachments. The aim of this study is to produce a working description of monovalent attachments formed by a model system, then to measure and interpret the behavior of divalent attachments under force. We investigated attachments between antibody-coated microspheres and surfaces coated with sparse monomeric or dimeric ligands. When bonds were subjected to a pulling force, they exhibited both a force-dependent dissociation consistent with Bell's empirical formula and a force- and time-dependent strengthening well described by a single parameter. Divalent attachments were stronger and less dependent on forces than monovalent ones. The proportion of divalent attachments resisting a force of 30 piconewtons for at least 5 s was 3.7 fold higher than that of monovalent attachments. Quantitative modeling showed that this required rebinding, i.e. additional bond formation between surfaces linked by divalent receptors forming only one bond. Further, experimental data were compatible with but did not require stress sharing between bonds within divalent attachments. Thus many ligand-receptor interactions do not behave as single-step reactions in the millisecond to second timescale. Rather, they exhibit progressive stabilization. This explains the high efficiency of multimerized or clustered receptors even when bonds are only subjected to moderate forces. Our approach provides a quantitative way of relating binding avidity to measurable parameters including bond maturation, rebinding and force sharing, provided these parameters have been determined. Also, this provides a quantitative description of the phenomenon of bond strengthening.
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 ∼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 ∼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 (∼20
s) compared to the no-cushion case. The average membrane height is ∼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
μJ/m
2) as compared to the thin-cushion case (0.54
μJ/m
2). In the thin-cushion case, the presence of short (∼4
nm) glyco-polymers on the vesicles results in a hitherto unreported stable partial adhesion state—the membrane height ranges from zero to ∼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.
We created APC-mimetic synthetic substrates to study the impact of ligand clustering on T cell activation and spreading. The substrates exhibit antibodies directed against the TCR-complex in the form ...of a patterned array of sub micrometric dots surrounded by a fluid supported lipid bilayer (SLB) which may itself be functionalized with another bio-molecule. We show that for T cell adhesion mediated by T cell receptor (TCR) alone, in the patterned, but not in the corresponding homogeneous controls, the TCR, ZAP-70 and actin are present in the form of clusters or patches that co-localize with the ligand-dots. However, global cell scale parameters like cell area and actin distribution are only weakly impacted by ligand clustering. In presence of ICAM-1 - the ligand of the T cell integrin LFA-1 - on the SLB, the TCR is still clustered due to the patterning of its ligands, but now global parameters are also impacted. The actin organization changes to a peripheral ring, resembling the classical actin distribution seen on homogeneous substrates, the patterned membrane topography disappears and the membrane is flat, whereas the cell area increases significantly. These observations taken together point to a possible pivotal role for LFA-1 in amplifying the effect of TCR-clustering. No such effect is evident for co-engagement of CD28, affected
its ligand B7.2. Unlike on ICAM-1, on B7.2 cell spreading and actin organization are similar for homogeneous and patterned substrates. However, TCR and ZAP-70 clusters are still formed in the patterned case. These results indicate complementary role for LFA-1 and CD28 in the regulation and putative coupling of TCR micro-clusters to actin. The engineered substrates presented here clearly have the potential to act as platform for fundamental research in immune cell biology, as well as translational analyses in immunotherapy, for example to screen molecules for their role in T cell adhesion/activation.
Actin networks cross-linked by natural linkers alpha-actinin and filamin are generated in giant vesicles by polymerization through ionophore-mediated influx of Mg2+. alpha-actinin induces the ...formation of randomly linked networks at 25 degrees C which transform at <15 degrees C into spiderweblike gels or ringlike bundles depending on the vesicle size. Muscle filamin forms ringlike structures under all experimental conditions which can supercoil by subsequent Mg2+ addition. The polymorphism is rationalized in terms of recent models of bivalent ion coupled semiflexible polyelectrolytes and by considering the topology of the linkers.
A method is presented for combining atomic force microscopy (AFM) force mode and fluorescence microscopy in order to (a) mechanically stimulate immune cells while recording the subsequent activation ...under the form of calcium pulses, and (b) observe the mechanical response of a cell upon photoactivation of a small G protein, namely Rac. Using commercial set-ups and a robust signal coupling the fluorescence excitation light and the cantilever bending, the applied force and activation signals were very easily synchronized. This approach allows to control the entire mechanical history of a single cell up to its activation and response down to a few hundreds of milliseconds, and can be extended with very minimal adaptations to other cellular systems where mechanotransduction is studied, using either purely mechanical stimuli or via a surface bound specific ligand.
•A signal coupling AFM and fluorescence microscopy was characterized for soft cantilevers.•It can be used as an intrinsic timer to synchronize images and forces.•Mechanical stimulation of single immune cells while recording calcium fluxes was detailed.•Light-induced mechanical modifications of lymphocytes using a PA-Rac protein were demonstrated.•The precautions and limitations of use of this effect were presented.
The rupture forces and adhesion frequencies of single recognition complexes between an affinity selected peptide/MHC complex and a TCR at a murine hybridoma surface were measured using Atomic Force ...Microscopy. When the CD8 coreceptor is absent, the adhesion frequency depends on the nature of the peptide but the rupture force does not. When CD8 is present, no effect of the nature of the peptide is observed. CD8 is proposed to act as a time and distance lock, enabling the shorter TCR molecule to bridge the pMHC and have time to finely read the peptide. Ultimately, such experiments could help the dissection of the sequential steps by which the TCR reads the peptide/MHC complex in order to control T cell activation.
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