Over the course of the project, we have addressed the function of lipid asymmetry in a number of complementary approaches, which are detailed below. We developed chemical tools for photochemically modulating lipid levels in individual membranes of living cells, including important lipid second messengers (Wagner et al, Angew. Chem. Int. Ed. 2018, Wagner et al. Chem. Eur J. 2019). Using a chemical biology approach, based on our ability to photochemically modulate lipid levels, we developed a methodology that now enables us to quantitatively measure the extent of lipid transbilayer movement in the plasma membrane of living cells (Schuhmacher et al. PNAS 2020). These technological developments have led to a number of unexpected findings. Specifically, we found that lipid transbilayer movement has an unexpected influence on cell signaling, in particular on the action of diacylglycerols, important lipid second messengers. Surprisingly, highly unsaturated species featuring many double bonds in their side chains greatly differ from more saturated species in their capacity to recruit cytosolic proteins to the plasma membrane. We expanded our methodology for quantitative lipid biology in living cells to include single cell data in collaboration with the laboratory of Christoph Zechner (Gonzales, Schumacher et al. 2023, Biophys. J.) and initiated collaborations with other researchers now incorporate our approaches to study cell signalling in their work (Stinchcombe et al., Science 2023).
In a second line of research, we established an asymmetric model membrane system that allows for rapid changes of transbilayer lipid distribution. To this end, we have generated a number of leaflet-specific, photo-caged phospholipid derivatives that can be incorporated into the outer leaflet of giant unilamellar vesicles, an important model membrane system. This method now allows us to photochemically modulate lipid levels in a controlled model membrane environment. We have developed a protocol to anchor these vesicles in microfluidic chambers, thus enabling straightforward analysis by fluorescence microscopy and the ability to rapidly change conditions in the surrounding medium (manuscript in preparation, expected date for preprint Q3 2024).
Thirdly, we generated a series of knockout cell lines that each lack one of the lipid translocases (enzymes that move lipids against a concentration gradient across the lipid bilayer) and scramblases (enzymes that equilibrate the lipid concentration gradient across the bilayer). We used the generated lipid translocase knock-out cell lines in a broad array of lipidomic, transcriptomic and proteomic screens. The obtained hits from all screening approaches and subsequent validation suggest a strong link between lipid asymmetry and energy metabolism. (Manuscript in preparation, expected date for preprint Q2 2024). To directly measure assess lipid internalization changes in translocase knockout lines we developed a new methodological approach for monitoring lipid transport in the retrograde direction. We genrated a large library of chemical probes that now allow us to monitor lipid transport in cells and conclude upon the mechanisms that maintain organelle membrane identity (manuscript in preparation, expected date for preprint Q2 2024). This technology has now developed into one of the main research lines of the laboratory and we will continue to work in this direction
A significant portion of the work carried out during the funding period of the ERC starting grant project ASYMMEM has already been published in prestigious journals. We anticipate that the key results of the second half of the funding period (detailed above in results sections 1-3) will be published over the course of the year 2024. In addition to publications in scientific journals, the PI and all members of the team have attended key meetings in the field of membrane biology (e.g. various Gordon conferences, FEBS meetings) and presented the obtained results. The presentations were very well received as evidenced by the number of poster and presentation awards by members of the team. Publications and conference participations were promoted on social media (X, Mastodon and Bsky) by the PI, team members and the media office of the host institution. To present our results to the general public, team members participated in the “Long night of Science” in Dresden, an occasion when all research institutions invite the general public into the laboratories and inform on their projects through lectures, workshops and experimental demonstrations.
