Nejvíce citovaný článek - PubMed ID 30362705
Membrane Lipid Nanodomains
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
Extracellular vesicles (EVs) are membranous particles released by cells and are considered to be promising sources of biomarkers for various diseases. Mass spectrometry (MS) analysis of EVs requires a sample of purified and detergent-lysed EVs. Purification of EVs is laborious, based on size, density, or surface nature, and requires large amounts of the source material (e.g., blood, spinal fluid). We have employed synthetically produced large unilamellar lipid vesicles (LUVs) as analogs of EVs to demonstrate an alternative approach to vesicle separation for subsequent mass spectrometry analysis of their composition. Mass-to-charge ratio m/z separation by frequency-controlled quadrupole was employed to filter narrow-size distributions of LUVs from a water sample. Lipid vesicles were positively charged with nanoelectrospray and transferred into a vacuum using two wide m/z-range frequency-controlled quadrupoles. The m/z, charges, and masses of individual vesicles were obtained by the nondestructive single-pass charge detector. The resolving mode of the second quadrupole with m/z RSD < 10% allowed to separate size selected distributions of vesicles with modal diameters of 88, 112, 130, 162, and 190 nm at corresponding quadrupole m/z settings of 2.5 × 105, 5 × 105, 8 × 105, 1.5 × 106, and 2.5 × 106, respectively with a rate of 20-100 counts per minute. The distributions of bioparticles with masses between 108 and 1010 Da were separated from human blood serum in the pilot experiment. The presented approach for lipid vesicle separation encourages the development of new techniques for the direct mass-spectrometric analysis of biomarkers in MS-separated EVs in a vacuum.
- MeSH
- extracelulární vezikuly * chemie MeSH
- hmotnostní spektrometrie * metody MeSH
- lidé MeSH
- unilamelární lipozómy * chemie analýza izolace a purifikace MeSH
- vakuum MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- unilamelární lipozómy * MeSH
Cholesterol is a central building block in biomembranes, where it induces orientational order, slows diffusion, renders the membrane stiffer, and drives domain formation. Molecular dynamics (MD) simulations have played a crucial role in resolving these effects at the molecular level; yet, it has recently become evident that different MD force fields predict quantitatively different behavior. Although easily neglected, identifying such limitations is increasingly important as the field rapidly progresses toward simulations of complex membranes mimicking the in vivo conditions: pertinent multicomponent simulations must capture accurately the interactions between their fundamental building blocks, such as phospholipids and cholesterol. Here, we define quantitative quality measures for simulations of binary lipid mixtures in membranes against the C-H bond order parameters and lateral diffusion coefficients from NMR spectroscopy as well as the form factors from X-ray scattering. Based on these measures, we perform a systematic evaluation of the ability of commonly used force fields to describe the structure and dynamics of binary mixtures of palmitoyloleoylphosphatidylcholine (POPC) and cholesterol. None of the tested force fields clearly outperforms the others across the tested properties and conditions. Still, the Slipids parameters provide the best overall performance in our tests, especially when dynamic properties are included in the evaluation. The quality evaluation metrics introduced in this work will, particularly, foster future force field development and refinement for multicomponent membranes using automated approaches.
- MeSH
- cholesterol chemie MeSH
- fosfatidylcholiny * chemie MeSH
- lipidové dvojvrstvy * chemie MeSH
- simulace molekulární dynamiky MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- 1-palmitoyl-2-oleoylphosphatidylcholine MeSH Prohlížeč
- cholesterol MeSH
- fosfatidylcholiny * MeSH
- lipidové dvojvrstvy * 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
Coexisting liquid ordered (Lo) and liquid disordered (Ld) lipid phases in synthetic and plasma membrane-derived vesicles are commonly used to model the heterogeneity of biological membranes, including their putative ordered rafts. However, raft-associated proteins exclusively partition to the Ld and not the Lo phase in these model systems. We believe that the difference stems from the different microscopic structures of the lipid rafts at physiological temperature and the Lo phase studied at room temperature. To probe this structural diversity across temperatures, we performed atomistic molecular dynamics simulations, differential scanning calorimetry, and fluorescence spectroscopy on Lo phase membranes. Our results suggest that raft-associated proteins are excluded from the Lo phase at room temperature due to the presence of a stiff, hexagonally packed lipid structure. This structure melts upon heating, which could lead to the preferential solvation of proteins by order-preferring lipids. This structural transition is manifested as a subtle crossover in membrane properties; yet, both temperature regimes still fulfill the definition of the Lo phase. We postulate that in the compositionally complex plasma membrane and in vesicles derived therefrom, both molecular structures can be present depending on the local lipid composition. These structural differences must be taken into account when using synthetic or plasma membrane-derived vesicles as a model for cellular membrane heterogeneity below the physiological temperature.
- Publikační typ
- časopisecké články 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.
T cells communicate with the environment via surface receptors. Cooperation of surface receptors regulates T-cell responses to diverse stimuli. Recently, finger-like membrane protrusions, microvilli, have been demonstrated to play a role in the organization of receptors and, hence, T-cell activation. However, little is known about the morphogenesis of dynamic microvilli, especially in the cells of immune system. In this review, I focus on the potential role of lipids and lipid domains in morphogenesis of microvilli. Discussed is the option that clustering of sphingolipids with phosphoinositides at the plasma membrane results in dimpling (curved) domains. Such domains can attract phosphoinositide-binding proteins and stimulate actin cytoskeleton reorganization. This process triggers cortical actin opening and bundling of actin fibres to support the growing of microvilli. Critical regulators of microvilli morphogenesis in T cells are unknown. At the end, I suggest several candidates with a potential to organize proteins and lipids in these structures.
- Klíčová slova
- T cell, dimpling domains, lipid rafts, membrane curvature, membrane-associated proteins, microvilli, phosphoinositides, sphingolipids,
- MeSH
- buněčná membrána chemie metabolismus MeSH
- fosfatidylinositoly metabolismus MeSH
- imunomodulace MeSH
- lidé MeSH
- membránové mikrodomény chemie metabolismus MeSH
- metabolismus lipidů * MeSH
- mikroklky metabolismus ultrastruktura MeSH
- morfogeneze MeSH
- sfingolipidy metabolismus MeSH
- signální transdukce MeSH
- T-lymfocyty cytologie fyziologie ultrastruktura MeSH
- vazba proteinů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
- Názvy látek
- fosfatidylinositoly MeSH
- sfingolipidy MeSH
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
