Role of Thrombospondins in interstitial Extracellular Matrix 3-Dimensional Organisation: Mechanisms and Functions

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In our bodies, our skin is supple and our bones are rigid because of the properties of extracellular matrix (ECM) that surrounds the cells in each tissue. ECM is built up from the beginning of development as a suite of large proteins released by cells that bind with each other to form insoluble fibrils and networks. These structures are maintained throughout life, are repaired after injury, and can cause health problems if disrupted in disease. The physical and chemical properties of ECM inform cells whether to be stationary, move, or divide, and modulate the expression of genes. These signals enable cells to function properly throughout life but how the 3-dimensional organisation of ECM is set up and tuned is poorly understood. For all these reasons, research into principles of ECM organisation is important and necessary to identify new therapeutic targets and materials. The most abundant proteins in ECM are collagens that form elongated fibrils. Many accessory proteins act to maintain the number, sizes, and packing of collagen fibrils, and how the fibrils interact with other ECM components. Accessory proteins are attractive as possible therapeutic targets because they are less abundant (so could be more easy to modulate with a small drug) and yet can act as "levers" to change ECM organisation. Our recent research identified that thrombospondin proteins (TSPs) are conserved with collagens and a few other ECM components throughout animals, from sponges to human. Because of this conservation we consider that the mechanism(s) by which TSPs bind into ECM and modulate collagen fibril organisation are likely to be important and a promising therapeutic target. In studying how TSPs bind into ECM, we identified a protein sequence motif that is widely conserved between TSPs and is required for them to accumulate into ECM made by cultured cells. TSPs mutated at this site are secreted normally by cells but are not held in the ECM. We discovered recently that this site works by mediating interactions between separate TSP molecules. These interactions cause TSP clusters to form small dot-like structures or "puncta". From this research we developed a model, (a hypothetical diagram), of how TSPs can enter and cluster in ECM to impact on collagen fibril organisation. Our model is novel because neither the molecular identity of the motif or its role in interactions between TSP molecules were known before. Our central goals are: 1), To further understand mechanisms of TSPs within cell culture ECM, to extend the model and strengthen its accuracy. 2), To test how the site we have identified in cell culture works in living animals. To make this test is essential to understand how our discoveries in cell culture relate to protein function in real tissues. With the mouse we can examine the role of the TSP interaction site in the skin of an animal close to human anatomy and physiology that is used widely in biomedical research. 3). From the new genetic strains of mice we will also isolate cells from the skin so that we can closely study their properties in culture and relate the mechanisms that we identify in goal 1 to effects of TSPs on cell functions and ECM properties. These experiments will generate additional materials from the experimental mice and help us understand how our new knowledge might be adapted for clinical materials.