Selectin projects
The laser trap is an ideal system to obtain data on the mechanical strength, off-rate, and stress-strain relationship of single adhesion receptor - ligand bonds. As part of a Bioengineering Research Partnership, we are studying the load-dependent kinetics of L-selectin bonds with PSGL-1.
Binding of biotin (space-filled representation, left) to avidin (ribbon representation, left), compared to carbohydrate ligand (space-filled, right) to mannose-binding protein (ribbon, right), which is closely related to selectins in structure.
Microspheres and/or giant lipid vesicles are being formed with ligand or selectin attached/incorporated. 10 ml of each suspension will be combined and immediately introduced into a 15 ml flow cell made from two coverslips separated by Mylar shim stock. Once on the microscope, two laser traps are created. In one, we will trap a microsphere bearing ligand or selectin, in the other a microsphere, vesicle, microsphere or cell bearing selectin and/or ligand. To illustrate, assume that we have trapped a microsphere bearing selectin in one trap and a cell in the other. The microsphere will be brought into contact with the trapped cell by moving the laser traps toward one another. The cell will then be moved away from the microsphere by deflecting its trap over a range of velocities. As bonds between the microsphere and cell are strained, the microsphere will be deflected from the center of its trap. The instantaneous deflection of the microsphere from trap center (d) is related to the force acting on the bond by f = d · 2atrap where atrap is the trap stiffness (assuming that the stiffnesses of the two traps are equal). In each experiment we note the loading rate (determined by separation velocity and atrap) and the force at which bond rupture occurs. The cumulative results of many such experiments results in a graph of bond strength versus loading rate, which is a reflection of the "energy landscape" for unbinding.
Several interesting hypotheses can be tested with respect to the significance of the length and the structure of the extracellular domain of selectins. In a cell-bead system in which PSGL-1 is expressed in its physiological position on the tip of microvilli, we will obtain an estimate of the cellular contribution to the elastic behavior. The spring constant of individual microvilli has recently been measured (Shao et al., 1998) and suggests that the force exerted by a few P-selectin-PSGL-1 bond can significantly extend a microvillus of an attached cell. The cell-bead system is ideally suited to directly measure this cellular compliance once the molecular compliance is known.
We are also building liposomes that use leukocyte adhesion molecules to bind to vessel walls under shear stress, with the intent of designing vehicles suitable for drug and gene delivery in vivo. We are presently incorporating L-selectin into liposomes. We have already produced liposomes containing P-selectin and have shown that they can roll on PSGL-1. For in vivo experiments, L-selectin containing liposomes will be made. …a surrogate cytoskeleton.
