Our long-term objective is to develop a detailed biophysical understanding of leukocyte adhesion at the molecular level, relating adhesive bond strength to chemical interactions and establishing how adhesive bond stress is transmitted through the cell membrane to the interior connections between receptor tails and the cell cytostructure where it can impact specific biochemical pathways. Our current research has provided significant insight into the mechanical strengths of leukocyte adhesion bonds and their kinetics under force, (i) first for interactions between the sialo mucin ligand PSGL-1 and selectins that initiate leukocyte attachments to vessel walls, (ii) second for interactions between super immunoglobulin (Ig)family ligands and integrins that signal and stabilize leukocyte adhesion enabling emigration into tissues. We have developed novel methods to immobilize ligands on the tip of ultrasensitive force probes and test the mechanical strengths of individual bonds to receptors either immobilized on microspheres or expressed on cells. Exploiting an exciting new approach and established cell lines with strategic alterations in receptor- cytoskeletal interactions, the principal objective of this application is to "move inside the cell", first establishing the extent to which cytostructural proteins regulate the mechanical strength of an adhesion complex, and then determining the mechanical role that these linkages play in the leukocyte signalling processes important for recruitment to sites of inflammation and injury. The studies are designed to test three hypotheses. Hypothesis: leukocyte selectin and integrin interactions have function-specific mechanical designs that govern bond formation and release under conditions of low stress interior to a cell- surface contact, thereby impacting bond proliferation and determining the fate of the initial adhesion event. Hypothesis: mechanical strengths of molecular adhesion complexes are governed by the weakest link in entire sequence of protein interactions from the outside adhesive bond to the inside bonds that connect receptor-tail domains to the cell cytostructure. Hypothesis: the linkages that anchor an integrin adhesion complex to the cell structure are key mechanical effectors of "outside-in" and "inside-out" signaling in leukocytes and represent an important feed-back process to regulate adhesion strength.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL065333-09
Application #
7990432
Study Section
Erythrocyte and Leukocyte Biology Study Section (ELB)
Program Officer
Kindzelski, Andrei L
Project Start
2000-07-01
Project End
2012-11-30
Budget Start
2010-12-01
Budget End
2012-11-30
Support Year
9
Fiscal Year
2011
Total Cost
$346,550
Indirect Cost
Name
Boston University
Department
Engineering (All Types)
Type
Schools of Engineering
DUNS #
049435266
City
Boston
State
MA
Country
United States
Zip Code
02215
Kuznetsov, Igor R; Evans, Evan A (2013) Brownian nanoimaging of interface dynamics and ligand-receptor binding at cell surfaces in 3-D. Methods 60:214-24
Chen, Wei; Lou, Jizhong; Evans, Evan A et al. (2012) Observing force-regulated conformational changes and ligand dissociation from a single integrin on cells. J Cell Biol 199:497-512
Evans, Evan; Smith, Benjamin A (2011) Kinetics of Hole Nucleation in Biomembrane Rupture. New J Phys 13:
Evans, Evan; Kinoshita, Koji; Simon, Scott et al. (2010) Long-lived, high-strength states of ICAM-1 bonds to beta2 integrin, I: lifetimes of bonds to recombinant alphaLbeta2 under force. Biophys J 98:1458-66
Kinoshita, Koji; Leung, Andrew; Simon, Scott et al. (2010) Long-lived, high-strength states of ICAM-1 bonds to beta2 integrin, II: lifetimes of LFA-1 bonds under force in leukocyte signaling. Biophys J 98:1467-75
Chen, Wei; Evans, Evan A; McEver, Rodger P et al. (2008) Monitoring receptor-ligand interactions between surfaces by thermal fluctuations. Biophys J 94:694-701
Evans, Evan; Kinoshita, Koji (2007) Using force to probe single-molecule receptor-cytoskeletal anchoring beneath the surface of a living cell. Methods Cell Biol 83:373-96
Herant, Marc; Heinrich, Volkmar; Dembo, Micah (2006) Mechanics of neutrophil phagocytosis: experiments and quantitative models. J Cell Sci 119:1903-13
Bayas, Marco V; Leung, Andrew; Evans, Evan et al. (2006) Lifetime measurements reveal kinetic differences between homophilic cadherin bonds. Biophys J 90:1385-95
Heinrich, Volkmar; Rawicz, Wieslawa (2005) Automated, high-resolution micropipet aspiration reveals new insight into the physical properties of fluid membranes. Langmuir 21:1962-71

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