Date

2016

Document Type

Dissertation

Degree

Doctor of Philosophy

Department

Bioengineering

First Adviser

Zhang, Xiaohui F.

Other advisers/committee members

Berdichevsky, Yevgeny; Berger, Bryan; Ou-Yang, Daniel; Zhou, Chao

Abstract

The precisely regulated lymphocytes trafficking plays very important roles in immune surveillance and host defense. The adhesive interaction between leukocyte integrin α4β7 and its endothelial ligand, mucosal addressin cell adhesion molecule-1 (MAdCAM-1), mediates the rolling and firm adhesion of leukocytes to the high endothelial venules of mucosal tissues. A key property of α4β7 is that it mediates rolling on resting leukocytes. Upon leukocyte activation, α4β7 is induced to adopt a high-affinity conformation and thus mediates firm adhesion. We used single-molecule force spectroscopy (SMFS) and single-cell force spectroscopy (SCFS) to determine the mechanical strength of the low- and high-affinity α4β7/MAdCAM-1 complexes. An atomic force microscope (AFM) was used to pull individual α4β7/MAdCAM-1 complexes to determine their mechanical strength and force-dependent dissociation kinetics under ionic conditions that produce low- and high-affinity complexes, which mediate leukocyte rolling and firm adhesion, respectively. Integrin α4β7 also binds to vascular cell adhesion molecule-1 (VCAM-1) expressed in other tissues. To regulate the adhesion of lymphocytes to target tissues, integrin α4β7 must be able to distinguish different ligands. Here we used SCFS to pull individual α4β7/MAdCAM-1 or α4β7/VCAM-1 complexes under different chemokine stimulations and demonstrated the chemokine CCL25 promotes α4β7-mediated lymphocyte adhesion to MAdCAM-1 but suppresses adhesion to VCAM-1, whereas the chemokine CXCL10 regulates adhesion in the opposite way at the single molecule level. In addition, Sin Nombre Hantavirus (SNVs) bind to the Plexin Semaphorin Integrin (PSI) domain of inactive, bent conformation β3 integrins. We used SMFS to pull individual integrin αIIbβ3 and P2Y2R complexes with or without SNVs binding and demonstrated that binding of SNV to the PSI domain induces an increase in the affinity of the integrin’s RGD binding site, and stimulates activation of several heterotrimeric G-proteins, Rho GTPases and infection.In conclusion, this work provided new insights into the kinetic mechanism of integrin-mediated leukocyte rolling and firm adhesion, but also a mechanism for lymphocyte homing through the unique ligand-specific regulation of integrin adhesion by different chemokines. Our findings are also fundamental to understanding integrin-GPCR transactivation, and Hantavirus pathogenesis.

Available for download on Sunday, June 10, 2018

Share

COinS