H. Mathilda Lennartz

Clathrin-mediated endocytosis is a major transport route for proteins from the plasma membrane to the interior of the cell. While the recruitment of cargo proteins to clathrin-coated pits is well understood, it remains an open question whether lipids are also sorted by this process. Here, to address this question, we combined super-resolution stimulated emission depletion imaging of bifunctional lipid probes with mathematical modelling. Quantification of ten different lipid species revealed significant differences in pit partitioning, ranging from slight enrichment to moderate exclusion. We find that the lipid asymmetry in the plasma membrane is sufficient to explain the observed trend. Taken together, our findings imply that clathrin-mediated endocytosis has a minor selectivity for cytoplasmic leaflet lipids, but overall does not substantially contribute to lipid sorting compared with non-vesicular trafficking.

Organelle membrane identity is encoded by protein and lipid composition. Human cells produce thousands of distinct lipid species for this purpose. Yet, the precise molecular functions of most lipids remain unknown, owing to limited methods for in situ investigations of individual lipid species. Here, we use minimally modified bifunctional lipid probes to detect and functionally characterize lipid-protein interactions across time and subcellular compartments. We leverage this approach to map time-resolved protein interactomes of individual lipids representing major membrane lipid classes as they are transported through the organelle system of human cells. We identify hundreds of unique lipid-protein interaction candidates, measure lipid-induced protein abundance changes, and distinguish between directly and indirectly interacting subunits of associated protein complexes. Informed by this multimodal dataset, we interrogate the lipid specificity of membrane organizing complexes, in particular the selective co-association of phosphatidylethanolamine with the ancestral subunits of the nuclear pore (NDC1) and MICOS (MIC60) complexes. Using super-resolution microscopy and molecular dynamics simulations, we demonstrate that phosphatidylethanolamine stabilizes both NDC1 and MIC60 in highly curved membrane nanodomains, indicating that major membrane organizing complexes are positioned by selective lipid-protein interactions. Altogether, our experimental strategy provides a blueprint for discovering and characterizing cellular functions of individual lipids.

Lipids and proteins compartmentalize biological membranes into nanoscale domains, which are crucial for signalling, intracellular trafficking and many other cellular processes. Studying nanodomain function requires the ability to measure protein and lipid localization at the nanoscale. Current methods for visualizing lipid localization do not meet this requirement. Here we introduce a correlative light and electron microscopy workflow to image lipids (Lipid-CLEM), combining near-native lipid probes and on-section labelling by click chemistry. This approach enables the quantification of relative lipid densities in membrane nanodomains. We find differential partitioning of sphingomyelin into intraluminal vesicles, recycling tubules and the boundary membrane of the early endosome, representing a degree of nanoscale organization previously observed only for proteins. We anticipate that our Lipid-CLEM workflow will greatly facilitate the mechanistic analysis of lipid functions in cell biology, allowing for the simultaneous investigation of proteins and lipids during membrane nanodomain assembly and function.

Bifunctional diazirine lipids are valuable tools for mapping protein–lipid interactions and cellular localization by photocrosslinking. Yet, the crosslinking efficiency of these probes has not been systematically evaluated. Here, we use the lipid transfer protein STARD10, which binds phospholipids in a 1:1 stoichiometry within a hydrophobic pocket, to measure the upper limit of the photo-crosslinking efficiency of bifunctional lipid probes. We characterize reaction products using native and denaturing mass spectrometry. Our results show that approximately 5% of photoactivated lipids form covalent protein–lipid crosslinks, while the majority follow intramolecular reaction trajectories, resulting in the formation of products featuring alkene, ketone and hydroxyl moieties. These findings provide essential context for the use of bifunctional probes to uncover the cell biology of lipids and highlight the need for continuous improvement to experimental workflows. Abstract Figure:

Zusammenfassung: Ether‐Glycerophospholipide enthalten einen langkettigen Alkohol, der über eine Alkyl‐ oder Vinyletherbindung an der sn1 ‐Position des Glycerin‐Grundgerüsts gebunden ist. Etherlipide sind für zelluläre Physiologie und menschliche Gesundheit hoch relevant. Ihre Zellbiologie ist jedoch weitgehend unklar, da es an Methoden zur Analyse ihrer subzellulären Lokalisierung und Identifikation interagierender Proteine fehlt. Als Ausgangspunkt zellbiologischer Studien wurden bifunktionelle Etherlipid‐Sonden mit Diazirin‐ und Alkin‐Gruppen synthetisiert. Um die Kinetik des intrazellulären Etherlipid‐Transports in Säugetierzellen zu untersuchen, wurde eine Kombination aus Fluoreszenzbildgebung, KI‐unterstützter Bildanalyse und mathematischer Modellierung verwendet. Wir stellen fest, dass alkylverknüpfte Etherlipide bis zu zweifach schneller transportiert werden als vinylverknüpfte Spezies (Plasmalogene). Dies deutet darauf hin, dass die zelluläre Lipidtransportmaschinerie in der Lage ist, zwischen Verknüpfungstypen zu unterscheiden, die sich nur durch zwei Wasserstoffatome unterscheiden. Der Transport von Etherlipiden erfolgt dabei überwiegend über nichtvesikuläre Mechanismen, wobei der Beitrag des vesikulären Transports je nach Zelltyp variiert. Insgesamt deuten unsere Ergebnisse darauf hin, dass die unterschiedliche Erkennung von Alkyl‐ und Vinyletherlipiden durch Lipidtransferproteine zu deren unterschiedlichen biologischen Funktionen beiträgt. Die hier vorgestellten Sonden werden es in Zukunft ermöglichen, die Biologie der Etherlipide genauer zu untersuchen, z.B. durch die Identifizierung der interagierenden Proteine.

Ether glycerophospholipids bear a long chain alcohol attached via an alkyl or vinyl ether bond at the sn1 position of the glycerol backbone. Ether lipids play a significant role in physiology and human health. However, their cellular functions remain largely unknown due to a lack of tools for identifying their subcellular localization and interacting proteins. Here, we address this methodological gap by synthesizing minimally modified bifunctional ether lipid probes by introducing diazirine and alkyne groups. To interrogate the subcellular kinetics of intracellular ether lipid transport in mammalian cells, we used a combination of fluorescence imaging, machine learning‐assisted image analysis, and mathematical modelling. We find that alkyl‐linked ether lipids are transported up to twofold faster than vinyl‐linked species (plasmalogens), pointing to yet undiscovered cellular lipid transport machinery able to distinguish between linkage types differing by as little as two hydrogen atoms. We find that ether lipid transport predominantly occurs via non‐vesicular pathways, with varying contributions from vesicular mechanisms between cell types. Altogether, our results suggest that differential recognition of alkyl‐ and vinyl ether lipids by lipid transfer proteins contributes to their distinct biological functions. In the future, the probes reported here will enable studying ether lipid biology in much greater detail through identification of interacting proteins and in‐depth characterization of intracellular ether lipid dynamics.

Biological membranes often feature unequal and actively maintained concentration gradients of lipids between bilayer leaflets, termed lipid asymmetry. ^1–3 Lipid asymmetry has been linked to many cellular processes ^4,5 , but the mechanisms that connect lipid trans-bilayer concentration gradients to cellular functions are poorly understood. Here we systematically map the cellular processes affected by dysregulation of lipid asymmetry by knocking out flippases, floppases, and scramblases in mammalian cells. We identify broad alterations to core metabolic pathways, including nutrient uptake, neutral lipid turnover, the pentose phosphate pathway, and glycolysis. Lipidomics, respirometry, lipid and metabolic imaging, and live-cell calorimetry revealed elevated neutral lipid and ATP consumption rates, coupled with increased heat loss per unit biomass synthesized despite slower growth. This suggests that compensatory maintenance of lipid asymmetry strains the cellular energy budget, shifting the cell from a growth-promoting anabolic state to a more energy-using catabolic state. Our data indicate that lipid asymmetry and its active maintenance are linked to cellular energetics, reflecting either the energetic cost of maintaining lipid asymmetry or its putative role as a cellular store of potential energy akin to proton and ion transmembrane gradients.

Clathrin-mediated endocytosis is a major transport route for proteins from the plasma membrane to the interior of the cell. While the recruitment of cargo proteins to clathrin-coated pits is well understood, it remains an open question if lipids are also sorted by this process. To address this question, we combined super-resolution STED imaging of bifunctional lipid probes with mathematical modeling. Quantification of 10 different lipid species revealed significant differences in pit partitioning, ranging from slight enrichment to moderate exclusion. We find that the lipid asymmetry in the plasma membrane is sufficient to explain the observed trend. Taken together, our findings imply that clathrin-mediated endocytosis has a minor selectivity for cytoplasmic leaflet lipids, but overall does not significantly contribute to lipid sorting compared to non-vesicular trafficking. More broadly, we believe that lipid super-resolution imaging will be a powerful approach to quantify lipid partitioning into membrane structures in cells.

Lipids and proteins compartmentalize biological membranes into nanoscale domains which are crucial for signaling, intracellular trafficking and many other cellular processes. Studying nanodomain function requires the ability to measure protein and lipid localization at the nanoscale. Current methods for visualizing lipid localization do not meet this requirement. Here, we introduce a correlative light and electron microscopy workflow to image lipids (Lipid-CLEM), combining near-native lipid probes and on-section labeling by click chemistry. This approach enables the quantification of relative lipid densities in membrane nanodomains. We find differential partitioning of sphingomyelin into intraluminal vesicles, recycling tubules, and the boundary membrane of the early endosome, representing a degree of nanoscale organization previously observed only for proteins. We anticipate that our Lipid-CLEM workflow will greatly facilitate the mechanistic analysis of lipid functions in cell biology, allowing for the simultaneous investigation of proteins and lipids during membrane nanodomain assembly and function.

Ether glycerophospholipids bear a long chain alcohol attached via an alkyl or vinyl ether bond at the sn1 position of the glycerol backbone. Emerging evidence suggests that ether lipids play a significant role in physiology and human health but their precise cellular functions remain largely unknown. Here, we introduce bifunctional ether lipid probes bearing diazirine and alkyne groups to study ether lipid biology. To interrogate the kinetics of intracellular ether lipid transport in mammalian cells we used a combination of fluorescence imaging, machine learning-assisted image analysis and mathematical modelling. We find that alkyl-linked ether lipids are transported up to twofold faster than vinyl-linked plasmalogens, suggesting that the lipid transport machinery can distinguish between linkage types differing by as little as two hydrogen atoms. We find that ether lipid transport predominantly occurs via non-vesicular pathways, with varying contributions from vesicular mechanisms between cell types. Altogether, our results suggest that differential recognition of alkyl- and vinyl ether lipids by lipid transfer proteins contributes to their distinct biological functions. In the future, the probes reported here will enable studying ether lipid biology in much greater detail through identification of interacting proteins and in-depth characterization of intracellular ether lipid dynamics.