Control of binding and movement of fibroblast growth factors by heparan sulfate in extracellular matrix

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. 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. 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. 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. 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.