Synthesis and application of novel caged-phospholipids for high resolution optical manipulation of lipid signaling events in single living cells

We want to study the significance of phospholipids as second messengers in living cells. To this end we aim at synthesizing novel photosensitive precursors of these phospholipid molecules, loading them into cells and generate the bioactive molecules after irradiation with UV or near-IR light. This will allow us to study the effect of local accumulation of the phospholipids with high specificity, sensitivity and spatial resolution in living cells with minimal perturbation. Phospholipids play an essential role in spatially confined signalling evens that regulate cell shape and cell migration. Instead of the well-known cleavage of PtdIns(4,5)P2 into the "classical" second messengers IP3 and DAG, it has been shown that local levels of PtdIns(4,5)P2 rise in certain signalling events which is essential for membrane ruffle formation. These intriguing new insights have led to the idea that PtdIns(4,5)P2 is a second messenger in its own right. Similarly, the product of phospholipase D: phosphatidic acid (or PtdOH) is emerging as an intracellular second messenger. In order to study the role of the lipid second messengers at the single cell level, it is of major interest to have direct control over local phospholipid concentrations. An elegant way to increase the concentration of specific phospholipid second messengers with high spatial and temporal resolution is by using photosensitive ("caged") precursors. Upon exposure to UV, photolysis takes place, effectively releasing the biologically active second messenger. We propose to synthesize novel caged phospholipid second messengers which can be used to study effects of the local generation of specific phospholipids in single living cells. Special attention will be given to novel photosensitive protecting groups which are compatible with photolysis upon two-photon excitation. This will allow the controlled release in truly three-dimensionally restricted spots, providing a unique tool to study spatially localized properties of phospholipid signalling in general and the role of PtdIns(4,5)P2 and PtdOH in regulating the cell shape in particular. The new class of caged second messengers will be used for studying the relevance of compartmentalized lipid signalling i) for inducing membrane ruffling and actin polymerization in HeLa and NIH3T3 cells, ii) for cell morphogenesis during (plant) root hair deformation and curling in legumes, and iii) for recruitment of signalling molecules with lipid binding domains (e.g. ARF, Cdc-42, PLD2, PI4P 5-kinase) to specific areas in the cell (e.g. the plasmamembrane and the cell nucleus). It is expected that the new caged compounds will be highly useful for addressing the second messenger function of phospholipid molecules as PtdIns(4,5)P2 and PtdOH in vivo, in particular if, where and to what extent PtdIns(4,5)P2 and PtdOH by themselves, are able to recruit and activate downstream effector-proteins at various specific locations in the living cell.