Periodic Reporting for period 1 - nano-Asymmetry (Nanoscale analysis of symmetry and molecular membrane topography)
Reporting period: 2023-01-01 to 2024-12-31
The plasma membrane is the structure separating interior of the cell from its exterior. Moreover, it is a hub for almost all signalling events for cells. Therefore, maintaining the molecular structure of the plasma membrane is of outermost importance for cell survival and homeostasis. The plasma membrane is composed of individual lipids assembled to a lipid bilayer, consisting of outer and inner leaflet, presenting their polar headgroups to the surrounding fluid while the hydrophobic acyl chains of each monolayer are facing each other to build a hydrophobic core, impermeable for polar or charged molecules. Besides its function as barrier, the plasma membrane serves as scaffold for transmembrane proteins, which make up around 30% of all human coded proteins and are major drug targets.
Lipids are a diverse class of molecules consisting of thousands of different molecular species which are distributed heterogeneously in the plasma membrane and between the inner and outer leaflet. For example, sphingomyelin is almost exclusively present in the outer leaflet whereas phosphatidylserine and phosphatidylethanolamine are mainly part of the inner leaflet . This transbilayer lipid asymmetry is maintained by a set of enzymes called scramblases, flippases and floppases which catalyse the inter-leaflet transfer of specific lipids. Lipid asymmetry is a key feature of the plasma membrane and enables cells to quickly respond to extracellular signals by transient local breakdown and its consequences on nearby membrane proteins. However, there is a significant knowledge gap on where and when changes in asymmetry occur in the plasma membrane during important cellular events, such as signalling, trafficking or host-pathogen interactions.
So far, the state-of-the-art to determine lipid asymmetry of biological membranes relies on time- and energy-consuming techniques, such as electron microscopy and/or mass spectrometry. Furthermore, the experiments are performed on population of cells without internal membranes (i.e. red blood cells) using an indirect readout system based on headgroup-cleaving enzymes which might introduce errors and hinders the cell types we can study. Using fluorescence microscopy as a direct readout technique would significantly improve the required time and resources and allow for extracting subcellular spatial information at the nanoscale, in health and disease. Moreover, it would allow to visualize transbilayer asymmetry of intracellular membranes, enclosing endoplasmic reticulum, Golgi apparatus, mitochondria, and nucleus, which is not possible with currently used techniques. I aim to fill this gap by developing a direct imaging-based technology to study membrane asymmetry in single cell level.
Lipids are a diverse class of molecules consisting of thousands of different molecular species which are distributed heterogeneously in the plasma membrane and between the inner and outer leaflet. For example, sphingomyelin is almost exclusively present in the outer leaflet whereas phosphatidylserine and phosphatidylethanolamine are mainly part of the inner leaflet . This transbilayer lipid asymmetry is maintained by a set of enzymes called scramblases, flippases and floppases which catalyse the inter-leaflet transfer of specific lipids. Lipid asymmetry is a key feature of the plasma membrane and enables cells to quickly respond to extracellular signals by transient local breakdown and its consequences on nearby membrane proteins. However, there is a significant knowledge gap on where and when changes in asymmetry occur in the plasma membrane during important cellular events, such as signalling, trafficking or host-pathogen interactions.
So far, the state-of-the-art to determine lipid asymmetry of biological membranes relies on time- and energy-consuming techniques, such as electron microscopy and/or mass spectrometry. Furthermore, the experiments are performed on population of cells without internal membranes (i.e. red blood cells) using an indirect readout system based on headgroup-cleaving enzymes which might introduce errors and hinders the cell types we can study. Using fluorescence microscopy as a direct readout technique would significantly improve the required time and resources and allow for extracting subcellular spatial information at the nanoscale, in health and disease. Moreover, it would allow to visualize transbilayer asymmetry of intracellular membranes, enclosing endoplasmic reticulum, Golgi apparatus, mitochondria, and nucleus, which is not possible with currently used techniques. I aim to fill this gap by developing a direct imaging-based technology to study membrane asymmetry in single cell level.
The overall methodology of this proposal relies on specific labeling of individual lipid species and the controllable combination of expansion microscopy (ExM) with super-resolution STED microscopy (Figure 01). Until now, we could not directly resolve the individual leaflets of biological membranes by fluorescence microscopy due to their proximity (membrane is ≈5nm thick). Covalent linkage of lipids into a swellable hydrogel and subsequent ten-fold expansion in combination with STED microscopy will allow to resolve each individual leaflet. To establish the method, I compared different membrane labelling approaches with the goal to identify a robust labelling approach for the outer and inner membrane leaflet, respectively. The most robust and efficient labelling approach for the outer membrane was based on metabolic glycan engineering and live-cell click chemistry labelling using reactive DBCO-dyes. For the inner membrane, I identified the transient expression of the plamitoylated tyrosin-protein kinase Lck fused to mScarlet (Lck-mScarlet) as method of choice. Of note, the labelling approaches were stable and suitable for subsequent production of giant plasma membrane vesicles (GPMVs).
Next, I tested whether membrane models can be used as simplified systems to establish the method based on combination of ExM and STED. Unfortunately, the tested membrane models (GUVs, GPMVs) were too fragile to be used in combination with ExM and did not withstand the tested expansion microscopy protocols. Therefore, future efforts focused on native cell membranes rather than simplified membrane model systems.
Next, I tested whether membrane models can be used as simplified systems to establish the method based on combination of ExM and STED. Unfortunately, the tested membrane models (GUVs, GPMVs) were too fragile to be used in combination with ExM and did not withstand the tested expansion microscopy protocols. Therefore, future efforts focused on native cell membranes rather than simplified membrane model systems.
Efficient and homogeneous lipid labeling represents a major hurdle impeding the widespread application of the technology. Ideally, the labeling should be specific for native lipid species and happen instantaneously on the outer and inner membrane leaflet without compromising the lipid’s function. However, the barrier function of the lipid bilayer and the relatively small size of individual lipids drastically challenges this reaction to happen. Future research should thus focus on developing efficient and homogeneous labeling strategies for simultaneous labeling of native lipids on the outer and inner membrane leaflet.