The impact of the glycan headgroup on the nanoscopic segregation of gangliosides.

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.

Quantitative FRET Microscopy Reveals a Crucial Role of Cytoskeleton in Promoting PI(4,5)P2 Confinement.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is an essential plasma membrane component involved in several cellular functions, including membrane trafficking and cytoskeleton organization. This function multiplicity is partially achieved through a dynamic spatiotemporal organization of PI(4,5)P2 within the membrane. Here, we use a Förster resonance energy transfer (FRET) approach to quantitatively assess the extent of PI(4,5)P2 confinement within the plasma membrane. This methodology relies on the rigorous evaluation of the dependence of absolute FRET efficiencies between pleckstrin homology domains (PHPLCδ) fused with fluorescent proteins and their average fluorescence intensity at the membrane. PI(4,5)P2 is found to be significantly compartmentalized at the plasma membrane of HeLa cells, and these clusters are not cholesterol-dependent, suggesting that membrane rafts are not involved in the formation of these nanodomains. On the other hand, upon inhibition of actin polymerization, compartmentalization of PI(4,5)P2 is almost entirely eliminated, showing that the cytoskeleton network is the critical component responsible for the formation of nanoscale PI(4,5)P2 domains in HeLa cells.

Structural and Functional Properties of the Capsid Protein of Dengue and Related Flavivirus.

Dengue, West Nile and Zika, closely related viruses of the Flaviviridae family, are an increasing global threat, due to the expansion of their mosquito vectors. They present a very similar viral particle with an outer lipid bilayer containing two viral proteins and, within it, the nucleocapsid core. This core is composed by the viral RNA complexed with multiple copies of the capsid protein, a crucial structural protein that mediates not only viral assembly, but also encapsidation, by interacting with host lipid systems. The capsid is a homodimeric protein that contains a disordered N-terminal region, an intermediate flexible fold section and a very stable conserved fold region. Since a better understanding of its structure can give light into its biological activity, here, first, we compared and analyzed relevant mosquito-borne Flavivirus capsid protein sequences and their predicted structures. Then, we studied the alternative conformations enabled by the N-terminal region. Finally, using dengue virus capsid protein as main model, we correlated the protein size, thermal stability and function with its structure/dynamics features. The findings suggest that the capsid protein interaction with host lipid systems leads to minor allosteric changes that may modulate the specific binding of the protein to the viral RNA. Such mechanism can be targeted in future drug development strategies, namely by using improved versions of pep14-23, a dengue virus capsid protein peptide inhibitor, previously developed by us. Such knowledge can yield promising advances against Zika, dengue and closely related Flavivirus.

Which Moiety Drives Gangliosides to Form Nanodomains?

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.

Organization of gangliosides into membrane nanodomains.

Gangliosides are glycosphingolipids consisting of a ceramide base and a bulky sugar chain that contains one or more sialic acids. This unique structure endows gangliosides with a strong tendency to self-aggregate in solution, as well as in cellular membranes, where they can form nanoscopic assemblies called ganglioside nanodomains. As gangliosides are important biological molecules involved in a number of physiological processes, characterization of their lateral organization in membranes is essential. This review aims at providing comprehensive information about the nanoscale organization of gangliosides in various synthetic models. To this end, the impact of the hydrophobic backbone and the headgroup on the segregation of gangliosides into nanodomains are discussed in detail, as well as the way in which the properties of nanodomains are affected by ligand binding. Small size makes the characterization of ganglioside nanodomains challenging, and we thus highlight the biophysical methods that have advanced this research, such as Monte Carlo Förster resonance energy transfer, atomic force microscopy and approaches based on molecular diffusion.

Fluorescence detection of lipid-induced oligomeric intermediates involved in lysozyme "amyloid-like" fiber formation driven by anionic membranes.

Recent findings implicate that "amyloid-like" fiber formation by several non-amyloidogenic proteins/peptides can be triggered by negatively charged lipid membranes. In order to elucidate the factors that govern the formation of these structures, the interaction of lysozyme with phosphatidylserine-containing lipid vesicles was studied by steady-state and time-resolved fluorescence measurements. Three consecutive stages in the interaction of Alexa488-fluorescently labeled lysozyme (Lz-A488) with acidic lipid vesicles were identified in ensemble average measurements. The variation of the mean fluorescence lifetime of Lz-A488 as a function of the surface coverage of the liposomes was quantitatively described by a cooperative partition model that assumes that monomeric lysozyme molecules partition into the bilayer surface and reversibly assemble into oligomers with k subunits (k ≥ 6). The global fit to the experimental data covering a wide range of experimental conditions was performed by taking into account electrostatic effects by means of the Gouy-Chapman theory using a single self-consistent pair of parameters (aggregation constant and stoichiometry). The lipid-protein supramolecular assemblies formed at a low lipid/protein molar ratio were further characterized by fluorescence lifetime imaging microscopy at the single-fiber level, which reported that quenched oligomers are the predominant species in these structures.