Kirkham, Matthew John (2006). THE ROLE OF CAVEOLIN IN ENDOCYTOSIS AND CAVEOLAE BIOGENESIS PhD Thesis, Institute for Molecular Bioscience, University of Queensland.

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Author Kirkham, Matthew John
School, Centre or Institute Institute for Molecular Bioscience
Institution University of Queensland
Publication date 2006
Thesis type PhD Thesis
Supervisor Rob Parton
Abstract/Summary Caveolae, flask-shaped invaginations of the plasma membrane, are a striking feature of many mammalian cells. Caveolae are defined by their morphology and by the presence of integral membrane proteins, called caveolins. In this study, we have investigated the formation of caveolae and the dynamics of cell surface caveolae. Caveolae can be generated de novo by the expression of caveolin-1 (Cav1), but the precise role Cav1 plays in this process is presently unclear. The caveolin family of proteins includes splice variants Cav1[alpha] and Cav1[beta], caveolin-2 (Cav2) and the muscle specific caveolin, caveolin-3 (Cav3). Caveolins are conserved in evolution. As well as being expressed in all mammals examined, caveolin are also found in lower eukaryotes such as the nematode, Caenorhabditis elegans (C. elegans), the zebrafish, Danio rerio and the amphibian, X.laevis laevis. Though both the trafficking and the biochemical properties of Cav1 and Cav3 have been extensively studied by the creation of a large number of deletion, truncation and substitution mutants, few of these studies have assessed the formation of caveolae. We have established an electron microscopy assay through the use of Cav1-null mouse embryo fibroblasts (MEFs) to study the biogenesis of caveolae. We found that caveolae produced by the transient expression of Cav1 and Cav3 had the same morphology and that the first 48-residues of the N-terminus and the last 31-residues of the C-terminus of human Cav1 could be individually removed without affecting the shape of the caveolae generated. More severe deletions caused the protein to be retained within the Golgi complex. Caveolae generated by a point mutation of Cav1 that has an increased association with cholesterol, and several point mutations of Cav3 shown to be involved in muscular disease, had normal morphology. The conservation between species of caveolin in caveolae formation was investigated. Interestingly, we showed that C. elegans caveolin is not functionally equivalent to mammalian Cav1 in mammalian cells, and does not cause caveolae biogenesis in Cav1- null MEFs. Furthermore, through the creation of a number of hybrid proteins between C. elegans and mammalian Cav1 we have showed that only the C-terminus of mammalian Cav1 could be exchanged with C. elegans Cav1 without affecting caveolae biogenesis. We demonstrated a fundamental role of two previously uncharacterised - vii - regions of mammalian Cav1 in caveolae formation. These regions of mammalian Cav1 are a leucine rich region in the C-terminus and an area of the N-terminus between residues 48 to 61. Through sequence analysis we have also highlighted two conserved tryptophan residues that when mutated slow caveolin exit from the Golgi complex, but do not inhibit caveolae formation. This has allowed us to propose a new model of caveolae formation. Caveolae have been described to take part in the endocytosis of certain ligands, but whether caveolae are involved in a dynamic constitutive endocytic pathway has been controversial for many years. The uptake of the bacterial toxin, cholera toxin, has been reported to be significantly inhibited in cells derived from Cav1 null mice, but other cell systems where Cav1 expression has been shown to be reduced showed no reduction in cholera toxin uptake. We therefore studied the uptake of cholera toxin in primary MEFs derived from WT and Cav1-null mice. We show that cholera toxin is internalised and transported to the Golgi complex in Cav1-null MEFs and that this occurs with the same dynamics as in WT MEFs. Through the use of a novel electron microscopy technique we have shown that cholera toxin in WT MEFs could be internalised by caveolae, although this was an infrequent process. Caveolae endocytosis could be stimulated by phosphorylation and the addition of lactosyl ceramide. Thus caveolae endocytosis is not a major dynamic constitutive pathway. In WT MEFs, cholera toxin can also be internalised by clathrin coated pits and a non-clathrin non-caveolae dependent pathway. We have shown that this pathway is present in Cav1-null MEFs and that it is sensitive to cholesterol, but not dependent on Arf6, dynamin or EPS 15. Moreover, we have described the morphology of the very first endocytic carriers of this pathway and found them to have a tubular or ring-shaped cisternal morphology with a diameter of 65nm. Finally we have shown that this non-clathrin non-caveolae pathway is a major endocytic pathway and that it is involved in both glycosylphosphatidylinositol (GPI) anchored proteins and fluid phase uptake.

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