The regulated interaction of leukocyte with endothelium is of key importance during normal immune surveillance and leukocyte infiltration to sites of infection in the inflammatory response. This thesis is concerned with the structure and function of CD31 (platelet-endothelial cell adhesion molecule-1), one of the adhesion molecules implicated in these processes. Previous work has shown both in vivo and in vitro that CD31 is involved in the final step of leukocyte recruitment, transmigration across the endothelial monolayer. CD31 mediated adhesion is complex, since it is capable of mediating multiple adhesive interactions, both to itself (homophilic adhesion) and to other ligands (heterophilic adhesion). In order to study homophilic adhesion, an heterologous cell-protein assay was used in combination with recombinant chimeric CD31Fc fusion proteins, ICAM-3/CD31 chimeras and chimeras between human and murine CD31. These reagents located the homophilic binding site to the NH₂-terminal domains 1 and 2, but also define a non-binding accessory role for the membrane proximal domains. Using site-directed mutagenesis to target all of the exposed charged residues in domain 1 and a subset of charged residues in domain 2, five residues were identified, mutations in which resulted in inhibition of homophilic adhesion. These residues map to both faces of the domain 1 immunoglobulinlike fold, suggesting that each molecule of CD31 interacts with two others. A novel zipper model of homophilic adhesion involving CD31 lateral association analogous to that seen amongst cadherins is proposed on the basis of these results. Evidence for lateral association of CD31 to form dimers was obtained from biophysical, biochemical and molecular biology techniques. These show that Cd31 exists in an equilibrium between monomeric and dimeric forms both in solution as soluble recombinant protein, and at the cell surface. In solution the affinity of the interaction was calculated to lie in the range 12-14μM. A large panel of anti-CD31 monoclonal antibodies were generated and tested for their ability to effect homophilic adhesion. Inhibitory antibodies were identified, mapping throughout the extracellular domain, away from the ligand binding site. In addition possible stimulating antibodies mapping to the membrane proximal domains were also identified. This indicates that CDS 1 may be induced to undergo conformational changes which effect homophilic adhesion, and it is proposed that these conformational changes may be linked to the ability of CD31 to form laterally associated dimers. Using the reagents described above, a screen of haematopoietic cell lines identified a novel heterophilic interaction, which was shown to be mediated by the integrin αvβ3. Proteinprotein assays were used to confirm a direct physical association between CD31 and αvβ3, and to map the integrin binding site to the third immunoglobulin-like fold of CD31. The functional significance of this interaction was assessed in neutrophil transmigration assays, in which both anti-CD31 and anti-αvβ3 antibodies were found to partially inhibit neutrophil transmigration.

Abstract Adhesive interactions between cells and extracellular matrix proteins play a vital role in biological processes such as cell proliferation, differentiation and survival. Integrins comprise a major family of cell surface receptors that mediate these interactions. Integrin engagement triggers adhesion-dependent intracellular signalling cascades that include the phosphorylation of tyrosines in intracellular signalling proteins. Integrin-dependent signals act in concert with signals from growth factors and other signalling receptors. The objective of this thesis was to study how cell adhesion and growth factors interact with intracellular components to regulate cell behavior in normal and transformed cells. One of the main proteins phosphorylated following integrin ligation in several different cell types is the docking protein p130Cas (Cas), which is tyrosine phosphorylated after stimulation of cells with low concentrations of epidermal growth factor (EGF). Tyrosine-phosphorylated Cas associates with an adapter protein c-Crk, the main binding protein for Cas, suggesting a novel role for EGF in Cas signalling. The interaction of cells with a variety of agonists such as growth factors and integrin ligation results in stimulation of mitogen-activated protein kinases (MAPKs), which control the expression of genes important for many cell functions. Expression of Cas and Crk induces activation of C-Jun N-terminal kinases (JNKs), which are members of MAPK family. JNK activation induced by integrin ligand binding is blocked by the expression of a dominant-negative mutant of Cas or Crk demonstrating an important role for the Cas-Crk complex in integrin-mediated JNK activation. The proto-oncogene product p120Cbl (Cbl) was identified as the main tyrosine-phosphorylated protein following integrin ligation in hematopoietic cells of myeloid lineage. Tyrosine-phosphorylated Cbl interacts with and activates other signalling proteins, such as Src tyrosine kinase and phosphatidylinositol 3"-kinase (PI 3-kinase), thereby mediating adhesion-dependent signals in hematopoietic cells. Unlike the cellular Cbl, the transforming mutants of Cbl were tyrosine-phosphorylated in an adhesion-independent manner and interacted with and activated signalling molecules both in suspended and in adherent cells. Further, the oncogenic forms of Cbl induced anchorage-independent but serum-dependent proliferation of cells. These results support the view that transformation by Cbl results from constitutive activation of integrin-dependent rather than growth factor-dependent signalling events.

No description available.

Yes / For the first time a series of functional hydrogels based on semi-interpenetrating networks with both branched and crosslinked polymer components have been prepared and we show the successful use of these materials as substrates for cell culture. The materials consist of highly branched poly(N-isopropyl acrylamide)s with peptide functionalised end groups in a continuous phase of crosslinked poly(vinyl pyrrolidone). Functionalisation of the end groups of the branched polymer component with the GRGDS peptide produces a hydrogel that supports cell adhesion and proliferation. The materials provide a new synthetic functional biomaterial that has many of the features of extracellular matrix, and as such can be used to support tissue regeneration and cell culture. This class of high water content hydrogel material has important advantages over other functional hydrogels in its synthesis and does not require post-processing modifications nor are functional-monomers, which change the polymerisation process, required. Thus, the systems are amenable to large scale and bespoke manufacturing using conventional moulding or additive manufacturing techniques. Processing using additive manufacturing is exemplified by producing tubes using microstereolithography. / EPSRC

Cell adhesion to extracellular matrix proteins is critical to physiological and pathological processes as well as biomedical and biotechnological applications. Cell adhesion is a highly regulated process involving initial receptor-ligand binding, and subsequent clustering of these receptors and rapid association with the actin cytoskeleton as focal adhesions are assembled. Focal adhesions enhance adhesion, functioning as structural links between the cytoskeleton and the extracellular matrix and triggering signaling pathways that direct cell function. The objective of this thesis research is to develop a mechanical and biochemical analysis of the adhesion strengthening response. Our central hypothesis was that focal adhesion size and position regulate cell adhesion strength by controlling the distribution of mechanical loading. We engineered micropatterned surfaces to control the size and position of focal adhesions in order to analyze the contributions of these specialized adhesive structures to adhesion strengthening. By applying surface micropatterning techniques, we showed robust control over cell-substrate contact area and focal adhesion assembly. Using a hydrodynamic shear assay to quantify adhesion strength to micropatterned substrates, we observed significant adhesive area- and time-dependent increases in adhesion strength. Complimentary biochemical assays allowed us to probe the role of structural proteins recruited to focal adhesions and examine the structure-function relationships between these adhesive structures and adhesion strength. These findings provide insights into the role of focal adhesions in adhesion strengthening, and may contribute to tissue engineering and biomaterials applications.

During hemostatic and inflammatory responses, cell adhesion molecules play a major role in regulating the leukocytes and platelets adhesion to vascular surfaces under the hydrodynamic environment of the circulation. Selectin-ligand interactions (bonds) mediate leukocyte rolling on vascular surfaces. The molecular basis for differential ligand recognition by selectins is poorly understood. Using atomic force microscopy (AFM), the kinetics of three mutants L-selectin interacting with surrogates of PSGL-1 and PNAd, is compared with those of wild-type L-selectin. The interaction between glycoprotein Ib (GPIb) and von Willebrand Factor (VWF) mediates platelet translocation at the vascular vessel damage sites, which plays a critical role in initiating the platelets adhesion and thrombus formation. Translocation of platelets on VWF requires a shear threshold, suggesting a possible catch bond at work there. We characterized the kinetics of GPIbα interacting with VWF A1 domain, confirming the catch bond existed. Two type 2B VWD A1 mutants eliminated the catch bond and gave longer low force lifetimes. The prolonged lifetimes at low force resulted in more agglutination of platelets with A1 coated microspheres in flow. During the process of hemostasis, the size of prothrombotic ULVWF affects the affinity of VWF to platelets bearing GPIbα on the membrane. ADAMTS13 has been identified and characterized as a multi-domain metalloprotease that regulate the size of ULVWF. We studied how force regulated the binding and cleavage of ADAMTS13 on VWF. We found the cleavage effects could only be observed after the catastrophic structural change of A1A2A3. The unfolding exposed the ADAMTS13 cleavage site and favored the cleavage. Two protocols using different stretching molecules (GPIbα and CR1) and A1A2A3 immobilization methods revealed the cleavage effects diminished with increasing stretching force. This study elucidated mechanisms of the binding kinetics of L-selectin with different structure components from PSGL-1 and PNAd by structural variants. It also provided new insights into our current knowledge of the dynamic adhesion and regulation of GPIbα-VWF interaction in vivo. Using single molecule method, the chemical catalytic reaction between enzyme and substrate has been targeted. These results help us understand this important enzyme-substrate interaction involved in the hemostasis.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 152-171).

Thesis (Ph.D.). / Written for the Dept. of Biology. Title from title page of PDF (viewed 2008/02/12). Includes bibliographical references.

Thesis (Ph. D.)--School of Pharmacy and School of Dentistry. University of Missouri--Kansas City, 1998. / eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 1996. / Vita. Includes bibliographical references (leaves [106]-127).