Disulfide bridge-dependent dimerization triggers FGF2 membrane translocation into the extracellular space.

Fibroblast growth factor 2 (FGF2) exits cells by direct translocation across the plasma membrane, a type I pathway of unconventional protein secretion. This process is initiated by phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2)-dependent formation of highly dynamic FGF2 oligomers at the inner plasma membrane leaflet, inducing the formation of lipidic membrane pores. Cell surface heparan sulfate chains linked to glypican-1 (GPC1) capture FGF2 at the outer plasma membrane leaflet, completing FGF2 membrane translocation into the extracellular space. While the basic steps of this pathway are well understood, the molecular mechanism by which FGF2 oligomerizes on membrane surfaces remains unclear. In the current study, we demonstrate the initial step of this process to depend on C95-C95 disulfide-bridge-mediated FGF2 dimerization on membrane surfaces, producing the building blocks for higher FGF2 oligomers that drive the formation of membrane pores. We find FGF2 with a C95A substitution to be defective in oligomerization, pore formation, and membrane translocation. Consistently, we demonstrate a C95A variant of FGF2 to be characterized by a severe secretion phenotype. By contrast, while also important for efficient FGF2 secretion from cells, a second cysteine residue on the molecular surface of FGF2 (C77) is not involved in FGF2 oligomerization. Rather, we find C77 to be part of the interaction interface through which FGF2 binds to the α1 subunit of the Na,K-ATPase, the landing platform for FGF2 at the inner plasma membrane leaflet. Using cross-linking mass spectrometry, atomistic molecular dynamics simulations combined with a machine learning analysis and cryo-electron tomography, we propose a mechanism by which disulfide-bridged FGF2 dimers bind with high avidity to PI(4,5)P2 on membrane surfaces. We further propose a tight coupling between FGF2 secretion and the formation of ternary signaling complexes on cell surfaces, hypothesizing that C95-C95-bridged FGF2 dimers are functioning as the molecular units triggering autocrine and paracrine FGF2 signaling.

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.

Determining the Functional Oligomeric State of Membrane-Associated Protein Oligomers Forming Membrane Pores on Giant Lipid Vesicles.

Several peripheral membrane proteins are known to form membrane pores through multimerization. In many cases, in biochemical reconstitution experiments, a complex distribution of oligomeric states has been observed that may, in part, be irrelevant to their physiological functions. This phenomenon makes it difficult to identify the functional oligomeric states of membrane lipid interacting proteins, for example, during the formation of transient membrane pores. Using fibroblast growth factor 2 (FGF2) as an example, we present a methodology applicable to giant lipid vesicles by which functional oligomers can be distinguished from nonspecifically aggregated proteins without functionality. Two distinct populations of fibroblast growth factor 2 were identified with (i) dimers to hexamers and (ii) a broad population of higher oligomeric states of membrane-associated FGF2 oligomers significantly distorting the original unfiltered histogram of all detectable oligomeric species of FGF2. The presented statistical approach is relevant for various techniques for characterizing membrane-dependent protein oligomerization.

Interleaflet organization of membrane nanodomains: What can(not) be resolved by FRET?

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.

Ionic Strength and Solution Composition Dictate the Adsorption of Cell-Penetrating Peptides onto Phosphatidylcholine Membranes.

Adsorption of arginine-rich positively charged peptides onto neutral zwitterionic phosphocholine (PC) bilayers is a key step in the translocation of those potent cell-penetrating peptides into the cell interior. In the past, we have shown both theoretically and experimentally that polyarginines adsorb to the neutral PC-supported lipid bilayers in contrast to polylysines. However, comparing our results with previous studies showed that the results often do not match even at the qualitative level. The adsorption of arginine-rich peptides onto 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) may qualitatively depend on the actual experimental conditions where binding experiments have been performed. In this work, we systematically studied the adsorption of R9 and K9 peptides onto the POPC bilayer, aided by molecular dynamics (MD) simulations and fluorescence cross-correlation spectroscopy (FCCS) experiments. Using MD simulations, we tested a series of increasing peptide concentrations, in parallel with increasing Na+ and Ca2+ salt concentrations, showing that the apparent strength of adsorption of R9 decreases upon the increase of peptide or salt concentration in the system. The key result from the simulations is that the salt concentrations used experimentally can alter the picture of peptide adsorption qualitatively. Using FCCS experiments with fluorescently labeled R9 and K9, we first demonstrated that the binding of R9 to POPC is tighter by almost 2 orders of magnitude compared to that of K9. Finally, upon the addition of an excess of either Na+ or Ca2+ ions with R9, the total fluorescence correlation signal is lost, which implies the unbinding of R9 from the PC bilayer, in agreement with our predictions from MD simulations.

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.

Hidden complexity in membrane permeabilization behavior of antimicrobial polycations.

A promising alternative to classical antibiotics are antimicrobial peptides and their synthetic mimics (smAMPs) that supposedly act directly on membranes. For a more successful design of smAMPs, we need to know how the type of interaction with the membrane determines the type of membrane perturbation. How this, in turn, transfers into selectivity and microbial killing activity is largely unknown. Here, we characterize the action of two smAMPs: MM:CO (a copolymer of hydrophobic cyclooctyl subunits and charged ß-monomethyl-α-aminomethyl subunits) and the highly charged poly-NM (a homopolymer of α-aminomethyl subunits). By thorough characterization of vesicle leakage experiments, we elucidate complex membrane perturbation behavior in zwitterionic or negatively charged vesicles. Vesicle leakage data does not entirely agree with the growth inhibition of microbes. Our ensemble of advanced membrane permeabilization approaches clarifies these discrepancies. Long cumulative leakage kinetics show that the two smAMPs act either by transient leakage or by rare stochastic leakage events that occur at charge neutralization in the sample. We determine the strengths of individual leakage events induced by the smAMPs in membranes of various compositions. These strengths indicate changes in leakage mechanism over time and concentration range. Thus, our sophisticated analysis of vesicle leakage experiments reveals a fine-tuned flexibility in membrane permeabilization mechanisms. These details are indispensable in judging and designing membrane-active compounds.

Functional Assay to Correlate Protein Oligomerization States with Membrane Pore Formation.

In-membrane oligomerization is decisive for the function (or dysfunction) of many proteins. Techniques were developed to characterize membrane-inserted oligomers and the hereby obtained oligomerization states were intuitively related to the function of these proteins. However, in many cases, it is unclear whether the obtained oligomerization states are functionally relevant or are merely the consequence of nonspecific aggregation. Using fibroblast growth factor 2 (FGF2) as a model system, we addressed this methodological challenge. FGF2 oligomerizes in a PI(4,5)P2-dependent manner at the inner plasma membrane leaflet. This process results in membrane insertion and the formation of a lipidic membrane pore, the key intermediate in unconventional secretion of FGF2. To tackle the problem of discriminating functional oligomers from irrelevant aggregates, we present a statistical single molecule and single vesicle assay determining the brightness of individually diffusing in-membrane oligomers and correlating their oligomerization state with membrane pore formation. Importantly, time-dependent membrane pore formation was analyzed with an ensemble of single vesicles providing detailed statistics. Our findings demonstrate that quantifying oligomeric states alone does not allow for a deep understanding of the structure-function relationship of membrane-inserted oligomers.

Regulation of Cell Death by Mitochondrial Transport Systems of Calcium and Bcl-2 Proteins.

Mitochondria represent the fundamental system for cellular energy metabolism, by not only supplying energy in the form of ATP, but also by affecting physiology and cell death via the regulation of calcium homeostasis and the activity of Bcl-2 proteins. A lot of research has recently been devoted to understanding the interplay between Bcl-2 proteins, the regulation of these interactions within the cell, and how these interactions lead to the changes in calcium homeostasis. However, the role of Bcl-2 proteins in the mediation of mitochondrial calcium homeostasis, and therefore the induction of cell death pathways, remain underestimated and are still not well understood. In this review, we first summarize our knowledge about calcium transport systems in mitochondria, which, when miss-regulated, can induce necrosis. We continue by reviewing and analyzing the functions of Bcl-2 proteins in apoptosis. Finally, we link these two regulatory mechanisms together, exploring the interactions between the mitochondrial Ca2+ transport systems and Bcl-2 proteins, both capable of inducing cell death, with the potential to determine the cell death pathway-either the apoptotic or the necrotic one.

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.

Interleaflet Coupling of Lipid Nanodomains - Insights From in vitro Systems.

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.

Controlled Peptide-Mediated Vesicle Fusion Assessed by Simultaneous Dual-Colour Time-Lapsed Fluorescence Microscopy.

We have employed a model system, inspired by SNARE proteins, to facilitate membrane fusion between Giant Unilamellar Vesicles (GUVs) and Large Unilamellar Vesicles (LUVs) under physiological conditions. In this system, two synthetic lipopeptide constructs comprising the coiled-coil heterodimer-forming peptides K4, (KIAALKE)4, or E4, (EIAALEK)4, a PEG spacer of variable length, and a cholesterol moiety to anchor the peptides into the liposome membrane replace the natural SNARE proteins. GUVs are functionalized with one of the lipopeptide constructs and the fusion process is triggered by adding LUVs bearing the complementary lipopeptide. Dual-colour time lapse fluorescence microscopy was used to visualize lipid- and content-mixing. Using conventional confocal microscopy, lipid mixing was observed on the lipid bilayer of individual GUVs. In addition to lipid-mixing, content-mixing assays showed a low efficiency due to clustering of K4-functionalized LUVs on the GUVs target membranes. We showed that, through the use of the non-ionic surfactant Tween 20, content-mixing between GUVs and LUVs could be improved, meaning this system has the potential to be employed for drug delivery in biological systems.

CD8 Binding of MHC-Peptide Complexes in cis or trans Regulates CD8+ T-cell Responses.

The coreceptor CD8αß can greatly promote activation of T cells by strengthening T-cell receptor (TCR) binding to cognate peptide-MHC complexes (pMHC) on antigen presenting cells and by bringing p56Lck to TCR/CD3. Here, we demonstrate that CD8 can also bind to pMHC on the T cell (in cis) and that this inhibits their activation. Using molecular modeling, fluorescence resonance energy transfer experiments on living cells, biochemical and mutational analysis, we show that CD8 binding to pMHC in cis involves a different docking mode and is regulated by posttranslational modifications including a membrane-distal interchain disulfide bond and negatively charged O-linked glycans near positively charged sequences on the CD8ß stalk. These modifications distort the stalk, thus favoring CD8 binding to pMHC in cis. Differential binding of CD8 to pMHC in cis or trans is a means to regulate CD8+ T-cell responses and provides new translational opportunities.

Force Field Comparison of GM1 in a DOPC Bilayer Validated with AFM and FRET Experiments.

The great physiological relevance of glycolipids is being increasingly recognized, and glycolipid interactions have been shown to be central to cell-cell recognition, neuronal plasticity, protein-ligand recognition, and other important processes. However, detailed molecular-level understanding of these processes remains to be fully resolved. Molecular dynamics simulations could reveal the details of the glycolipid interactions, but the results may be influenced by the choice of the employed force field. Here, we have compared the behavior and properties of GM1, a common, biologically important glycolipid, using the CHARMM36, OPLS, GROMOS, and Amber99-GLYCAM06 (in bilayers comprising SLIPIDS and LIPID14 lipids) force fields in bilayers comprising 1,2-dioleoyl-sn-glycero-3-phosphocholine lipids and compared the results to atomic force microscopy and fluorescence resonance energy transfer experiments. We found discrepancies within the GM1 behavior displayed between the investigated force fields. Based on a direct comparison with complementary experimental results derived from fluorescence and AFM measurements, we recommend using the Amber99-GLYCAM force field in bilayers comprising LIPID14 or SLIPIDS lipids followed by CHARMM36 and OPLS force fields in simulations. The GROMOS force field is not recommended for reproducing the properties of the GM1 head group.

Experimental Evidence of the Existence of Interleaflet Coupled Nanodomains: An MC-FRET Study.

Plasma membranes of living cells are compartmentalized into small submicroscopic structures (nanodomains) having potentially relevant biological functions. Despite this, structural features of these nanodomains remain elusive, primarily due to the difficulties in characterizing such small dynamic entities. It is unclear whether nanodomains found in the upper bilayer leaflet are transversally registered with those found in the lower leaflet. Experiments performed on larger microscopic domains indicate that the coupling between the leaflets is strong, forcing the domains to be in perfect registration, but can the same thing be said about the biologically more relevant nanodomains? This work provides experimental evidence that even small nanodomains of variable sizes between 10 and 160 nm are interleaflet coupled. Importantly, the alternative scenarios of partially registered, independent, or antiregistered nanodomains could be excluded.

Arginine-rich cell-penetrating peptides induce membrane multilamellarity and subsequently enter via formation of a fusion pore.

Arginine-rich cell-penetrating peptides do not enter cells by directly passing through a lipid membrane; they instead passively enter vesicles and live cells by inducing membrane multilamellarity and fusion. The molecular picture of this penetration mode, which differs qualitatively from the previously proposed direct mechanism, is provided by molecular dynamics simulations. The kinetics of vesicle agglomeration and fusion by an iconic cell-penetrating peptide-nonaarginine-are documented via real-time fluorescence techniques, while the induction of multilamellar phases in vesicles and live cells is demonstrated by a combination of electron and fluorescence microscopies. This concert of experiments and simulations reveals that the identified passive cell penetration mechanism bears analogy to vesicle fusion induced by calcium ions, indicating that the two processes may share a common mechanistic origin.

Membrane Lipid Nanodomains.

Lipid membranes can spontaneously organize their components into domains of different sizes and properties. The organization of membrane lipids into nanodomains might potentially play a role in vital functions of cells and organisms. Model membranes represent attractive systems to study lipid nanodomains, which cannot be directly addressed in living cells with the currently available methods. This review summarizes the knowledge on lipid nanodomains in model membranes and exposes how their specific character contrasts with large-scale phase separation. The overview on lipid nanodomains in membranes composed of diverse lipids (e.g., zwitterionic and anionic glycerophospholipids, ceramides, glycosphingolipids) and cholesterol aims to evidence the impact of chemical, electrostatic, and geometric properties of lipids on nanodomain formation. Furthermore, the effects of curvature, asymmetry, and ions on membrane nanodomains are shown to be highly relevant aspects that may also modulate lipid nanodomains in cellular membranes. Potential mechanisms responsible for the formation and dynamics of nanodomains are discussed with support from available theories and computational studies. A brief description of current fluorescence techniques and analytical tools that enabled progress in lipid nanodomain studies is also included. Further directions are proposed to successfully extend this research to cells.

Distinct roles of SNARE-mimicking lipopeptides during initial steps of membrane fusion.

A model system for membrane fusion, inspired by SNARE proteins and based on two complementary lipopeptides CPnE4 and CPnK4, has been recently developed. It consists of cholesterol (C), a poly(ethylene glycol) linker (Pn) and either a cationic peptide K4 (KIAALKE)4 or an anionic peptide E4 (EIAALEK)4. In this paper, fluorescence spectroscopy is used to decipher distinct but complementary roles of these lipopeptides during early stages of membrane fusion. Molecular evidence is provided that different distances of E4 in CPnE4 and K4 in CPnK4 from the bilayer represent an important mechanism, which enables fusion. Whereas E4 is exposed to the bulk and solely promotes membrane binding of CPnK4, K4 loops back to the lipid-water interface where it fulfills two distinct roles: it initiates bilayer contact by binding to CPnE4 containing bilayers; and it initiates fusion by modulating the bilayer properties. The interaction between CPnE4 and CPnK4 is severely down-regulated by binding of K4 to the bilayer and possible only if the lipopeptides approach each other as constituents of different bilayers. When the complementary lipopeptides are localized in the same bilayer, hetero-coiling is disabled. These data provide crucial insights as to how fusion is initiated and highlight the importance of both peptides in this process.

Biomembrane Permeabilization: Statistics of Individual Leakage Events Harmonize the Interpretation of Vesicle Leakage.

The mode of action of membrane-active molecules, such as antimicrobial, anticancer, cell penetrating, and fusion peptides and their synthetic mimics, transfection agents, drug permeation enhancers, and biological signaling molecules (e.g., quorum sensing), involves either the general or local destabilization of the target membrane or the formation of defined, rather stable pores. Some effects aim at killing the cell, while others need to be limited in space and time to avoid serious damage. Biological tests reveal translocation of compounds and cell death but do not provide a detailed, mechanistic, and quantitative understanding of the modes of action and their molecular basis. Model membrane studies of membrane leakage have been used for decades to tackle this issue, but their interpretation in terms of biology has remained challenging and often quite limited. Here we compare two recent, powerful protocols to study model membrane leakage: the microscopic detection of dye influx into giant liposomes and time-correlated single photon counting experiments to characterize dye efflux from large unilamellar vesicles. A statistical treatment of both data sets does not only harmonize apparent discrepancies but also makes us aware of principal issues that have been confusing the interpretation of model membrane leakage data so far. Moreover, our study reveals a fundamental difference between nano- and microscale systems that needs to be taken into account when conclusions about microscale objects, such as cells, are drawn from nanoscale models.

Lipid Driven Nanodomains in Giant Lipid Vesicles are Fluid and Disordered.

It is a fundamental question in cell biology and biophysics whether sphingomyelin (SM)- and cholesterol (Chol)- driven nanodomains exist in living cells and in model membranes. Biophysical studies on model membranes revealed SM and Chol driven micrometer-sized liquid-ordered domains. Although the existence of such microdomains has not been proven for the plasma membrane, such lipid mixtures have been often used as a model system for 'rafts'. On the other hand, recent super resolution and single molecule results indicate that the plasma membrane might organize into nanocompartments. However, due to the limited resolution of those techniques their unambiguous characterization is still missing. In this work, a novel combination of Förster resonance energy transfer and Monte Carlo simulations (MC-FRET) identifies directly 10 nm large nanodomains in liquid-disordered model membranes composed of lipid mixtures containing SM and Chol. Combining MC-FRET with solid-state wide-line and high resolution magic angle spinning NMR as well as with fluorescence correlation spectroscopy we demonstrate that these nanodomains containing hundreds of lipid molecules are fluid and disordered. In terms of their size, fluidity, order and lifetime these nanodomains may represent a relevant model system for cellular membranes and are closely related to nanocompartments suggested to exist in cellular membranes.