The pleiotropic cellular function of phosphatidic acid in the secretory pathway deciphered by a novel PA-toolbox – PA-Box

Techniques that enable the manipulation of the cellular levels of specific lipids are of great importance in the exploration of their cellular function, but the tools currently available for PA analysis are not optimal due to the lack of subcellular specificity and to inadequate timing of activity. Indeed, good pharmacological agent to modulate the activity of PA producing and metabolizing enzymes are currently lacking, at the exception of PLD inhibitors. Second, at moment the molecular analysis of lipids is largely limited to gene knock-out (KO), knock-down (KD) or knock-in (KI) techniques, as well as to gene overexpression, which is associated to several caveats, such as compensatory adaptive changes since these manipulations occur in the range of hours to days and often in the entire organism.

To circumvent these shortcomings we now propose to engineer molecular tools that will allow expression of PA-metabolizing enzymes in specific subcellular compartments by using expression vectors encoding specific targeting peptides and this in a timely controlled manner. At first, we will focus on ER, mitochondria, Golgi, secretory granule, endosomes, and plasma membrane targeting, intracellular compartments containing detectable levels of PA (unpublished observations and see preliminary data). Hence, we propose to generate tools that will allow local synthesis of PA by PLDs, DAGKs, LPAATs or its conversion into other lipids by PA-phosphatases, CDP-DAG synthetases, PLAs. Of note we have previously engineered a rapamycin sensitive PI4P-5kinase that allowed us to efficiently detect changes in PIP2 levels in chromaffin cells minutes after rapamycin addition.

Another approach to manipulate PA levels is the direct addition of membrane permeable PA analogs. Indeed, our preliminary data indicate that provision of commercial PA through the extracellular medium allows rescue defects on secretion due to PLD inhibition. Relying on the know-how of Partner 2 in fluorescence, organophosphorus chemistry, bioconjugate chemistry, and click chemistry (6 recent ANR grants in the field) PA analogues bearing a caged phosphate head group that can be uncaged after UV illumination will be synthesized in vitro. Taking advantage of a versatile grafting function introduced on the PA analogues, we will also generate PA analogues labeled with a fluorophore or inducible for selective light-induced cross-linking to study timely the physical link between PA and interactors into subcellular membranes through the formation of covalent bonds.

Having established experimental conditions where addition of exogenous PA can reach the inner leaflet of the plasma membrane, we were able to show that individual PA species and among them unsaturated forms are playing specific roles in distinct steps along the secretion of stress hormones. For instance, PA produced by PLD1 contributed with chromogranin A to the biogenesis of secretory vesicles at the TGN level and to their transport towards the cell periphery. More specifically, mono-unsaturated PA regulate the docking of vesicles at exocytotic sites while poly-unsaturated forms of PA control the dynamics of the fusion pore. The synthesis of PA at the plasma membrane requires the activation of PLD1 by a combination of kinases and GTPases, including Arf6. Interestingly, we have shown that the dissociation of the V1 and V0 segments of the V-ATPase on secretory granules is required for the interaction of the V0a subunit with the ARNO, an Arf6 activator.

We have generated inducible manipulators of PA metabolism by focusing on three enzymes involved in PA synthesis, namely PLD1, DGKk, and the B domain of RIBEYE, and on LIPIN1 and PLA1 for the hydrolysis of PA. We have obtained rapamycin and light-induced versions for each of these enzymes that were developed in versions with or without fluorescent tag that can be used in combination with addressing vectors to specific subcellular compartments. Due to the fast recruitment of the enzymes to specific membrane subcompartments, the light-induced tools called opto-metabo-PA were used to probe the contribution of PA found either in the membranes of the TGN, the secretory granules, the mitochondria, and the plasma membrane to the distinct steps of exocytosis (manuscript in preparation).

We have developed a new synthetic pathway of PA bearing a discrete function allowing the grafting of fluorescent probes in cellulo or in vivo through click chemistry in order to let the structure of the modified PA as close as possible of the original PA. The mono- and polyunsaturated synthetic PA obtained were extensively characterized and reproduced the cellular activity of their natural counterparts during neurosecretion. The dynamics of these synthetic molecules was assessed in live cells and revealed that they accumulate in specific sub-compartments depending on their fatty acyl chain composition. Furthermore, using a fishing strategy in live cells, we were able to identify several hundreds of PA interactants some of which were known, but others new (manuscript in preparation). Interestingly, most of the interactant were specific for the species of PA and depend on the cell status (see illustration). These results open new avenue to better understand the multiple function of PA in neurosecretion and to phospholipid at large in the biology of cells.

We believe that this study will bring major conceptual advances in understanding various membrane traffic events, since PA has been suggested to be a major contributor in key cellular functions relevant beyond the field of neurosecretion, with implication in cell division and migration, immunological synapse formation or phagocytosis of pathogens and even host-pathogens interaction. Finally, the novel synthetic PA developed in this proposal and the chemistry to obtain them has been patented (WO2023180457), with discussion with private compagnies on-going and the potential for a start-up creation focused on the development of medically relevant phospholipid sensors for clinical diagnostics.

Despite increasing evidence that lipids play key cellular functions and are involved in an increasing number of human diseases, little information is available on their exact function. This is especially the case for phosphatidic acid (PA) that has been shown to be involved in many normal and pathological cellular functions. PA lies in the middle of three-enzymatic pathways, which makes it an ideal signaling integrator. Based on our latest lipidomic approach, we postulate that intracellular compartments have a specific PA composition, but we still do not understand the respective roles of PA species. We now propose to develop a complete PA toolbox to acutely play with PA levels in subcellular compartments, to follow its membrane dynamics and identify partners with a specific focus on secretory vesicle biogenesis and exocytosis, the two faces of neuro(endocrine) secretion for which PA involvement begins to be firmly established but still largely non-understood.