- Title
- Molecular mechanisms of cell migration
- Creator
- Tooney, Paul Anthony
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 1994
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- To understand the basis of cellular migration it is important to characterise the molecular event(s) of this biological process. It is clear that cell migration depends on the ability of cells to identify, attach to and detach from components of the extracellular matrix (ECM). One of these components, thrombospondin (TSP) is a multidomain glycoprotein that has been shown to have a number of functions including a role in the detachment and migration of normal and tumour cells. Adhesive interactions between a cell and its ECM occur via specific cellular receptors. Recent evidence indicates that members of the integrin family of cell surface receptors that mediate cellular adhesion to the ECM are also involved in cell migration, and in particular, the integrin ανβ₃ has recently been established to have a fundamental role in mediating cellular migration both in vitro and in vivo. The ανβ₃ integrin functions as a receptor for several glycoproteins of the ECM including TSP, however, whether the TSP-mediated effects on cell migration are through this interaction is not known. A number of migrating cells and tumour cell lines also express another cell surface receptor for TSP, termed CD36. Whether CD36 has a role in cellular migration has yet to be determined. In this thesis, Chapter 1 provides an overview of the historical models of cellular migration, TSP, CD36 and the integrin family of adhesion receptors. Discussed at the end of Chapter 1 is a "monkey" model of cell migration which proposes a role for TSP and its receptors (CD36 and ανβ₃) and formed the initial focus of the investigations presented in this thesis. The materials and methods employed are outlined in Chapter 2, and the next four chapters present the major experimental findings. Chapter 3 presents an initial study using colon fibroblasts undergoing cell spreading on collagen matrices. TSP was shown to be produced by these cells in culture and in an in vitro wound assay, but it did not localise along actin filaments, as did another ECM glycoprotein, fibronectin. A tyrosine kinase inhibitor is shown to affect cell spreading by disruption of actin filaments. In Chapter 4, the process of cellular de-adhesion of a number of cell types was investigated using human platelet thrombospondin, termed TSP1. TSP1 exhibited no adhesive properties for a variety normal and tumor cell lines, and when plated together with adhesive glycoprotein substrates, it was strongly anti-adhesive, inhibiting cell attachment to several extracellular matrix (ECM) components. When the TSP1 was reduced with dithiothreitol, anti-adhesive activity was lost and the molecule served as an adhesive substrate. Cell attachment to VN with or without phorbol 12-myristate-13-acetate (PMA) treatment, was inhibited by arginine-glycine-aspartate-serine (RGDS) peptides and antibodies to the ανβ₃ integrin. Significantly lower concentrations of peptide and antibody were necessary to inhibit adhesion to VN when TSP was incorporated into the matrix, and flow cytometry confirmed that this was not due to a downregulation of cell surface expression of integrin receptors. The anti-adhesive activity of TSP1 was also dependent on the nature of bound cations, and a novel mechanism involving cation exchange is proposed to mediate the downregulation of integrin function. Chapter 5 shows that a proportion of CD36 resists detergent extraction in unstimulated platelets and WM115 melanoma cells suggesting that this molecule associates with the cytoskeleton. Differential detergent extraction, affinity chromatography, equilibrium/partition co-sedimentation and site directed mutagenesis are used to show that CD36 associates with microtubules via positively charged amino acids in its carboxy-terminus. It is proposed that the microtubule-binding capacity of CD36 could facilitate the internalisation and/or transport of its ligands. In Chapter 6, CD36 and TSP are shown to colocalise to cytoplasmic organelles involved in intracellular transport pathways and it is proposed that CD36 plays a role in the intracellular transport of TSP to the cell surface, a function that may be facilitated by its capacity to associate with microtubules. In Chapter 7, the major findings of this thesis are discussed. Taken together these findings indicate that TSP1 has a role in cellular de-adhesion through down regulation of integrin function and the CD36 receptor may facilitate the delivery of this ECM component to the cell surface. These findings could have important implications for directed cell migration.
- Subject
- cell migration; extracellular matrix; biological processes; thrombospondin
- Identifier
- http://hdl.handle.net/1959.13/1416640
- Identifier
- uon:37086
- Rights
- Copyright 1994 Paul Anthony Tooney
- Language
- eng
- Full Text
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| Thumbnail | File | Description | Size | Format | |||
|---|---|---|---|---|---|---|---|
| View Details Download | ATTACHMENT01 | Thesis | 184 MB | Adobe Acrobat PDF | View Details Download | ||
| View Details Download | ATTACHMENT02 | Abstract | 8 MB | Adobe Acrobat PDF | View Details Download |