| Abstract |
Polyphophoinositol lipids are minor components (about 1%) of the phospholipids that make up mammalian cell membranes, but they have a physiological and pathological importance way out of proportion to their quantity. There are seven of them in all, each with at least one (most with more than one) physiological action to regulate how cells function - and these actions take place in almost every conceivable location within a cell. When these processes are not appropriately regulated, pathologies can occur: polyphosphoinositol lipids are implicated in, for example, cancer, inflammation, neurological disorders and cardiovascular disease. One subject of this application is the most recently discovered polyphosphoinositol lipid, phosphatidylinositol 5-phophate (PI5P), which is present in many locations of the cell and which has, by a sigificant body of circumstantial evidence, been implicated in the way in which cells respond to stress (uv light, heat, oxidising radicals), responses which self-evidently must act as part of their defence against environmentally-induced disease. The levels of PI5P (and thus its functions, because its actions are regulated primarily by how much is present at a given location) are regulated by a family of enzymes: the PI5P 4-kinases (PI5P4Ks). Humans have three PI5P4K genes (and thus three different PI5P4Ks), and one of these, PI5P4K gamma, has been ignored almost completely so far (except by us). Yet we have found that it has a striking expression pattern, being found at very high levels in precise and specific locations in epithelial cells in the kidney, and also in neurons within particular parts of the brain and the spinal cord. Inside the cells that express PI5P4K gamma it appears to be attached to membrane vesicles within the cytoplasm, but what these vesicles are, and what they are doing is unclear (and clarification is one of the major aims of this study). To further improve on our paucity of understanding of PI5P4K gamma we plan to apply many of the cutting edge tools of molecular cell biology to its study. We are in the final stages of generating a mouse lacking the PI5P4K gamma gene, and how this mouse functions in its kidneys and nervous system may give us new insights. Even if they do not, the availability of tissues and embryonic stem cells from these mice will give us powerful new tools to understand exactly where this protein is functioning and what it is doing. Our collaborators in this aspect will also investigate how removal of the PI5P4K gamma in zebra fish embryos alters the form and function of kidneys and neurons in this organism. In parallel we will use cell lines derived from the same part of the kidney that expresses PI5P4K gamma highly and also we will make full use of a cell line called Nalm-6 cells, which is derived from a human B cell lymphoma. The unique properties of Nalm-6 cells (it is extremely easy to manipulate genes in them) will enable us to 'tag', remove, and change PI5P4K gamma in order to find out who it talks to, and where, and what it says. |
