Periodic Reporting for period 4 - ASYMMEM (Lipid asymmetry: a cellular battery?)
Reporting period: 2022-07-01 to 2023-12-31
The project ASYMMEM aims at elucidating the function of lipid asymmetry by combining chemical, biochemical and cell biological approaches. The core hypothesis of the project, that lipid asymmetry serves as a store of potential energy, necessitates that cellular function is associated with lipid transbilayer movement. Understanding better how asymmetric cellular membranes function in cell biological processes would constitute a breakthrough in membrane biology.
During the course of the project, a number of key developments relating to lipid and membrane biology were made. We developed approaches to perform quantitative lipid biology in living cells, study lipid driven cell signaling and established a platform for monitoring lipid transport in living cells. In our cell biological work, we found compelling evidence that lipid asymmetry is linked to both energy metabolism and lipid transport. In a second line of research, we developed asymmetric model membrane systems that can be tuned with light and link changes of lipid asymmetry to membrane phase behavior. The methodological developments driven by this proposal are the aspects most likely to be beneficial for society at large. Lipid mislocalisation and ectopic accumulation in cells and tissues is a hallmark of many hereditary and lifestyle diseases and better ways for monitoring lipids are in high demand, both for drug development and diagnostic purposes.
During the course of the project, a number of key developments relating to lipid and membrane biology were made. We developed approaches to perform quantitative lipid biology in living cells, study lipid driven cell signaling and established a platform for monitoring lipid transport in living cells. In our cell biological work, we found compelling evidence that lipid asymmetry is linked to both energy metabolism and lipid transport. In a second line of research, we developed asymmetric model membrane systems that can be tuned with light and link changes of lipid asymmetry to membrane phase behavior. The methodological developments driven by this proposal are the aspects most likely to be beneficial for society at large. Lipid mislocalisation and ectopic accumulation in cells and tissues is a hallmark of many hereditary and lifestyle diseases and better ways for monitoring lipids are in high demand, both for drug development and diagnostic purposes.
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.
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.
Taken together, we have significantly progressed beyond the state of the art: Lipid transbilayer movement of native lipids is now measurable and our photochemical probes allow to alter lipid levels on millisecond timescales, a huge improvement over less precise and temporally not well-defined pharmacological interventions. We have generated a platform of CRISPR-knockout lines of lipid translocases and scramblases that will serve as valuable resource for the membrane biology community in the future. We developed a technology to monitor lipid transport in cells with unprecedented precision. Finally, we created a model membrane system that allows to photochemically trigger changes in the asymmetric lipid composition, and thus mimic the physiological situation. These tools have enabled us to demonstrate links between lipid asymmetry and energy metabolism as well as lipid asymmetry and lateral membrane organization, both of which were not known before.