Eukaryotic cells produce over 1,000 different lipid species that tune organelle membrane properties, control signalling and store energy1,2. How lipid species are selectively sorted between organelles to maintain specific membrane identities is largely unclear, owing to the difficulty of imaging lipid transport in cells3. Here we measured the retrograde transport and metabolism of individual lipid species in mammalian cells using time-resolved fluorescence imaging of bifunctional lipid probes in combination with ultra-high-resolution mass spectrometry and mathematical modelling. Quantification of lipid flux between organelles revealed that directional, non-vesicular lipid transport is responsible for fast, species-selective lipid sorting, in contrast to the slow, unspecific vesicular membrane trafficking. Using genetic perturbations, we found that coupling between energy-dependent lipid flipping and non-vesicular transport is a mechanism for directional lipid transport. Comparison of metabolic conversion and transport rates showed that non-vesicular transport dominates the organelle distribution of lipids, while species-specific phospholipid metabolism controls neutral lipid accumulation. Our results provide the first quantitative map of retrograde lipid flux in cells4. We anticipate that our pipeline for mapping of lipid flux through physical and chemical space in cells will boost our understanding of lipids in cell biology and disease.

• Publication type
• Journal Article MeSH

Galectin-1 (Gal-1) is a galactose-binding protein involved in various cellular functions. Gal-1's activity has been suggested to be connected to two molecular concepts, which are, however, lacking experimental proof: a) enhanced binding affinity of Gal-1 toward membranes containing monosialotetrahexosylganglioside (GM1) over disialoganglioside GD1a and b) cross-linking of GM1's by homodimers of Gal-1. We provide evidence about the specificity and the nature of the interaction of Gal-1 with model membranes containing GM1 or GD1a, employing a broad panel of fluorescence-based and label-free experimental techniques, complemented by atomistic biomolecular simulations. Our study demonstrates that Gal-1 indeed binds specifically to GM1 and not to GD1a when embedded in membranes over a wide range of concentrations (i.e., 30 nM to 20 μM). The apparent binding constant is about tens of micromoles. On the other hand, no evidence of Gal-1/GM1 cross-linking was observed. Our findings suggest that cross-linking does not result from sole interactions between GM1 and Gal-1, indicating that in a physiological context, additional triggers are needed, which shift the GM1/Gal-1 equilibria toward the membrane-bound homodimeric Gal-1.

• Keywords
• GD1a, GM1, Kd determination, cross-linking, galectin-1, neuraminidase,
• Publication type
• Journal Article MeSH

The formation of functional nanoscopic domains is an inherent property of plasma membranes. Stimulated emission depletion combined with fluorescence correlation spectroscopy (STED-FCS) has been previously used to identify such domains; however, the information obtained by STED-FCS has been limited to the presence of such domains while crucial parameters have not been accessible, such as size (Rd), the fraction of occupied membrane surface (f), in-membrane lipid diffusion inside (Din) and outside (Dout) the nanodomains as well as their self-diffusion (Dd). Here, we introduce a quantitative approach based on a revised interpretation of the diffusion law. By analyzing experimentally recorded STED-FCS diffusion law plots using a comprehensive library of simulated diffusion law plots, we extract these five parameters from STED-FCS data. That approach is verified on ganglioside nanodomains in giant unilamellar vesicles, validating the Saffman-Delbrück assumption for Dd. STED-FCS data in both plasma membranes of living PtK2 cells and giant plasma membrane vesicles are examined, and a quantitative framework for molecular diffusion modes in biological membranes is presented.

• MeSH
• Cell Membrane * chemistry   metabolism   MeSH
• Diffusion MeSH
• Spectrometry, Fluorescence MeSH
• Unilamellar Liposomes chemistry   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Unilamellar Liposomes MeSH

Gangliosides are important glycosphingolipids involved in a multitude of physiological functions. From a physicochemical standpoint, this is related to their ability to self-organize into nanoscopic domains, even at molar concentrations of one per 1000 lipid molecules. Despite recent experimental and theoretical efforts suggesting that a hydrogen bonding network is crucial for nanodomain stability, the specific ganglioside moiety decisive for the development of these nanodomains has not yet been identified. Here, we combine an experimental technique achieving nanometer resolution (Förster resonance energy transfer analyzed by Monte Carlo simulations) with atomistic molecular dynamic simulations to demonstrate that the sialic acid (Sia) residue(s) at the oligosaccharide headgroup dominates the hydrogen bonding network between gangliosides, driving the formation of nanodomains even in the absence of cholesterol or sphingomyelin. Consequently, the clustering pattern of asialoGM1, a Sia-depleted glycosphingolipid bearing three glyco moieties, is more similar to that of structurally distant sphingomyelin than that of the closely related gangliosides GM1 and GD1a with one and two Sia groups, respectively.

• MeSH
• G(M1) Ganglioside MeSH
• Gangliosides * chemistry   MeSH
• Glycosphingolipids MeSH
• Sphingomyelins * MeSH
• Molecular Dynamics Simulation MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• G(M1) Ganglioside MeSH
• Gangliosides * MeSH
• Glycosphingolipids MeSH
• Sphingomyelins * MeSH

Plasma membranes as well as their simplified model systems show an inherent nanoscale heterogeneity. As a result of strong interleaflet interactions, these nanoheterogeneities (called here lipid nanodomains) can be found in perfect registration (i.e., nanodomains in the inner leaflet are registered with the nanodomains in the outer leaflet). Alternatively, they might be interleaflet independent, antiregistered, or located asymmetrically in one bilayer leaflet only. To distinguish these scenarios from each other appears to be an experimental challenge. In this work, we analyzed the potential of Förster resonance energy transfer to characterize interleaflet organization of nanodomains. We generated in silico time-resolved fluorescence decays for a large set of virtual as well as real donor/acceptor pairs distributed over the bilayer containing registered, independent, antiregistered, or asymmetrically distributed nanodomains. In this way, we were able to identify conditions that gave satisfactory or unsatisfactory resolution. Overall, Förster resonance energy transfer appears as a robust method that, when using donor/acceptor pairs with good characteristics, yields otherwise difficult-to-reach characteristics of membrane lipid nanodomains.

• MeSH
• Models, Biological MeSH
• Cell Membrane metabolism   MeSH
• Lipid Bilayers metabolism   MeSH
• Membrane Lipids * MeSH
• Membranes metabolism   MeSH
• Fluorescence Resonance Energy Transfer * methods   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Lipid Bilayers MeSH
• Membrane Lipids * MeSH