The plasma membrane (PM) of live cells has a striking phospholipid asymmetry between bilayer leaflets, yet the purpose of this fundamental structure remains elusive. It is also unknown whether and how this phospholipid asymmetry impacts on the lateral organization of the PM, such as microdomains or lipid rafts that are thought to facilitate specific protein-protein interactions. Here, we generated asymmetric giant unilamellar vesicles (GUVs) and found that microdomain formation is inhibited by outer leaflet very long acyl chain (24:0) sphingomyelin (SM), the primary sphingolipid species in mammalian cells. Interestingly, although cholesterol is believed to associate favourably with SM, molecular dynamic (MD) simulations of asymmetric membranes indicate a strong preference for cholesterol in the inner leaflet when 24:0 SM is in the outer leaflet, as well as, interdigitation of 24:0 SM acyl chain across the centre of the bilayer. We thus hypothesized that the outer leaflet-localized 24:0 SM interdigitates across the leaflets of the bilayer and facilitates cholesterol enrichment in the inner leaflet. Indeed, we obtained evidence that, in asymmetric unilamellar vesicles with 24:0 SM exclusively in the outer leaflet, 75-80% of cholesterol was partitioned into the inner leaflet, which was correlated with the disappearance of microdomains in GUVs. Importantly, in live cell PM, where 24:0 sphingolipids are the predominant species and exclusively in the outer leaflet, cholesterol was similarly enriched in the cytoplasmic leaflet. SM with shorter acyl chains such as 16:0, a minor species in mammalian cells, failed to generate cholesterol asymmetry and promoted microdomains in both symmetric and asymmetric GUVs. Furthermore, we generated live mammalian cells with either 16:0 or 24:0 SM and analyzed submicron domains in these cells, using density-dependent FRET of GPI-anchored proteins. Indeed, 16:0 SM cells are capable of forming submicron domains. The 24:0 SM cells, by contrast, are nearly devoid of submicron domains, as are unmodified control cells. Moreover, we silenced ceramide synthase 2 (CerS2), the enzyme that generates very long acyl chain sphingolipids. We found that silencing CerS2 alters diffusional properties of membrane proteins, consistent with enhanced microdomain formation. Together, our results establish a surprising and central role of very long acyl chain sphingolipids in regulating membrane lateral organization, including in the native plasma membrane, by creating cholesterol asymmetry. We propose that sphingolipid asymmetry functions to dynamically regulate microdomains in live cells.

Endozytose erfordert hohe Membrankrümmung und führt zu Flächenänderungen der Membranhälften. Dies kann durch eine Oberflächendifferenz zwischen den Schichten initiiert werden, die durch geänderte Lipidzusammensetzungen hervorgerufen werden kann. Daher wurde die Hypothese aufgestellt, dass Lipid-Transporter zu Beginn der Endozytose für veränderte Flächenverhältnisse verantwortlich sind. Um den Einfluss veränderter Flächen auf Endozytose zu untersuchen, wurden die Oberflächenverhältnisse der Membran durch Zugabe von Phospholipiden verändert und anschließend Endozytose gemessen. Abhängig von der Sorte wurden die Lipide nur in die äußere Schicht eingebaut oder auch auf die innere Seite transportiert, wodurch die entsprechende Seite vergrößert wurde. Die Zugabe verschiedener Aminophospholipide, die auf die innere Membranseite transportiert werden, führte zu gesteigerter „bulk flow“ Endocytose in K562-Zellen. Darüber hinaus deuten die Ergebnisse darauf hin, dass Clathrin-vermittelte Endozytose von Hep2-Zellen ebenfalls stimuliert wurde. Umgekehrt hatte die Zugabe von Lipiden, die auf der äußeren Hälfte bleiben, reduzierte „bulk flow“- oder Clathrin-vermittelte Endozytose in verschiedenen Zelllinien zur Folge. Bemerkenswert ist, dass auch Clathrin-vermittelte Endozytose durch die Lipidzugabe beeinflusst wurde, obwohl gerade in diesem Weg viele Proteine involviert sind, die Krümmung induzieren können. Dies passt zu einem neuen Modell wie Lipidtransporter in Endozytose involviert sind. Durch den Transport von Lipiden und die zusätzliche Interaktion mit Endozytoseproteinen, könnten diese Transporter zwei Mechanismen zur Erzeugung von Krümmung miteinander verbinden: Membrankrümmung induziert durch eine Flächenasymmetrie zwischen den Membranhälften und durch Wechselwirkung mit Proteinen. Die Ergebnisse dieser Arbeit deuten darauf hin, dass die für Endozytose notwendige Krümmung durch die durch Lipidtransport induzierte Flächenasymmetrie der Membranschichten unterstützt wird. / Endocytic engulfment requires high local membrane curvature and causes significant area changes of the membrane leaflets. This can be initiated by differences between the surface areas of the two monolayers related to leaflet specific modulation of lipid composition. Thus, it was proposed that lipid translocators, pumping phospholipids from the outer to the inner leaflet, account for monolayer area asymmetry as an early step in endocytosis. To elucidate the influence of this asymmetry on endocytosis, surface area relation was altered by adding exogenous phospholipids to living cells and changes in endocytic activity were quantified. Depending on the lipid species, exogenous lipids were only incorporated into the outer layer or subsequently translocated across the plasma membrane thereby increasing either the outer or inner surface area. Addition of different analogues of aminophospholipids, which are translocated to the inner leaflet, led to an enhancement of bulk flow endocytosis in K562 cells. Moreover, our data indicate that clathrin-mediated endocytosis of Hep2 cells was stimulated as well. Inversely, addition of phospholipids, which remain on the outer layer, reduced bulk flow or clathrin-mediated endocytosis in various cell lines. Notably, also clathrin-mediated endocytosis was influenced by the addition of lipids, although many proteins noted for their ability to induce membrane curvature are known to be implicated in this pathway. This corroborates a recent model how aminophospholipid translocases are implicated in endocytosis. Upon translocating lipids and additionally interacting with endocytic accessory proteins, lipid translocators could integrate two processes to generate curvature: membrane bending based on monolayer area asymmetry and protein-related mechanisms. Collectively, findings in the present study suggest that curvature generation in endocytosis is supported by the induction of monolayer area asymmetry mediated by the translocation of lipids.

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