Our long-term goal is to enable quantitative prediction and control of leukocyte adhesion to vascular surface during inflammation and trafficking. This multistep process requires interactions of selectins, which mediate tethering and rolling adhesion, and integrins, which mediate stable adhesion. The focus of the previous grant period was selectins. We used the micropipette, atomic force microscopy, and flow chamber experiments to analyze their kinetic and mechanical properties. We propose to extend this work to alphaLbeta2 integrin in the present application. Compared to selectins, integrins are a larger family of adhesion molecules, have a broader tissue distribution, and mediate a wider variety of biological functions. Integrins provide mechanical linkage between cells so their ligand binding kinetics and affinity are relevant. Such kinetics and affinity are regulated by force and conformation, so mechanics is important. Yet there have only been a handful of studies reporting the single molecule kinetic and mechanical measurements on integrins. Using a variety of mutants provided by the Springer lab, we propose to employ kinetic and mechanical measurements as well as molecular modeling and molecular dynamics simulations to elucidate the regulation of integrin function by conformational changes. Specifically, we will measure and simulate the effects of structural variations in alphaL I domain on its 2D binding kinetics and affinities, to characterize the global conformational states of alphaLbeta2 and induced transitions among different states using kinetic and mechanical measurements, and to quantify the regulation of 2D kinetics of WT and mutant aL I domains and alphaLbeta2 integrin by activating/inhibitory agent. Not only will the information generated from this research advance our understanding of the workings of integrins, but it may also provide guidance to the development of integrin-based drugs that is currently being actively pursued by many biotechnological and pharmaceutical companies.
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