Visualization of HRas Domains in the Plasma Membrane of Fibroblasts

The plasma membrane is a highly complex, organized structure where the lateral organization of signaling proteins is tightly regulated. In the case of Ras proteins, it has been suggested that the differential activity of the various isoforms is due to protein localization in separate membrane compartments. To date, direct visualization of such compartmentalization has been achieved only by electron microscopy on membrane sheets. Here, we combine photoactivated light microscopy with quantitative statistical analysis to visualize protein distribution in intact cells. In particular, we focus on the localization of HRas and its minimal anchoring domain, CAAX. We demonstrate the existence of a complex partitioning behavior, where small domains coexist with larger ones. The protein content in these domains varied from two molecules to tens of molecules. We found that 40% of CAAX and 60% of HRas were localized in domains. Subsequently, we were able to manipulate protein distributions by inducing coalescence of supposedly cholesterol-enriched domains. Clustering resulted in an increase of the localized fraction by 15%.     

Hydrophobic Compounds Reshape Membrane Domains

Cell membranes have a complex lateral organization featuring domains with distinct composition, also known as rafts, which play an essential role in cellular processes such as signal transduction and protein trafficking. In vivo, perturbations of membrane domains (e.g., by drugs or lipophilic compounds) have major effects on the activity of raft-associated proteins and on signaling pathways, but they are difficult to characterize because of the small size of the domains, typically below optical resolution. Model membranes, instead, can show macroscopic phase separation between liquid-ordered and liquid-disordered domains, and they are often used to investigate the driving forces of membrane lateral organization. Studies in model membranes have shown that some lipophilic compounds perturb membrane domains, but it is not clear which chemical and physical properties determine domain perturbation. The mechanisms of domain stabilization and destabilization are also unknown. Here we describe the effect of six simple hydrophobic compounds on the lateral organization of phase-separated model membranes consisting of saturated and unsaturated phospholipids and cholesterol. Using molecular simulations, we identify two groups of molecules with distinct behavior: aliphatic compounds promote lipid mixing by distributing at the interface between liquid-ordered and liquid-disordered domains; aromatic compounds, instead, stabilize phase separation by partitioning into liquid-disordered domains and excluding cholesterol from the disordered domains. We predict that relatively small concentrations of hydrophobic species can have a broad impact on domain stability in model systems, which suggests possible mechanisms of action for hydrophobic compounds in vivo.     

Membrane-Mediated Interactions Measured Using Membrane Domains

Cell membrane organization is the result of the collective effect of many driving forces. Several of these, such as electrostatic and van der Waals forces, have been identified and studied in detail. In this article, we investigate and quantify another force, the interaction between inclusions via deformations of the membrane shape. For electrically neutral systems, this interaction is the dominant organizing force. As a model system to study membrane-mediated interactions, we use phase-separated biomimetic vesicles that exhibit coexistence of liquid-ordered and liquid-disordered lipid domains. The membrane-mediated interactions between these domains lead to a rich variety of effects, including the creation of long-range order and the setting of a preferred domain size. Our findings also apply to the interaction of membrane protein patches, which induce similar membrane shape deformations and hence experience similar interactions.     

Glycosphingolipid Domains on Cell Plasma Membrane

In this study we analyzed by immunofluorescence, laser confocal microscopy, immunoelectron microscopy and label fracture technique the ganglioside distribution on the plasma membrane of several different cell types: human peripheral blood lymphocytes (PBL), Molt-4 lymphoid cells, and NIH 3T3 fibroblasts, which mainly express monosialoganglioside GM3, and murine NS20Y neuroblastoma cells, which have been shown to express a high amount of monosialoganglioside GM2. Our observations showed an uneven distribution of both GM3 and GM2 on the plasma membrane of all cells, confirming the existence of ganglioside-enriched microdomains on the cell surface. Interestingly, in lymphoid cells the clustered immunolabeling appeared localized over both the microvillous and the nonvillous portions of the membrane. Similarly, in cells growing in monolayer, the clusters were distributed on both central and peripheral regions of the cell surface. Therefore, glycosphingolipid clusters do not appear confined to specific areas of the plasma membrane, implying general functions of these domains, which, as structural components of a cell membrane multimolecular signaling complex, may be involved in cell activation and adhesion, signal transduction and, when associated to caveolae, in endocytosis of specific molecules.     

Glycolipid-Enriched Membrane Domains Are Assembled into Membrane Patches by Associating with the Actin Cytoskeleton

Nonionic detergent lysates of cells contain a glycolipid-enriched membrane (GEM) fraction. It has been proposed that the GEM fraction represents poorly solubilized GEM microdomains, or lipid rafts. However, the properties of GEM domains in intact cells remain controversial. To study the properties of a GEM-associated protein using confocal microscopy, GFP was targeted to GEM domains using the N-terminal domain of p56(lck) (LckNT). Imaging of HeLa cells expressing LckNT-GFP showed that it was targeted to large actin-rich patches in the plasma membrane that contained up to a fivefold enrichment of protein. Double-labeling experiments showed that the patches were selectively enriched with other GEM-associated molecules. Furthermore, the patches were resistant to extraction by TX-100, and disrupting GEM domains by extracting cholesterol also disrupted colocalization of LckNT-GFP with F-actin. Analogous to the actin-rich patches in HeLa cells, LckNT-GFP colocalized with actin-rich membrane caps in stimulated T cells. Furthermore, disrupting the GEM-targeting signal of LckNT-GFP also inhibited its targeting to membrane caps. Altogether, these findings extend previous studies by showing that association of GEM domains with the actin cytoskeleton provides a mechanism for targeting signaling molecules to membrane patches and caps.     

Yeast Lipids Can Phase-separate into Micrometer-scale Membrane Domains

The lipid raft concept proposes that biological membranes have the potential to form functional domains based on a selective interaction between sphingolipids and sterols. These domains seem to be involved in signal transduction and vesicular sorting of proteins and lipids. Although there is biochemical evidence for lipid raft-dependent protein and lipid sorting in the yeast Saccharomyces cerevisiae, direct evidence for an interaction between yeast sphingolipids and the yeast sterol ergosterol, resulting in membrane domain formation, is lacking. Here we show that model membranes formed from yeast total lipid extracts possess an inherent self-organization potential resulting in liquid-disordered-liquid-ordered phase coexistence at physiologically relevant temperature. Analyses of lipid extracts from mutants defective in sphingolipid metabolism as well as reconstitution of purified yeast lipids in model membranes of defined composition suggest that membrane domain formation depends on specific interactions between yeast sphingolipids and ergosterol. Taken together, these results provide a mechanistic explanation for lipid raft-dependent lipid and protein sorting in yeast.     

Adaptive Lipid Packing and Bioactivity in Membrane Domains

Lateral compositional and physicochemical heterogeneity is a ubiquitous feature of cellular membranes on various length scales, from molecular assemblies to micrometric domains. Segregated lipid domains of increased local order, referred to as rafts, are believed to be prominent features in eukaryotic plasma membranes; however, their exact nature (i.e. size, lifetime, composition, homogeneity) in live cells remains difficult to define. Here we present evidence that both synthetic and natural plasma membranes assume a wide range of lipid packing states with varying levels of molecular order. These states may be adapted and specifically tuned by cells during active cellular processes, as we show for stimulated insulin secretion. Most importantly, these states regulate both the partitioning of molecules between coexisting domains and the bioactivity of their constituent molecules, which we demonstrate for the ligand binding activity of the glycosphingolipid receptor GM1. These results confirm the complexity and flexibility of lipid-mediated membrane organization and reveal mechanisms by which this flexibility could be functionalized by cells.     

Nanoscale Imaging of Caveolin-1 Membrane Domains In Vivo

Light microscopy enables noninvasive imaging of fluorescent species in biological specimens, but resolution is generally limited by diffraction to ~200–250 nm. Many biological processes occur on smaller length scales, highlighting the importance of techniques that can image below the diffraction limit and provide valuable single-molecule information. In recent years, imaging techniques have been developed which can achieve resolution below the diffraction limit. Utilizing one such technique, fluorescence photoactivation localization microscopy (FPALM), we demonstrated its ability to construct super-resolution images from single molecules in a living zebrafish embryo, expanding the realm of previous super-resolution imaging to a living vertebrate organism. We imaged caveolin-1 in vivo, in living zebrafish embryos. Our results demonstrate the successful image acquisition of super-resolution images in a living vertebrate organism, opening several opportunities to answer more dynamic biological questions in vivo at the previously inaccessible nanoscale.     

Visualization of Dynamic Sub-microsecond Changes in Membrane Potential

Direct observation of rapid membrane potential changes is critical to understand how complex neurological systems function. This knowledge is especially important when stimulation is achieved through an external stimulus meant to mimic a naturally occurring process. To enable exploration of this dynamic space, we developed an all-optical method for observing rapid changes in membrane potential at temporal resolutions of ～25 ns. By applying a single 600-ns electric pulse, we observed sub-microsecond, continuous membrane charging and discharging dynamics. Close agreement between the acquired results and an analytical membrane-charging model validates the utility of this technique. This tool will deepen our understanding of the role of membrane potential dynamics in the regulation of many biological and chemical processes within living systems.     

Mapping of the Coronavirus Membrane Protein Domains Involved in Interaction with the Spike Protein

The coronavirus membrane (M) protein is the key player in virion assembly. One of its functions is to mediate the incorporation of the spikes into the viral envelope. Heterotypic interactions between M and the spike (S) protein can be demonstrated by coimmunoprecipitation and by immunofluorescence colocalization, after coexpression of their genes in eukaryotic cells. Using these assays in a mutagenetic approach, we have mapped the domains in the M protein that are involved in complex formation between M and S. It appeared that the 25-residue luminally exposed amino-terminal domain of the M protein is not important for M-S interaction. A 15-residue deletion, the insertion of a His tag, and replacement of the ectodomain by that of another coronavirus M protein did not affect the ability of the M protein to associate with the S protein. However, complex formation was sensitive to changes in the transmembrane domains of this triple-spanning protein. Deletion of either the first two or the last two transmembrane domains, known not to affect the topology of the protein, led to a considerable decrease in complex formation, but association was not completely abrogated. Various effects of changes in the part of the M protein that is located at the cytoplasmic face of the membrane were observed. Deletions of the extreme carboxy-terminal tail appeared not to interfere with M-S complex formation. However, deletions in the amphipathic domain severely affected M-S interaction. Interestingly, changes in the amino-terminal and extreme carboxy-terminal domains of M, which did not disrupt the interaction with S, are known to be fatal to the ability of the protein to engage in virus particle formation (C. A. M. de Haan, L. Kuo, P. S. Masters, H. Vennema, and P. J. M. Rottier, J. Virol. 72:6838-6850, 1998). Apparently, the structural requirements of the M protein for virus particle assembly differ from the requirements for the formation of M-S complexes.     

Acute and Chronic Effects of Ethanol on Transbilayer Membrane Domains

Alcohols, including ethanol, have a specific effect on transbilayer and lateral membrane domains. Recent evidence has shown that alcohols in vitro have a greater effect on fluidity of one leaflet as compared to the other. The present study examined effects of chronic ethanol consumption on fluidity of synaptic plasma membrane (SPM) exofacial and cytofacial leaflets using trinitrobenzenesulfonic acid (TNBS) labeling and differential polarized fluorometry of 1,6-diphenyl-1,3,5-hexatriene (DPH). Mice were administered ethanol or a control liquid diet for 3 weeks. Animals were killed and SPM prepared. The exofacial leaflet of SPM was significantly more fluid than the cytofacial leaflet in both groups, as indicated by limiting anisotropy of DPH. However, differences between the two leaflets were much smaller in the ethanol-treated group. Ethanol at concentrations seen clinically had a greater effect in vitro on the more fluid exofacial leaflet. This asymmetric effect of ethanol was significantly diminished in the exofacial leaflet of the ethanol-treated mice. Chronic ethanol consumption has a specific effect on membranes. Membrane functions that may be regulated by asymmetry of fluidity and lipid distribution may be altered by chronic ethanol consumption.     

Protein Partitioning into Ordered Membrane Domains: Insights from Simulations

Cellular membranes are laterally organized into domains of distinct structures and compositions by the differential interaction affinities between various membrane lipids and proteins. A prominent example of such structures are lipid rafts, which are ordered, tightly packed domains that have been widely implicated in cellular processes. The functionality of raft domains is driven by their selective recruitment of specific membrane proteins to regulate their interactions and functions; however, there have been few general insights into the factors that determine the partitioning of membrane proteins between coexisting liquid domains. In this work, we used extensive coarse-grained and atomistic molecular dynamics simulations, potential of mean force calculations, and conceptual models to describe the partitioning dynamics and energetics of a model transmembrane domain from the linker of activation of T cells. We find that partitioning between domains is determined by an interplay between protein-lipid interactions and differential lipid packing between raft and nonraft domains. Specifically, we show that partitioning into ordered domains is promoted by preferential interactions between peptides and ordered lipids, mediated in large part by modification of the peptides by saturated fatty acids (i.e., palmitoylation). Ordered phase affinity is also promoted by elastic effects, specifically hydrophobic matching between the membrane and the peptide. Conversely, ordered domain partitioning is disfavored by the tight molecular packing of the lipids therein. The balance of these dominant drivers determines partitioning. In the case of the wild-type linker of activation of T cells transmembrane domain, these factors combine to yield enrichment of the peptide at L_o/L_d interfaces. These results define some of the general principles governing protein partitioning between coexisting membrane domains and potentially explain previous disparities among experiments and simulations across model systems.     

Dissecting the Functional Domains of a Nonenveloped Virus Membrane Penetration Peptide

Recent studies have established that several nonenveloped viruses utilize virus-encoded lytic peptides for host membrane disruption. We investigated this mechanism with the “gamma” peptide of the insect virus Flock House virus (FHV). We demonstrate that the C terminus of gamma is essential for membrane disruption in vitro and the rescue of immature virus infectivity in vivo, and the amphipathic N terminus of gamma alone is not sufficient. We also show that deletion of the C-terminal domain disrupts icosahedral ordering of the amphipathic helices of gamma in the virus. Our results have broad implications for understanding membrane lysis during nonenveloped virus entry.The presence of membrane lytic peptides in many nonenveloped viruses is well established (3, 16), but how these peptides are deployed from the virus capsid during host cell entry and disrupt membranes remains unclear. These peptides are typically generated by a postassembly proteolytic processing event (1, 11) and are exposed from a previously buried position during conformational alterations in the capsid triggered by host cell conditions (2, 18). Flock House virus (FHV), an insect nodavirus, contains a 4-kDa peptide called “gamma” (γ), which shares many of the characteristics of other nonenveloped virus lytic peptides (3). The FHV capsid is made from 180 copies of a single-coat protein (α) enclosing a single-stranded bipartite RNA genome (9). Gamma is generated by the autocatalytic cleavage of α during virus maturation (α → β + γ) (15), remains localized in the capsid interior (9) with occasional externalization or “breathing” (6), and is exposed under low-pH conditions in the endosomes during entry (Odegard et al., submitted for publication). Covalently independent gamma is necessary for virus infection, since maturation-defective FHV (D75N/N363T FHV), which does not undergo the autocleavage of α, is not infectious (15, 17). The N-terminal ∼21 residues of gamma (corresponding to residues 364 to 384 of α) constitute an amphipathic helix which can disrupt membranes in vitro when synthetically produced (4, 5) and is recognized as the host membrane-interacting region of FHV during entry. The hydrophobic, ∼23-residue-long C terminus of gamma, especially certain phenylalanine residues (at positions 402, 405, and 407), is responsible for specifically packaging viral RNA into capsids during assembly (14).It was recently demonstrated that a supply of full-length gamma from noninfectious virus-like particles (VLPs) of FHV (13) during entry can restore infectivity to maturation-defective FHV (17), suggesting that gamma can function in trans to mediate access into host cells. This trans-complementation assay (17) was utilized to provide a quantitative readout of the effect of gamma mutations specifically on virus entry and to thus assess the region(s) of gamma required during virus entry. To determine the minimal sequence of gamma required for trans-complementation, FHV VLPs were produced that included the first 384, 390, or 395 amino acids of capsid protein α (designated Δγ384, Δγ390, and Δγ395, respectively). These VLPs contained only the amphipathic region of gamma, or that and additional parts of the C-terminal region (Fig. ​(Fig.1A),1A), and underwent normal assembly and maturation cleavage (Fig. ​(Fig.1B).1B). The amount of progeny virus produced by trans-complementing maturation-defective D75N/N363T FHV with these mutated VLPs was negligible (∼5%) compared to that produced by the wild-type (WT) VLPs (considered 100%) (Fig. ​(Fig.1C).1C). Thus, the amphipathic region of gamma was not sufficient by itself to restore infectivity to maturation-defective FHV, and the C terminus of gamma, beyond residue 395, was essential for rescue. Three phenylalanines located beyond residue 395 in gamma were separately mutated to alanines, and mature VLPs were generated (Fig. 1A and B) and tested in the trans-complementation assay. The relative efficiencies of rescue demonstrated by the F402A, F405A, and F407A VLPs were 15%, 29%, and 43%, respectively (Fig. ​(Fig.1C),1C), compared to that by the WT VLPs (100%). This rescue efficiency was similar to the relative infectivity previously determined for FHV containing the same point mutations (14). This suggests that the phenylalanines at the gamma C terminus are not only involved in the specific packaging of genomic RNA during FHV assembly (14) but are also required during virus entry.Open in a separate windowFIG. 1.C-terminal region of gamma required for trans-complementation during the entry of maturation-defective FHV. (A) Schematic of truncations or single mutations in the gamma region of FHV capsid protein α in VLPs. The sequence of gamma in each of the mutated VLPs is shown, with the N-terminal amphipathic region boxed. The single F→A mutations in F402A, F405A, and F407A are indicated in boldface. (B) A total of 5 μl of WT, Δγ384, phenylalanine mutant, or D75N VLPs, at a concentration of 5 mg/ml, was subjected to SDS-PAGE on a 4 to 20% Tris-glycine gel (Invitrogen) and stained with Coomassie brilliant blue. The position of gamma is indicated. The cleavage-defective D75N VLPs do not have gamma. (C) Drosophila DL-1 cells (1 × 10⁸) were coinfected with 1.5 × 10³ particles/cell of D75N/N363T FHV and 9 × 10³ particles/cell of WT, Δγ384, Δγ390, Δγ395, F402A, F405A, or F407A VLPs. [³⁵S]methionine-cysteine-labeled progeny virus was quantified, with the amount of progeny produced during coinfection with D75N/N363T FHV and WT VLPs normalized at 100%. The standard deviation was calculated from three replicates.Since the primary function of gamma during FHV entry is expected to be host membrane disruption, the ability of the mutated VLPs to disrupt DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine)-treated liposomes and release enclosed fluorescent dye was determined in comparison to the WT VLPs. We found that the in vivo rescue behavior of VLPs containing truncated gamma correlated with their in vitro membrane disruption activities. WT VLPs, at a concentration of 0.1 mg/ml (6.37 × 10¹¹ particles), released dye from liposomes at pH 7.0 (Fig. ​(Fig.2A)2A) and at a much higher rate at pH 6.0 (Fig. ​(Fig.2B),2B), which mimics the acidic endosomal environment. In contrast, the Δγ384, Δγ390, and Δγ395 VLPs were severely impaired in disrupting liposomes at both pH conditions (Fig. 2A and B), indicating that diminished endosomal membrane lysis by truncated gamma peptides could be responsible for the inability of mutated VLPs to rescue the infectivity of maturation-defective particles. The maturation-defective D75N VLP, which is unable to rescue infection (17), was also inefficient in liposome disruption at a neutral or low pH (Fig. 2A and B), indicating that in vitro membrane disruption by VLPs is a reliable indicator of their in vivo entry behavior.Open in a separate windowFIG. 2.Disruption of liposomes and fluorescent dye release by VLPs in vitro. In each case, total fluorescence is normalized to dye release achieved by the addition of 0.1% Triton X-100 to liposomes under the same conditions. In the case of panels A, B, and D, closely similar results were obtained in three different experiments, whereas the standard deviations in panel C were calculated from three replicates. Fluorescence measurements were carried out at excitation/emission maxima of 492/514 nm for 6-carboxyfluorescein and 535/585 nm for SulfoB. (A) Kinetic study of 6-carboxyfluorescein release from DOPC-treated liposomes upon the addition of 6.37 × 10¹¹ particles (lipid/particle molar ratio, 481:1) of WT, gamma-truncated, or maturation-defective D75N VLPs to liposomes in 50 mM HEPES (pH 7.0). (B) SulfoB release from DOPC-treated liposomes in 50 mM Bis-Tris (pH 6.0) upon the addition of 6.37 × 10¹¹ particles of VLPs. (C) SulfoB release from DOPC-treated liposomes by 6.37 × 10¹¹ particles of WT, F402A, F405A, or F407A VLPs after 1 h at pH 7.0 (black) or by 2 × 10¹¹ particles (lipid/particle molar ratio, 1,387:1) of each of the VLPs after 15 min at pH 6.0 (gray). (D) SulfoB fluorescence upon the addition of heat-released gamma peptide with the WT sequence or with phenylalanine mutations at F402, F405, and F407 to dye-filled liposomes.VLPs containing single phenylalanine mutations in gamma were able to disrupt liposomes but were overall less effective than the WT. At a concentration of 0.1 mg/ml at pH 7.0, F402A and F405A VLPs displayed ∼60% liposome disruption (1 h postincubation) compared to that of the WT (Fig. ​(Fig.2C).2C). At pH 6.0, these VLPs caused significantly less liposome disruption than the WT at an early time point (15 min postincubation) at a concentration of 0.035 mg/ml (2 × 10¹¹ particles) (Fig. ​(Fig.2C),2C), although after 45 min of incubation, the extent of the liposome disruption approached the WT levels (data not shown). The F407A mutant VLPs, approximately half as competent as the WT VLPs in rescuing infection (Fig. ​(Fig.1C),1C), were nonetheless as efficient as the WT VLPs in membrane disruption at low pH, although they were ∼80% as competent at a neutral pH (Fig. ​(Fig.2C).2C). When gamma was isolated from the WT and phenylalanine mutant VLPs, by heating equal amounts (80 μg) at 65°C in Bis-Tris (pH 6.0) and centrifuging the heated material on a 30% sucrose cushion, the top fraction, which contained WT or mutated gamma peptides, caused the immediate release of fluorescent dye from liposomes (Fig. ​(Fig.2D).2D). Given the dissimilar behavior of particle-associated phenylalanine mutant gamma and free mutant gamma, we hypothesized that the mutations in the C terminus could affect the organization of gamma within the particle.To test this hypothesis, cryoelectron microscopy (CryoEM) and image reconstruction were carried out to locate and compare the gamma peptides of WT FHV VLPs and Δγ384 VLPs, which had the most drastic truncation in gamma and could provide robust structural evidence. Data were collected on frozen hydrated samples of the VLPs (concentrated to 11 mg/ml in 50 mM HEPES [pH 7.0]) at the National Resource for Automated Molecular Microscopy on an FEI Tecnai F20 electron microscope operating at 120 kV. Images were processed using the EMAN suite (10), with a map calculated from the protein atomic coordinates of FHV (PDB entry no. 2Z2Q) as the starting model for image reconstructions. Real space and rigid body refinement was carried out using Chimera (12) and CNS (7), respectively. The estimated resolution for each image reconstruction was 8.8 Å, and the final R/R_free were 28.4%/28.3% for WT and 29.5%/29.3% for Δγ384 VLPs. While the WT and Δγ384 reconstructions were nearly indistinguishable at the surface, striking changes were detected in the gamma region (Fig. ​(Fig.3B).3B). To quantify these changes, the electron densities for the pseudoatomic models of FHV, lacking the interior RNA, the gamma helices, and the capsid protein N termini, and calculated at the resolution of the CryoEM image reconstructions, were subtracted from the WT and Δγ384 VLP image reconstructions. The volume of the density associated with the gamma helices and the capsid protein N termini was calculated above a threshold of 0.4σ. While the densities of the capsid protein N termini (residues 59 to 72), calculated as controls, appeared similar for the A, B, and C subunits in the icosahedral asymmetric unit (iASU) between the two reconstructions, the densities of the gamma helices (residues 364 to 381) in the A and B subunits were increased approximately fourfold and twofold, respectively, in the WT reconstruction, whereas the density of C-subunit gamma was fourfold stronger in the Δγ384 reconstruction (Fig. ​(Fig.3C).3C). The magnitude of the difference in density represents a clear structural dissimilarity between the WT and Δγ384 VLPs, with the gamma amphipathic helices becoming less icosahedrally ordered overall in the Δγ384 reconstruction. Interestingly, the pentameric helical bundles formed by gamma N termini at the fivefold axis of symmetry of the FHV capsid (Fig. ​(Fig.3A),3A), and thought to be primarily involved in membrane interaction (8), were visible in the WT VLP reconstruction, but the corresponding density weakened significantly in the Δγ384 VLP (Fig. ​(Fig.3B),3B), indicating significant disorder.Open in a separate windowFIG. 3.CryoEM image reconstructions of WT and Δγ384 VLPs. (A) Model of WT FHV showing the iASU consisting of the A, B, and C subunits, as well as the A subunits at the fivefold axis of symmetry. An expanded view of the fivefold axis shows the amphipathic region of the gamma peptides (red) forming a pentameric helical bundle. (B) Panel I, inner surface of WT VLP and Δγ384 VLP image reconstructions contoured at 0.4σ. The coloring is as follows: density for the capsid protein N termini (residues 59 to 72) in the iASU is in yellow, density for the gamma amphipathic helices (residues 364 to 381) is in cyan, and a potential pocket factor is in purple. Modeled into the density for the gamma amphipathic helices are the corresponding residues from the FHV crystal structure (PDB entry 2Z2Q) in red. Panel II, inner view, looking down on the fivefold axis of symmetry of WT and Δγ384 VLP image reconstructions. The density for the gamma amphipathic helices from the A subunits is in cyan, with the corresponding residues from the FHV crystal structure modeled in red. A pocket factor is in purple. (C) Volume ratios corresponding to the gamma amphipathic helices and the capsid protein N termini for subunits A, B, and C in the iASU. The reported ratio for each volume pair is the WT VLP density to the corresponding Δγ384 VLP density.Our data show that the N-terminal amphipathic helix of gamma, previously thought to be the key to infection, is not sufficient for membrane permeabilization during FHV entry and is coincidentally less icosahedrally ordered in the capsid interior in the absence of the C terminus. Although the role of the gamma C terminus in entry is not clear, one interesting possibility is that the phenylalanine residues in this region interact with other capsid elements or packaged RNA to maintain the N-terminal helices in a structural conformation essential for biological activity. We demonstrate that other regions of nonenveloped virus lytic peptides, in addition to the membrane-interacting domains, might be essential in order to gain maximum leverage during entry.     

Bile Acids Modulate Signaling by Functional Perturbation of Plasma Membrane Domains

Eukaryotic cell membranes are organized into functional lipid and protein domains, the most widely studied being membrane rafts. Although rafts have been associated with numerous plasma membrane functions, the mechanisms by which these domains themselves are regulated remain undefined. Bile acids (BAs), whose primary function is the solubilization of dietary lipids for digestion and absorption, can affect cells by interacting directly with membranes. To investigate whether these interactions affected domain organization in biological membranes, we assayed the effects of BAs on biomimetic synthetic liposomes, isolated plasma membranes, and live cells. At cytotoxic concentrations, BAs dissolved synthetic and cell-derived membranes and disrupted live cell plasma membranes, implicating plasma membrane damage as the mechanism for BA cellular toxicity. At subtoxic concentrations, BAs dramatically stabilized domain separation in Giant Plasma Membrane Vesicles without affecting protein partitioning between coexisting domains. Domain stabilization was the result of BA binding to and disordering the nonraft domain, thus promoting separation by enhancing domain immiscibility. Consistent with the physical changes observed in synthetic and isolated biological membranes, BAs reorganized intact cell membranes, as evaluated by the spatial distribution of membrane-anchored Ras isoforms. Nanoclustering of K-Ras, related to nonraft membrane domains, was enhanced in intact plasma membranes, whereas the organization of H-Ras was unaffected. BA-induced changes in Ras lateral segregation potentiated EGF-induced signaling through MAPK, confirming the ability of BAs to influence cell signal transduction by altering the physical properties of the plasma membrane. These observations suggest general, membrane-mediated mechanisms by which biological amphiphiles can produce their cellular effects.     

Phosphatidylserine Membrane Translocation in Human Spermatozoa: Topography in Membrane Domains and Relation to Cell Vitality

The complex structure of the human spermatozoa membrane comprises five topographic domains. Transmembrane asymmetry of the distribution of phospholipids including phosphatidylserine (PS) is considered a marker of cell activity. The objective of the study was to determine which cytomembrane domains of human spermatozoa are involved in PS membrane translocation and to identify the possible relationship of PS translocation with spermatozoa morphology and vitality. In normozoospermic semen of 35 donors, annexin-V labeling with fluorescein determined PS translocation. Propidium iodide staining distinguished between vital and dead spermatozoa. Three types of PS membrane translocation have been distinguished: (1) in the midpiece, (2) in the acrosomal part and (3) simultaneously in the midpiece and acrosomal part. In morphologically normal vital spermatozoa, PS translocation occurred in the midpiece but never in the equatorial region. In dead spermatozoa, simultaneous PS translocation in the midpiece and acrosomal part was most often observed. The difference between proportions of, respectively, vital and dead spermatozoa presenting PS translocation located in different domains was significant (P < 0.0001). In vital cells, there was no difference in PS translocation prevalence between morphologically normal and abnormal spermatozoa (P > 0.05). The strict relation of PS translocation to specific membrane domains indicates functional specificity. It seems doubtful to include this phenomenon in physiological mechanisms of elimination of abnormal spermatozoa.     

In Situ Determination of Structure and Fluctuations of Coexisting Fluid Membrane Domains

Biophysical understanding of membrane domains requires accurate knowledge of their structural details and elasticity. We report on a global small angle x-ray scattering data analysis technique for coexisting liquid-ordered (L_o) and liquid-disordered (L_d) domains in fully hydrated multilamellar vesicles. This enabled their detailed analysis for differences in membrane thickness, area per lipid, hydrocarbon chain length, and bending fluctuation as demonstrated for two ternary mixtures (DOPC/DSPC/CHOL and DOPC/DPPC/CHOL) at different cholesterol concentrations. L_o domains were found to be ∼10 Å thicker, and laterally up to 20 Å²/lipid more condensed than L_d domains. Their bending fluctuations were also reduced by ∼65%. Increase of cholesterol concentration caused significant changes in structural properties of L_d, while its influence on L_o properties was marginal. We further observed that temperature-induced melting of L_o domains is associated with a diffusion of cholesterol to L_d domains and controlled by L_o/L_d thickness differences.