Nejvíce citovaný článek - PubMed ID 28710349
Lipid Driven Nanodomains in Giant Lipid Vesicles are Fluid and Disordered
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.
- Publikační typ
- časopisecké články 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
- buněčná membrána * chemie metabolismus MeSH
- difuze MeSH
- fluorescenční spektrometrie MeSH
- unilamelární lipozómy chemie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- unilamelární lipozómy 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.
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
- biologické modely MeSH
- buněčná membrána metabolismus MeSH
- lipidové dvojvrstvy metabolismus MeSH
- membránové lipidy * MeSH
- membrány metabolismus MeSH
- rezonanční přenos fluorescenční energie * metody MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- lipidové dvojvrstvy MeSH
- membránové lipidy * MeSH
Gangliosides form an important class of receptor lipids containing a large oligosaccharide headgroup whose ability to self-organize within lipid membranes results in the formation of nanoscopic platforms. Despite their biological importance, the molecular basis for the nanoscopic segregation of gangliosides is not clear. In this work, we investigated the role of the ganglioside headgroup on the nanoscale organization of gangliosides. We studied the effect of the reduction in the number of sugar units of the ganglioside oligosaccharide chain on the ability of gangliosides GM1, GM2, and GM3 to spontaneously self-organize into lipid nanodomains. To reach nanoscopic resolution and to identify molecular forces that drive ganglioside segregation, we combined an experimental technique, Förster resonance energy transfer analyzed by Monte-Carlo simulations offering high lateral and trans-bilayer resolution with molecular dynamics simulations. We show that the ganglioside headgroup plays a key role in ganglioside self-assembly despite the negative charge of the sialic acid group. The nanodomains range from 7 to 120 nm in radius and are mostly composed of the surrounding bulk lipids, with gangliosides being a minor component of the nanodomains. The interactions between gangliosides are dominated by the hydrogen bonding network between the headgroups, which facilitates ganglioside clustering. The N-acetylgalactosamine sugar moiety of GM2, however, seems to impair the stability of these clusters by disrupting hydrogen bonding of neighboring sugars, which is in agreement with a broad size distribution of GM2 nanodomains. The simulations suggest that the formation of nanodomains is likely accompanied by several conformational changes in the gangliosides, which, however, have little impact on the solvent exposure of these receptor groups. Overall, this work identifies the key physicochemical factors that drive nanoscopic segregation of gangliosides.
The plasma membrane is a complex system, consisting of two layers of lipids and proteins compartmentalized into small structures called nanodomains. Despite the asymmetric composition of both leaflets, coupling between the layers is surprisingly strong. This can be evidenced, for example, by recent experimental studies performed on phospholipid giant unilamellar vesicles showing that nanodomains formed in the outer layer are perfectly registered with those in the inner leaflet. Similarly, microscopic phase separation in one leaflet can induce phase separation in the opposing leaflet that would otherwise be homogeneous. In this review, we summarize the current theoretical and experimental knowledge that led to the current view that domains are - irrespective of their size - commonly registered across the bilayer. Mechanisms inducing registration of nanodomains suggested by theory and calculations are discussed. Furthermore, domain coupling is evidenced by experimental studies based on the sparse number of methods that can resolve registered from independent nanodomains. Finally, implications that those findings using model membrane studies might have for cellular membranes are discussed.
- Klíčová slova
- biomembranes, domain registration, interleaflet coupling, membrane asymmetry, nanodomains, phase separation, plasma membranes,
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
The coexistence of lipid domains with different degrees of lipid packing in the plasma membrane of mammalian cells has been postulated, but direct evidence has so far been challenging to obtain because of the small size and short lifetime of these domains in live cells. Here, we use fluorescence spectral correlation spectroscopy in conjunction with a probe sensitive to the membrane environment to quantify spectral fluctuations associated with dynamics of membrane domains in live cells. With this method, we show that membrane domains are present in live COS-7 cells and have a lifetime lower bound of 5.90 and 14.69 ms for the ordered and disordered phases, respectively. Comparisons to simulations indicate that the underlying mechanism of these fluctuations is complex but qualitatively described by a combination of dye diffusion between membrane domains as well as the motion of domains within the membrane.
- MeSH
- benzoxaziny chemie MeSH
- buněčná membrána chemie MeSH
- Cercopithecus aethiops MeSH
- COS buňky MeSH
- fluorescenční spektrometrie MeSH
- kvartérní amoniové sloučeniny chemie MeSH
- membránové lipidy chemie MeSH
- viabilita buněk MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- benzoxaziny MeSH
- kvartérní amoniové sloučeniny MeSH
- membránové lipidy MeSH
- NR12S compound MeSH Prohlížeč
