Research objectives and content
Lipid translocation to the outer leaflet of the plasma membrane The objective is to determine how phospholipids reach their destination within the cell. In contrast with proteins the final localisation of a phospholipid molecule depends on it's intracellular transport between organelles and it's translocation (or 'flipping') between the two leaflets of the bilayer. The first stage of the project will characterise the translocation of natural lipid by the human multidrug resistance proteins MDR1 and MDR3. Methods already developed in the host laboratory will be used to permeabilize selectively one of the two plasma membrane domains of LLC PK1 cells grown on filters. Addition of liposomes of natural radiolabelled phospholipid in the presence of the appropriate transfer protein to the non-permeabilized surface will make it possible to assay translocation across the plasma membrane to the permeabalized side. This can be achieved by either the use of an appropriate bacterial phospholipase C in a kinetic assay or by using an appropriate transfer protein and a liposome acceptor. Further experiments, in primary cells from knock out mice, will determine the role of phospholipid transfer proteins in intracellular transport in intact cells.
Training content (objective, benefit and expected impact)
The objective is to do excellent science in one of the leading laboratories in the field of phospholipid traffic in polarised cells. The proposed project provides me with training in molecular biology, cell biology and lipid biochemistry which is necessary for the long-term goal of my research, namely to contribute to the elucidation of how lipids are transported and sorted on route to the plasma membrane and the sites and regulation of synthesis of phospholipids. I am very confident that I will be able to publish in high impact journals and to establish myself as a recognised research fellow in the field.
Links with industry / industrial relevance (22)