Structure of the pericellular matrix

The notion of gradients of morphogens and of epithelial-mesenchymal signal relays are common currency in developmental biology. Moreover, organism homeostasis often depends on similar transport of effector proteins, such as growth factors, cytokines and chemokines from source to target cell, for example, in wound repair, in the regulation of immune responses and in cancer. Such transport occurs in the extracellular and pericellular matrices that lie between cells where the heparan sulfate (HS) chains of proteoglycans (PG) are a dominant molecular species, due to their size (~40 nm to160 nm long), amount and the number of proteins they bind to (over 435).
Recent work using single molecule tracking of fibroblast growth factor-2 (FGF2) demonstrates that the HS binding sites for FGF2 in the pericellular matrix of fibroblasts are organised into discrete structures from the nanoscale (corresponding to individual FGF2 binding sites, ~4 nm and HS chains) to the mesoscale (many HS chains on different PG, up to several um). This spatial organisation determines the diffusive properties of the FGF2.
The hypothesis that the project will test is that effectors possessing a structural binding specificity in HS different to FGF2’s will “see” a different spatial organisation of HS and will, therefore, have different diffusive properties.
This will entail:
i) Mastering the synthesis of 1:1 conjugates of nanoparticles and recombinant proteins.
ii) Analysis by electron microscopy of the spatial distribution of selected effectors in the pericellular matrix of cells.
iii) Analysis by photothermal microscopy of the spatial distribution of these effectors in the pericellular matrix of cells.
iv) Applying an analysis of the distribution of the effectors in the pericellular matrix to predict their diffusive properties.
v) Testing the prediction in (iv) by single molecule tracking.

Training:
The project is at the interface of cutting edge biomolecular science and translational medicine. It offers research training in a key growth area of science, the interface of biochemistry/molecular cell biology and bionanotechnology, applied to an important clinical problem, pancreatic disease. Specific research training will include:
Biochemistry/Molecular Cell Biology: isolation and culture of cells from normal pancreas, and diseased tissue, specifically pancreatitis and pancreatic adenocarcinoma, DNA cloning, expression and purification of recombinant proteins.
Bionanotechnology: synthesis of nanoparticle ligand shells, the specific conjugation of nanoparticles to proteins.
Single molecule microscopy: photothermal microscopy of individual nanoparticle labelled proteins in living cells, analysis of the dynamics of these proteins.
Electron microscopy.
Modelling of the large datasets acquired through imaging.