EUROCORES EuroMEMBRANE_OXPL-Single molecule tracking of oxidized phospholipids in the live cell plasma membrane

Oxidized phospholipids (oxPL) are now known to be involved in several major pathological conditions, such as atherosclerosis, inflammation, cancer, type 2 diabetes, and Alzheimer`s disease. However, a coherent overall view of the causalities and mechanisms is lacking, mainly because of insufficient understanding of the cellular as well as molecular mechanisms. The OXPL consortium represents an integrated interdisciplinary approach of European research laboratories already active in this field, together with additional skills brought into the project by groups representing state-of-the-art expertise in method and instrumentation development for the characterization of biomolecules, their interactions and localization in cells. The project will pave the way to the development of improved diagnostics, therapies and preventive measures to combat the above diseases, and will take European research to the leading edge in this emerging and important research field. The OXPL consortium aims at unravelling the molecular level view of the metabolism, roles and mechanisms of action of oxPL in cells, both under physiological conditions and in diseased states. Accordingly, we assembled the leading European research groups active in research on chemical biology, biochemistry, cell biology, biophysics, and molecular pathology of oxPL, as well as lipidomics, mass spectrometry, single molecule measurements, high resolution imaging, fluorescence spectroscopy, NMR, ESR, and computer simulations. Our consortium is strongly interdisciplinary, from medical investigators to theoretical physicists. A major part of this proposal will be devoted to the development of new assays and methodologies, and to novel applications of state-of-the-art technologies. Throughout the project a number of new oxPL analogues need to be synthesized and characterized, to aid monitoring their behaviour in cells and to be able to isolate their adducts with other biomolecules. Because of the dimensions involved, high resolution imaging techniques need to be adapted to visualize these lipids in cells. Of particular concern is that traditional fluorescence microscopy readily generates reactive oxygen species, so approaches to circumvent such issues are mandatory. It is particularly important to introduce these problems to physicists and biophysicists with a track record in the instrumentation and method development. Likewise, an active dialogue with theoretical physicists allows the use of computer simulations for the prediction of properties, which can then be analyzed in model systems as well as cells. In our subproject we will follow the motion of individual fluorescently labeled oxidized phospholipid molecules in the plasma membrane of living cells. Diffusion constants will be determined for different molecular species in different cell lines. In addition, we will address deviations from Brownian motion to understand the various forces acting on the diffusing species at a nanoscopic length scale; in particular, we will characterize in detail sites of immobilization, which were identified in preliminary experiments. For this, we will employ novel variants of ultra- sensitive fluorescence microscopy, based on light sheet excitation and combination with photoactivation microscopy. Finally, modification of plasma membrane structure by the addition of the unlabeled oxPL will be measured by single molecule tracking of trace amounts of the fluorescent probe.

Phospholipids containing polyunsaturated fatty acids are highly prone to modification by reactive oxygen species, thereby generating a plethora of biologically active oxidized phospholipids (oxPL). oxPL are now known to be involved in major pathophysiological conditions, such as atherosclerosis, inflammation, cancer, type 2 diabetes, and Alzheimers disease. However, a coherent view of the causalities and underlying cellular and molecular mechanisms was lacking. For this we started the O XPL consortium, which represented an integrated interdisciplinary approach of European research laboratories already active in this field, together with additional skills brought into the project by groups representing state-of-the-art expertise in method- and instrumentation-development. In our subproject we investigated via ultrasensitive fluorescence microscopy the behavior of oxPL embedded in synthetic membranes and the cellular plasma membrane. The study can be grouped into two main questions: iii) How do oxPL move in membranes? For this question we used the fluorescently labelled oxPL PGPE-Alexa647, where a hydrophilic carboxyl group replaces one acyl chain of the phospholipid; the compound was synthesized by our collaboration partner Albin Hermetter, Graz University of Technology. In phase-separated model membranes we found out that PGPE has access to virtually all membrane phases, ranging from fluid phases to gel phases. This was not surprising, as PGPE has only a single hydrophobic chain and thus requires less free area within a membrane. Consistently, we observed much faster diffusion dynamics compared to standard phospholipids, again mainly due to PGPEs smaller area. Live cell data also showed a higher mobility of oxPL compared to conventional phospholipids. A closer look onto the PGPE diffusion behavior, however, revealed interesting interactions with cholesterol: The higher the cholesterol content of the membrane, the better PGPE was integrated in the lipid bilayer and approached the mobility of a conventional phospholipid. Molecular Dynamics simulation carried out in collaboration with Thomas Stockner (Medical University of Vienna) revealed that PGPE gets shifted significantly into the membrane core, where the interaction energy with other lipids increases. iv) How does the presence of oxPL influence general properties of membranes? Given the spectrum of effects of oxPL on cell signaling, it appeared likely that some of these effects are exerted directly at the plasma membrane. Indeed, using advanced fluorescence microscopy techniques we found out that protein-enriched plasma membrane nanodomains are dissolved by the presence of oxPL. To our surprise, however, it turned out that this effect was not direct, but initiated indirectly by activation of an enzyme which modifies the lipid composition of the plasma membrane.