Molecular species variants of phospholipids: a code through which cells distinguish phosphoinositide signals and their synthetic intermediates

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Phospholipids are components of cellular membranes that keep contents partitioned within and between cells. Phosphoinositides (PIPn) are a specific family of phospholipids whose study has revolutionised the way in which we now think about these molecules; they do not merely represent a biophysical barrier, but also adopt a range of important regulatory roles in which their presence at a particular membrane location triggers the activity of proteins with a specific job to perform. These jobs include communicating the presence of hormones and growth factors at the cell surface and co-ordinating appropriate cellular responses, such as growth and secretion (called the 'PLC' and 'PI3K' signalling pathways). The levels of PIPn must therefore be tightly and independently regulated.One of the major problems the cell needs to solve is how to segregate common biosynthetic intermediates used in the synthesis of PIPn from those that are also used in the synthesis of other lipids. Our preliminary data suggests one of the ways they might do this is to differentiate PIPn and molecules derived from them on the basis of their acyl chain composition. Phospholipids typically have two acyl chains; these are the hydrophobic parts of the molecule that anchor a phospholipid in a biological membrane. Most types of phospholipids comprise a wide variety of different acyl chains but for the most part the function of this diversity is unknown. PIPn however, particularly in mammalian cells, are relatively molecularly homogeneous, with a characteristic composition of one stearoyl chain (abbreviated C18:0) and one arachidonoyl chain (C20:4). When the PLC signalling pathway is activated by hormones at the plasma membrane (the outer membrane that surrounds a cell), PIPn are converted into a molecule called diacylglycerol and then into phosphatidic acid (PA). We hypothesise that phospholipid 'transfer proteins' selectively extract this PA from the plasma membrane on the basis of its C18:0/C20:4 composition and then deliver it to a neighbouring region of a separate membrane system (a 'PM/ER contact site') which is designed for PIPn resynthesis. These transfer proteins then transport the newly made PIPn back into the plasma membrane, forming a 'PI cycle'. We hypothesise that an efficient PI cycle is necessary to preserve the levels of PIPn at the plasma membrane and sustain chronic signalling. We plan to test these hypotheses by chemically synthesising PIPn with different acyl chains and then tracking what happens to them when they are delivered to cells by a technique called mass spectrometry. Mass spectrometry allows us to distinguish between the molecules we have delivered to the cells and the ones that are already there (on the basis of labelling them with heavy isotopes). We also plan to use several cutting-edge types of microscopy to track fluorescently tagged PAs and PIPn and the enzymes which make them to see if we can distinguish areas in the cell where PLC-stimulated phosphoinositide synthesis takes place. We will also directly test the hypothesis that selected transfer proteins can distinguish the acyl chain composition of PAs and PIPn. Finally, we will attempt to interfere with the cell's ability to enrich its PIPn with C18:0/C20:4 to see if this alters the efficiency of a PI cycle and chronic signalling through PLC and PI3K pathways.The results of this project will contribute greatly to our understanding of the specific function of acyl chains in PIPn and highlight a potentially widespread role for acyl chains as molecular signatures to distinguish closely related lipid pools. Our results may also uncover a potentially novel point in the chronic regulation of PLC and PI3K signalling pathways, with therapeutic implications. Overactive PLC signalling has been hypothesised as a cause of mania (based on the treatment of bipolar disorders with lithium) and there is overwhelming evidence that high levels of PI3K signalling drive many cancers