During inflammation, leukocytes modulate α4β1(VLA-4) integrin avidity in order to rapidly
stabilize nascent adhesive contacts to VCAM-1-expressing endothelial cells and resist
detachment forces imparted by the flowing blood. Linkage to the actin cytoskeleton is critical for
integrin function, yet the exact role of the actin cytoskeleton in leukocyte adhesion stabilization
under conditions of fluid flow remains poorly understood. We modeled leukocyte (U937 cell,
mouse lymphocyte and human monocyte) arrest and adhesion stabilization through the use of a
parallel plate flow chamber and visualized cells by phase contrast or fluorescent confocal
microscopy. Live cell imaging with Lifeact-transfected U937 cells revealed that mechanical
forces imparted by fluid flow induced formation of upstream tension-bearing anchors attached to
the VCAM-1-coated surface. Scanning electron microscopy confirmed that flow-induced
mechanical force culminates in the formation of structures that anchor monocyte adhesion. These
structures are critical for adhesion stabilization, since disruption of actin polymerization
dramatically inhibited VLA-4-dependent resistance to detachment, but did not affect VLA-4
expression, affinity modulation, and clustering or constitutive linkage to F-actin. Transfection of dominant-negative constructs and inhibition of kinase function or expression revealed key
signaling steps required for upstream actin polymerization and adhesion stabilization. Rap1 was
shown to be critical for resistance to flow-induced detachment and accumulated in its GTP form
at the sites of anchor formation. A key mediator of force-induced Rac activation and actin
polymerization is PI3K. Live cell imaging revealed accumulation of PIP3 within tension-bearing
anchors and blockade of PI3K or deficiency of PI3Kγ isoform reproduced the adhesion defect
produced by inhibition of actin polymerization. Thus, rapid signaling and structural adaptations
enable leukocytes to stabilize adhesion and resist detachment forces; these included activation of
Rap1, phosphoinositide 3-kinase γ-isoform and Rac, but not Cdc42.
| Identifer | oai:union.ndltd.org:TORONTO/oai:tspace.library.utoronto.ca:1807/34870 |
| Date | 19 December 2012 |
| Creators | Rullo, Jacob |
| Contributors | Cybulsky, Myron |
| Source Sets | University of Toronto |
| Language | en_ca |
| Detected Language | English |
| Type | Thesis |
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