Characterisation of PLD activity in real-time

Abstract

PLD catalyses hydrolysis of phosphatidylcholine (PtdCho) to produce phosphatidic acid (PtdOH) and choline. PtdOH is a second messenger responsible for a multitude of cell processes, ranging from cytoskeletal rearrangement to cell proliferation. Antigenic stimulation of RBL-2H3 mast cells and growth factor stimulation of endothelial HeLa cells results in PLD-dependent exocytosis and endocytosis, respectively. A novel fluorescent PtdCho (fPtdCho) was used to label both cell lines and Bligh-Dyer lipid extraction of fPtdCho-labelled RBL-2H3 cells showed the lipid was intact post-labelling. fPtdCho co-localised up to 50% with the lysosomal marker LysoTracker Red in RBL- 2H3 cells, and was not secreted in response to antigenic stimulation as recorded using real-time confocal microscopy. Primary alcohol treatment of fPtdCho-labelled RBL- 2H3 cells altered fPtdCho-labelling to diffuse from punctate distribution, suggesting PLD-generated PtdOH is responsible for retention of punctate fPtdCho staining. PLD isoforms 1b and 2a were labelled with Cherry (a red fluorescent protein) and transiently expressed in fPtdCho-labelled HeLa cells. Localisation was assessed using FRET by FRAP technology in live cells and showed that substrate and lipase were in close proximity. These findings will facilitate future development of a live real-time in vivo PLD assay. Furthermore, localisation of PLD and its activator Rac1 was assessed at rest and in EGF-stimulated HeLa cells in real-time. This showed co-localisation between PLD and Rac1 following stimulation. The fluorescent PtdCho was also used to develop a novel real-time in vitro PLD assay, monitoring fPtdCho metabolism at two second intervals. This in vitro assay is more sensitive than traditional end-point assays and will help clarify the relative rate of PLD activation in response to small G-protein activators and other co-factors in real-time