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CELL MEMBRANE
Research InterestsResearch in the Cell Membrane Group centres on investigations into the function of eukaryotic cell membranes, particularly those of trypanosomes. The experimental approach is interdisciplinary and uses the techniques of protein chemistry, bioenergetics, immunology, enzymology, electron and fluorescence microscopy and molecular biology. Work is focused currently on six project areas: 1. Signalling systems responsible for regulating the expression and switching of variant surface glycoproteins (VSGs). IgM and IgG anti-VSG antibodies have been found to stimulate an endocytotic / exocytotic cycle in trypanosomes. A new continuous assay for endocytosis / exocytosis developed in our lab has allowed investigations of the timing of events in the process, the characterization of the site of action of inhibitors and the identification of cellular signals generated by the cycle that may act on gene switching. The goal of this research is to determine how VSG gene switching is regulated. 2. Mechanisms that establish and regulate membrane potentials and cation gradients. Work in our lab has established that the mitochondrion of bloodstream forms of the African trypanosomes is energized by a novel system: in the cytoplasm acetyl phosphate is generated by a new pathway for the metabolism of glucose and, following its translocation to the mitochondrial matrix, is used at substrate level to generate ATP, which then energizes the H+ - pumping F1/F0-ATPase. Current work involves studying the enzymes of this new pathway, investigating the mechanism of the mitochondrial transport of acetyl phosphate and defining the changes that occur in the pathway and in mitochondrial energization during cellular differentiation between stages in the life cycle of these organisms. In addition the effect of tumor necrosis factor on the energy metabolism of trypanosomes is a current interest. 3. Invariant stage-specific surface glycoproteins. Although the predominant surface protein of bloodstream form trypanosomes is the phosphatidylinositol-anchored VSG (107 copies / cell), a number of other stage specific surface glycoproteins are present at more usual levels (104 - 105 copies / cell) and are of unknown function. We have purified and characterized three of these proteins, one of which has been shown to have a surface distribution suggesting a role in capping and in endocytosis / exocytosis. The genes for these proteins have either been cloned and sequenced (ISG70) or are currently being cloned (ISG100) or sequenced (ISG64) in order to gain further insights into their function. From a practical standpoint these proteins are being used to develop a new diagnostic test for African trypanosomiasis and antibodies against them are currently being used to study other aspects of their function. 4. Life cycle and distribution of Trypanosoma theileri in theileri and Trypanosoma granulosum in Ireland. Recently we have observed the presence of T. theileri in Ireland in association with a febrile illness in cattle. In addition we have found a very high infection rate within the Irish population of the European Eel with T. granulosum. Currently, we are assessing the incidence of distribution of this parasitic protozoon in Ireland, using the appearance of antibodies directed against invariant surface glycoproteins. It appears that the vector of T. theilleri is the horse-fly and that of T. granulosum is the leech. However, the life cycle of this protozoon is unknown and is currently being investigated in the laboratory with particular attention to the biochemical mechanism of differentiation. An additional interest is whether these trypanosomes undergo classical antigenic variation. 5. Function and structure of tubulin-based cytoskeletal systems that interact with cell membranes. Microtubule associated proteins (MAPS) are known to regulate the assembly / disassembly of microtubules and their attachment / detachment from the plasma membrane. Tau is one of these MAPS and occurs in six common forms in man that result from differential splicing of the primary transcript of a single gene. We are investigating the specific functions of each tau transcript. This work has attracted commercial support and several projects in this area are currently in progress. 6. The mechanism of intracellular pH regulation in trypanosomatids and it's relation to leishmanial and trypanosomal virulence. We have previously demonstrated that bloodstream forms of Trypanosoma brucei are not capable of regulating their intracellular H+ concentration and, consequently, their cytosolic pH follows a linear relationship to that found in their external environment. These cells rely upon their host's ability to maintain a constant pH in their extracellular compartment. However, following transformation into the forms found in the insect vector's gut, the parasite maintains a constant cytosolic pH that is independent of their extracellular pH over a broad range. The mechanism does not appear to depend upon an H+/Na+ exchanger but may be sensitive to inhibitors of the general anion channel. In Leishmania spp. the intra-endosomal forms of the parasite maintain their cytosolic pH via an H+-pumping ATPase but nothing is known about pH regulation in the invasive promastigote form of the parasite which leads an extracellular existence. Research StaffThomas Dowling, B.A.(Mod) Robert O'Byrne (Jointly with Dr Derek Nolan) Financial SupportEU Science and Technology for Development Programme, EU International Scientific Co-operation Programme, World Health Organization - World Bank, American Biogenetic Sciences Inc. Major CollaboratorsDepartment of Biochemistry, University of Cambridge (Drs. Gerry Smith & James Metcalfe, Dr. Rick Martin) Publications by the Cell Membrane Group |
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Contact: bbutler@tcd.ie. |
Last updated: Nov 24 2011.
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