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In animal development and tissue repair, gradients of extracellular signalling proteins, such as fibroblast growth factors (FGFs), are formed in the extracellular matrix (ECM) to conduct cell-cell communication. It has been argued that the heparan sulfate (HS) chains of proteoglycans are important in controlling the diffusion of these signalling proteins and the paracrine FGFs are classic HS binding proteins. There are over 883 proteins that bind to HS in the ECM, yet the extent to which protein binding is specific and selective is not clear. Moreover, the HS chains and the ECM in general are often considered to be a hydrated gel and have no structure, beyond the individual components. It is difficult to reconcile the regulation of critical and complex events in cell communication by HS and ECM with this view.\ud \ud In this thesis, the production of a number of recombinant FGFs is described. This led to the discovery that HaloTag, a mutated chloroalkane dehalogenase conventionally used as a means to chemically label fusion proteins, is also an excellent solubilisation tag for FGFs. Thus, HaloTag fusions allowed the production and purification of members of the FGF family not generally available. These FGFs were used to characterise the interactions of FGFs with HS in vitro and in cells.\ud \ud The interactions of FGF10 and FGF20 with HS were characterised by differential scanning fluorimetry, using a library of model polysaccharides. The data show that in terms of their preferred binding structures, FGF10 is most like its closely related subfamily member, FGF7. Likewise, binding preferences of FGF20 are most similar to those of FGF9, which is in the same subfamily. These results support the idea that the specificity and selectivity of the interactions between FGFs and HS have been driven by the same selection pressures that led to the diversification of the FGF family and to their binding selectivity for isoforms of their receptor tyrosine kinase.\ud \ud To study the interaction with HS in cells, fixed Rama 27 fibroblasts were used, so that cell biochemistry would not confound the results. Fluorescent Halo-FGF-1, -2, -6, -10 and -20 were produced by reacting the HaloTag with a chloroalkane-TMR ligand. The binding of the fluorescent FGFs to Rama 27 cell pericellular matrix was measured by confocal microscopy. This showed that the binding sites for these FGFs were heterogeneously distributed and the number of the binding sites for each FGF was different. Diffusion of these FGFs was measured by fluorescence recovery after photobleaching (FRAP). This demonstrated that the diffusion speed and the relative proportion of mobile versus immobile FGF were different for each FGF. The data indicate that the HS in pericellular matrix is selective in its binding of FGFs and determining how they diffuse. \ud \ud In conclusion, the pericellular matrix exhibits high binding selectivity for different FGFs, which is consistent with the in vitro characterisation of their interactions with model polysaccharides. Moreover, cells spatially segregate the different binding structures in HS. This indicates that pericellular matrix is highly organised over length scales equivalent to hundreds or more HS chains, suggesting that it is formed by the self-organisation of its constituents though their extracellular interactions and, for transmembrane components, intracellular interactions as well. Such structure would then lie at the heart of how ECM performs its varied functions, including the selective binding and transport of FGFs.

The range of biological outcomes generated by many signalling proteins in development and homeostasis is increased by their interactions with glycosaminoglycans, particularly heparan sulfate (HS). This interaction controls the localization and movement of these signalling proteins, but whether such control depends on the specificity of the interactions is not known. We used five fibroblast growth factors with an N-terminal HaloTag (Halo-FGFs) for fluorescent labelling, with well-characterized and distinct HS-binding properties, and measured their binding and diffusion in pericellular matrix of fixed rat mammary 27 fibroblasts. Halo-FGF1, Halo-FGF2 and Halo-FGF6 bound to HS, whereas Halo-FGF10 also interacted with chondroitin sulfate/dermatan sulfate, and FGF20 did not bind detectably. The distribution of bound FGFs in the pericellular matrix was not homogeneous, and for FGF10 exhibited striking clusters. Fluorescence recovery after photobleaching showed that FGF2 and FGF6 diffused faster, whereas FGF1 diffused more slowly, and FGF10 was immobile. The results demonstrate that the specificity of the interactions of proteins with glycosaminoglycans controls their binding and diffusion. Moreover, cells regulate the spatial distribution of different protein-binding sites in glycosaminoglycans independently of each other, implying that the extracellular matrix has long-range structure.

The interaction of a large number of extracellular proteins with heparan sulfate (HS) regulates their transport and effector functions, but the degree of molecular specificity underlying protein–polysaccharide binding is still debated. The 15 paracrine fibroblast growth factors (FGFs) are one of the paradigms for this interaction. Here, we measure the binding preferences of six FGFs (FGF3, FGF4, FGF6, FGF10, FGF17, FGF20) for a library of modified heparins, representing structures in HS, and model glycosaminoglycans, using differential scanning fluorimetry. This is complemented by the identification of the lysine residues in the primary and secondary binding sites of the FGFs by a selective labelling approach. Pooling these data with previous sets provides good coverage of the FGF phylogenetic tree, deduced from amino acid sequence alignment. This demonstrates that the selectivity of the FGFs for binding structures in sulfated polysaccharides and the pattern of secondary binding sites on the surface of FGFs follow the phylogenetic relationship of the FGFs, and so are likely to be the result of the natural selection pressures that led to the expansion of the FGF family in the course of the evolution of more complex animal body plans.