Photolysis of caged phosphatidic acid induces flagellar excision in Chlamydomonas.

Phosphatidic (PtdOH) acid formation is recognized as an important step in numerous signaling pathways in both plants and mammals. To study the role of this lipid in signaling pathways, it is of major interest to be able to increase the amount of this lipid directly. Therefore, "caged" PtdOH was synthesized, which releases the biologically active PtdOH upon exposure to UV. Analysis of the product revealed that two 2-nitrophenylethyl (NPE) caging groups were coupled to the phosphate headgroup of PtdOH. To measure the quantum efficiency of uncaging, a fluorimetric assay, based on the notion that the NPE cage is an efficient quencher of pyrene fluorescence, was developed. Consequently, after NPE-caged PtdOH and (N-pyrene)-PtdEtn had been mixed in DOPC vesicles, the extent of photolysis of caged PtdOH can be quantified by monitoring the increase in pyrene fluorescence. Using this assay, a quantum yield of 9.6% was determined for the uncaging reaction. The swimming green alga Chlamydomonas moewusii deflagellates upon addition of PtdOH. This response was used to study the release of PtdOH in vivo. Algae incubated with caged PtdOH only arrested swimming after exposure to UV, indicative of PtdOH release. This effect was not observed in the absence of the caged compound or when a control caged compound (caged acetic acid) was added. Fluorescein diacetate staining was used to show that the cells remained viable after UV exposure. The anticipated effect of PtdOH release is confirmed by phase contrast images of UV-exposed algae showing excision of flagella. Together, these results show that caged PtdOH can be used to efficiently increase PtdOH levels, demonstrating that it is a promising precursor for studying PtdOH-dependent signaling.

Release of gelatinase A (matrix metalloproteinase 2) induced by photolysis of caged phosphatidic acid in HT 1080 metastatic fibrosarcoma cells.

Phosphatidic acid (PA) is a putative novel messenger in signal transduction and membrane traffic. We have synthesized a photolyzable derivative of PA, termed caged PA (cPA), which may be utilized as a new tool in studies of PA-mediated cellular events. 1-(2-Nitrophenyl)diazoethane, synthesized from 2-nitroacetophenone, was reacted with dipalmitoyl-PA to yield a 1-(2-nitrophenyl)ethyl ester of PA. Photolysis of the compound by ultraviolet light resulted in the formation of phosphatidic acid. The structure of the compound and of its photolytic products was verified by NMR spectroscopy. The utility of cPA was examined in HT 1080 metastatic fibrosarcoma cells, in which the formation of PA by phospholipase D was implicated in laminin-induced release of gelatinase A (matrix metalloproteinase 2 (MMP-2)). The uptake of cPA by HT 1080 cells reached a plateau after 120 min of incubation. Ultraviolet illumination of cPA-loaded cells for 5 s resulted in photolysis of 1.8% of the cell-incorporated cPA. The photolysis of cPA caused a 2-fold elevation in the release of MMP-2 to the medium, whereas nonphotolyzed cPA caused no change in MMP-2 release. Moreover, the effect of cPA photolysis was significantly higher than that obtained with extracellularly introduced PA. Thus, the effect of laminin on MMP-2 secretion can be mimicked by photolysis of cPA, suggesting a pivotal role for phospholipase D in laminin-induced cancer cell invasiveness and metastasis. These results indicate that cPA could serve as a unique tool for studying the cellular roles of PA.