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

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

Phosphoinositides (PIPn) play key roles in the definition of membrane identity and signal transduction. The levels of these lipids must therefore be tightly regulated. Our preliminary data indicates diacylglycerol (DG) generated from PIPn by receptor-activated PLC generates a pool of phosphatidic acid (PA) that is selectively used for re-synthesis of PI and is segregated from DG and PA generated by PLD or bulk phospholipid synthesis. This 'PI cycle' is characterised by a distinct acyl chain composition (enriched in 'C38:4') and is more active than synthesis of PIs de novo. The molecular mechanisms that allow this cycle to operate are unknown.

Our overall aims are to test the hypotheses that:
1. Molecular species of PA are selected for PI re-synthesis based on their acyl chain compositions and their spatio-temporal localisations.
2. Inefficient selection of appropriate PAs for PI re-synthesis will significantly reduce signalling through PLC and PI3K pathways.

Our high-level objectives are:
1. To define how the acyl chain compositions of PI and PA influence their routes of metabolism.
2. To determine where in the cell PLC-dependent and PLD-dependent PA synthesis occur.
3. To define where in the cell basal and stimulated PI synthesis occur.
4. To define how phospholipid transfer proteins contribute to selective re-synthesis of different PI molecular species.
5. To understand how altering the acyl chain compositions of PIs influences acute and sustained PLC and PI3K signalling.

We propose a multi-disciplinary programme of work combining genetic manipulation by CRISPR/Cas9-directed mutagenesis in cultured cells, primary cells, chemical synthesis (to create isotopologues of PIs and PA with different acyl chain compositions), mass spectrometry (to track the levels and fate of relevant lipids), a range of fluorescence and EM imaging modalities (including TIRF, confocal, super-resolution and FIBS SEM) and in vitro assays with recombinant proteins.

The main beneficiaries:

1. The global academic community: This proposal will benefit the large UK and international community working on the cellular functions of an important class of phospholipids, the phosphoinositides. Further, this proposal seeks to establish a new paradigm for the role of acyl chain composition in the discrimination and sub-cellular segregation of lipids with identical head-groups, a concept with widespread repercussions in the fields of intracellular signalling and lipid metabolism.

2. Staff employed on the grant: The PDRA on the project will develop specific skills in lipidomics, microscopy and signalling research and transferable skills in executing and communicating a curiosity-led, but target-driven, project that will be relevant to several potential careers, including the pharmaceutical sector, scientific project management & leadership.

3. Biotech & pharmaceutical commercial partners: We hope to identify new therapeutic targets to treat diseases driven by the de-regulation of phosphoinositide metabolism e.g. cancer and bi-polar disorders. This will directly impact the competitiveness of our collaborators in the commercial sector and their wider organisations.

4. Patients & public health: Results from this project may indirectly impact heath care through the development of new strategies to create novel therapeutic reagents and a further level of understanding in the potential effects of fats in the diet.

5. Funders: The BBSRC/UKRI will deliver value for money and world-class research within their strategic remit of 'Bioscience for Health' and in line with the UK Industrial Strategy Grand Challenge of an Ageing Society

The main potential impacts

1. The creation of new knowledge and methodology for academic research; the specific role of acyl chains in phospholipids is under-researched and the communication of the results of this project (though publications, presentations and meetings) will have a significant impact on the accumulated academic knowledge in the area, encouraging other academic groups to explore this field.

2. New R&D investment from academics and industry to study phospholipid metabolism and to identify new therapeutic opportunities.

3. Enhancement of specific research capacity, through the creation and dissemination of new tools and reagents to study lipid metabolism by mass spectrometry and the training of the PDRA.

4. Our public engagement & communications activities will contribute to increasing public awareness and understanding of the science that underpins current biological research.