Elena Lomakina - Current Research
Dynamics of bond formation between cells and substrates
A significant advantage of single cell micromanipulation is the ability to control the timing and duration of contact between cells and other surfaces, and also to control the chemistry of the surfaces with which the cell interacts. We have taken advantage of this capability, first to characterize the rate at which bonds form between a cell and a surface presenting a specific adhesion molecule, and second, to examine the dynamics of the cell response when it encounters specific stimulatory molecules immobilized on a surface.
This work revealed an important limitation in bond formation at interfaces. Unlike chemical reactions between molecules in solution, or even the binding of molecules in solution to a surface, the rate at which bonds form at a cell-substrate interface depends critically on there being a molecule capable of binding the substrate ligand in close proximity with the substrate. Thus the irregular topography of the cell surface, and the localization of active molecules in relation to that topography are critically important in determining rates at which cells can form adhesive bonds. We have looked at two different cases. In both cases, adhesion molecules on the surface of the neutrophil (integrins) were activated using divalent ions in the external solution. In the first case, the binding target on the substrate (bead) was ICAM-1, and the active integrin type was LFA-1. In this case, the number of molecules on the surface is plentiful, and nearly all of them are in a high affinity conformation. Under these conditions, the rate of bond formation follows simple bi-molecular reaction kinetics, and the rate of bond formation can be characterized in terms of forward and reverse rate constants that reflect the full complexity of the bond formation process, including, not only the intrinsic reactivity of the molecules themselves, but also the additional effects of having the molecules distributed over an irregular topography1
Figure 1. Simple theory predictions fail to match measurements of neutrophil adhesion to VCAM-1. (The solid line was fit to the solid symbols, and the parameters were used to predict results for a different case (dashed line) which fell far from the measurements (open symbols).
Figure 2. Reaction zones are regions of close contact between a cell and a substrate where bonds (ZB) can form. Potential reaction zones are regions of the membrane containing adhesion molecules that cannot form a bond. (A. Potential reaction zones can form by a variety of mechanisms, B. denotes the membrane as it moves closer to surface, C. is the molecule near the surface changing affinity, and D. is the active molecule diffusing into a region of close contact.)
In a related study, we examined the case of a different integrin (VLA-4) binding to a different endothelial ligand (VCAM-1). In this case there are ten times fewer integrins on the neutrophil surface, and the effect of the divalent ions was such that no all of the molecules were necessarily in the high affinity conformation. In this case, results contradicted the predictions of simple bi-molecular reaction theory. We developed a new theoretical framework that had an explicit kinetic step involving the appearance of an active molecule in close proximity to the substrate. This more complex model resulted in predictions for bond formation rate that were consistent with the data, and shed light on the conformational activity of the VLA-4 molecules at the interface.23
Figure 3. The new theory predicts that the rate of reaction depends on both the concentration of target molecules (VCAM-1) on the substrate and the contact time.
Lomakina, E., and R.E. Waugh (2004) Micromechanical tests of adhesion dynamics between neutrophils and immobilized ICAM-1. Biophysical Journal 86:1223-1233
Waugh, R.E. and Lomakina, E.B. (2008). Active site formation, not bond kinetics, limits adhesion rate between human neutrophils and immobilized VCAM-1. Biophys. J. (in press).
Lomakina, E.B. and Waugh, R. E. (2008). Adhesion between human neutrophils and immobilized endothelial ligand VCAM-1: Divalent ion effects. Biophys. J. (revised, resubmitted).