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Well-defined biomimetic surfaces to characterize glycosaminoglycan-mediated interactions on the molecular, supramolecular and cellular levels

  • Thakar, Dhruv
Publication Date
Sep 07, 2015
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The oriented migration and controlled adhesion of cells is fundamental to many physiological and pathological processes. A family of linear polysaccharides, known as glycosaminoglycans (GAGs), help organizing and presenting signaling proteins, so-called chemokines, on the cell surface and in the extracellular matrix thus regulating cellular behavior. The objective of this PhD thesis was to develop biomimetic surfaces that are highly defined and tunable, for mechanistic studies of GAG-protein interactions on the molecular and supramolecular levels, and to probe cellular responses to defined biochemical and biophysical cues to better understand GAG-mediated cell-cell and cell-matrix communications.Applying oxime ligation, GAGs could be stably functionalized with biotin at the reducing end, and these features proved crucial for the reliable preparation of GAG-functionalized surfaces. A streptavidin monolayer served as a ‘molecular breadboard' to sequentially assemble biotinylated molecules with controlled orientation and surface densities. GAGs (heparan sulfate (HS) in particular), chemokines and other ECM components (e.g. integrin ligands promoting cell adhesion, RGD) were assembled into multifunctional surfaces that recapitulate selected aspects of the in vivo situation. Quartz crystal microbalance (QCM-D) and spectroscopic ellipsometry permitted us to characterize and control the supramolecular presentation of HS and RGD. These model surfaces were used to study the supramolecular interactions between HS and the selected chemokine stromal derived factor SDF-1α/CXCL12α and to analyze cellular responses to extracellular cues. Our data provide evidence that CXCL12α binding rigidifies HS assemblies, and that this effect is due to protein-mediated cross-linking of HS chains. The kinetics of chemokine binding to HS was quantified using surface plasmon resonance (SPR). We also demonstrate that the way in which the chemokine is presented, and in particular the presence of HS, is important for regulating myoblast behavior. Our data shows that the cell surface receptors CXCR4 (the CXCL12α receptor) and integrins (the RGD receptor) can act synergistically in controlling cellular adhesion and migration. These surfaces can generate novel insights in the field of glycobiology, e.g. in dissecting the function of GAGs in chemokine-mediated cellular migration.

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