Analysing diffusion of biological colloids in glycan rich extracellular matrices –experimental and theoretical tools

The extracellular matrix provides a crucial framework in multicellular organisms. Matrix composition varies between tissues and different physiological states, but glycosaminoglycans are a quintessential feature. Glycosaminoglycans are polymer chains consisting of repeated disaccharides with a high degree of polydispersity and negative charge. Biological colloids distribute within the extracellular matrix and are impacted by glycosaminoglycans, either through site-specific interactions or by generation of a physical and electrostatic barrier. For example, chemokines and growth factors have well documented interactions with glycosaminoglycans required for their biological functions. Other glycans, as part of glycosphingolipids, interact with toxins, such as shiga toxin, as they similarly traverse the extracellular matrix. Our current mechanistic understanding of diffusion processes in the extracellular matrix is limited, however, largely due to the lack of methods to probe such processes in a controlled environment. The aim of this thesis was to develop experimental and theoretical tools to probe and understand the molecular mechanisms that define the diffusion of biological colloids in glycan rich extracellular matrices.
Model extracellular matrices were generated and refined to recreate features to investigate the interaction between biological colloids and glycosaminoglycans. These colloids include the chemokine CXCL12, shiga toxin, and unilamellar vesicles. Glycan-binding ability was adversely affected by fluorescent labelling of the glycan binding molecules. Furthermore, the bespoke method of Plane sphere confinement microscopy (PSCM) was developed to directly probe the rate of diffusion of biological colloids within glycan-rich model matrix films without interference of exchange processes with the bulk solution. Finally, an analytical model to describe diffusion of biological colloids within a sticky polymer matrix was derived using knowledge of diffusion in solution and binding kinetics. These methods can be used to analyse diffusion and mobility of polymer binding molecules in polymer rich extracellular matrices and the impact of diffusion on functions in biological systems.