Real-time visualization of phosphatidic acid in live cells using novel sensors based on fret

Laburpena

Inglés: This thesis project focused on the development of fluorescent indicators able to report changes in the levels of phosphatidic acid (PA) in live cells. PA is the simplest naturally occurring diacylglycerophospholipid and exists in cell membranes of all living organisms. It is at the center of the metabolic routes of structural, signaling and energy storage lipids. In addition, PA itself is a structural and signaling lipid. Its small cone shape provides flexibility and negative curvature to lipid bilayers, and its signaling functions are granted by its remarkable phosphomonoester headgroup, which distinguishes PA from other lipid mediators and provides a special environment for docking of PA binding proteins. PA has attracted considerable attention because of its role as a lipid mediator, and for the large number of effectors, which include proteins involved in cytoskeleton rearrangement, vesicle trafficking, cell growth, spreading, proliferation, and survival. Traditionally, PA levels have been measured with thin-layer chromatography and liquid chromatography coupled to mass spectrometry. Recent advances in the latter technique have allowed detailed characterization of the content and the acyl chain composition of PA in cells and tissues, but at the cost of losing its intracellular spatial distribution. To reveal PA production at the cellular and subcellular levels, fluorescent indicators featuring PA binding domains tagged to GFP have been reported. These chimeras translocate to the plasma membrane upon PA production and provide sufficient specificity. However, such probes often only provide qualitative information. In addition, they do not allow imaging PA changes in particular membrane subdomains or organelles. To overcome these limitations, we have constructed genetically-encoded Förster Resonance Energy Transfer (FRET) biosensors for PA detection. We developed constructs using the PA binding domains of two PA binding proteins, neurogranin and Spo20, and the fluorescent proteins ECFP and several variants of Venus. In the chimeras that were responsive to PA, we found an inverse relation between PA levels and the FRET efficiency of the sensor. The construct with the larger change was further optimized to target it to the plasma membrane and the outer mitochondrial membrane to report PA fluctuations therein, in live mammalian cells. The studies carried out with the plasma membrane targeted sensor indicated that both basal PA levels and phospholipase D activity varied in different cell types. In addition, we found that stimuli that activate PA phosphatases, leading to lower PA levels, increased lamellipodia and filopodia formation. Lower PA levels were observed at the leading edge than at the trailing edge of migrating HeLa cells. In cell lines derived from Schwann cells and oligodendrocytes, the membrane processes involved in myelination showed lower PA levels than the cell body. The sensor targeted to the outer mitochondrial membrane allowed the measurement of PA fluctuations for the first time in this membrane. It revealed that phospholipase D and diacylglycerol kinase activities contribute to mitochondrial PA pools. Finally, serum starvation decreased PA levels in HeLa cells, both in the outer mitochondrial membrane and the plasma membrane. In summary, the FRET probes for PA reported here allow imaging the spatio-temporal dynamics of the PA pools in the plasma membrane and the outer mitochondrial membrane. The probes responded to both increases and decreases of PA, and displayed a wide dynamic range. We also reported PA fluctuations during cell migration, mitosis, membrane process extension or upon serum deprivation. These new biosensors, and modified versions targeted to other membranes, will be valuable tools to expand our knowledge of the role of PA in intracellular signaling and to study the subcellular heterogeneity of PA formation in situ.