Genetically encoded probes for phosphatidic acid

During the last two decades, it has become evident that cellular lipids not only form bilayers to separate compartments, but also participate in vesicular trafficking and signal transduction. Phosphoinositides have received major attention at this respect, but other phospholipids such as phosphatidic acid (PA) are quickly emerging as signaling molecules. The spatiotemporal distribution of cellular PA appears tightly regulated by localized synthesis and quick degradation. Although it has been reported that PA plays key roles in key cellular functions, it remains unknown when and where PA is produced in the living cells. Hence we have decided to characterize biosensors that specifically bind to PA through relatively compact binding domains. After protein expression and purification, their interaction with PA was characterized using PIP strips and liposomes assays. The results confirm that they have high specificity for PA, but we also found that chain length and saturation affected interaction with membranes. Finally we used these probes to visualize the dynamic of PA in various cell models and found that the various probes had also different affinity for PA in cells. In conclusion, the use of a large choice of probes may be required to adequately follow the complex dynamic of PA in various cell types, subcellular compartments and cellular processes.