An informative short-term study on the impacts of a triclocarban/weathered multi-walled carbon nanotube-adsorbed complex to benthic organisms.

Freshwater organisms are suitable models to study the fate of environmental pollutants. Due to their versatile and everyday use, many environmental pollutants such as triclocarban (TCC) or multi-walled carbon nanotubes (MWCNTs) enter environmental compartments very easily. TCC is known as a disinfectant and is declared as a highly aquatic toxicant. Multi-walled carbon nanotubes are used, e.g., in the automotive industry to improve plastic properties. Both TCCs and MWCNTs can pose major pollution hazards to various organisms. In addition, these substances can bind to each other due to their tendency to interact via strong hydrophobic interactions. Therefore, a short-term test was conducted to investigate the effects of the individual chemicals TCC and weathered MWCNTs (wMWCNTs) on a benthic biofilm and a grazing organism, Lymnaea stagnalis. Furthermore, the two compounds were coupled by an adsorption experiment resulting in a coupled complex formation (TCC + wMWCNTs). L. stagnalis showed no effects in terms of mortality. For benthic biofilm, the coupling test (TCC + wMWCNTs) showed a decrease of 58% in chlorophyll a (Chl-a) concentration. The main effect could be attributed to the wMWCNTs' exposure alone (decrease of 82%), but not to presence of TCC. The concentration range of Chl-a upon TCC exposure alone was comparable to that in the control group (32 and 37 µg/cm2). With respect to the particulate organic carbon (POC) concentration, very similar results were found for the solvent control, the TCC, and also for the TCC + wMWCNTs group (3, 2.9, and 2.9 mg/cm2). In contrast to the control, a significant increase in POC concentration (100%) was observed for wMWCNTs, but no synergistic effect of TCC + wMWCNTs was detected.

Impact of weathered multi-walled carbon nanotubes on the epithelial cells of the intestinal tract in the freshwater grazers Lymnaea stagnalis and Rhithrogena semicolorata.

Freshwater grazers are suitable organisms to investigate the fate of environmental pollutants, such as weathered multi-walled carbon nanotubes (wMWCNTs). One key process is the uptake of ingested materials into digestive or absorptive cells. To address this, we investigated the localization of wMWCNTs in the intestinal tracts of the mud snail Lymnaea stagnalis (L. stagnalis) and the mayfly Rhithrogena semicolorata (R. semicolorata). In L. stagnalis, bundles of wMWCNTs could be detected in the midgut lumen, whereas only single wMWCNTs could be detected in the lumina of the digestive gland. Intracellular uptake of wMWCNTs was detected by transmission electron microscopy (TEM) but was restricted to the cells of the digestive gland. In larvae of R. semicolorata, irritations of the microvilli and damages in the apical parts of the epithelial gut cells were detected after feeding with 1 to 10 mg/L wMWCNTs. In both models, we detected fibrillar structures in close association with the epithelial cells that formed peritrophic membranes (PMs). The PM may cause a reduced transmission of wMWCNT bundles into the epithelium by forming a filter barrier and potentially protecting the cells from the wMWCNTs. As a result, the uptake of wMWCNTs into cells is rare in mud snails and may not occur at all in mayfly larvae. In addition, we monitor physiological markers such as levels of glycogen or triglycerides and the RNA/DNA ratio. This ratio was significantly affected in L. stagnalis after 24 days with 10 mg/L wMWCNTs, but not in R. semicolorata after 28 days and 10 mg/L wMWCNTs. However, significant effects on the energy status of R. semicolorata were analysed after 28 days of exposure to 1 mg/L wMWCNTs. Furthermore, we observed a significant reduction of phagosomes per enterocyte cell in mayfly larvae at a concentration of 10 mg/L wMWCNTs (p < 0.01).

Multiwalled Carbon Nanotubes Promote Bacterial Conjugative Plasmid Transfer.

Multiwalled carbon nanotubes (MWCNTs) regularly enter aquatic environments due to their ubiquity in consumer products and engineering applications. However, the effects of MWCNT pollution on the environmental microbiome are poorly understood. Here, we evaluated whether these carbon nanoparticles can elevate the spread of antimicrobial resistance by promoting bacterial plasmid transfer, which has previously been observed for copper nanomaterials with antimicrobial properties as well as for microplastics. Through a combination of experimental liquid mating assays between Pseudomonas putida donor and recipient strains with plasmid pKJK5::gfpmut3b and mathematical modeling, we here demonstrate that the presence of MWCNTs leads to increased plasmid transfer rates in a concentration-dependent manner. The percentage of transconjugants per recipient significantly increased from 0.21 ± 0.04% in absence to 0.41 ± 0.09% at 10 mg L-1 MWCNTs. Similar trends were observed when using an Escherichia coli donor hosting plasmid pB10. The identified mechanism underlying the observed dynamics was the agglomeration of MWCNTs. A significantly increased number of particles with >6 µm diameter was detected in the presence of MWCNTs, which can in turn provide novel surfaces for bacterial interactions between donor and recipient cells after colonization. Fluorescence microscopy confirmed that MWCNT agglomerates were indeed covered in biofilms that contained donor bacteria as well as elevated numbers of green fluorescent transconjugant cells containing the plasmid. Consequently, MWCNTs provide bacteria with novel surfaces for intense cell-to-cell interactions in biofilms and can promote bacterial plasmid transfer, hence potentially elevating the spread of antimicrobial resistance. IMPORTANCE In recent decades, the use of carbon nanoparticles, especially multiwalled carbon nanotubes (MWCNTs), in a variety of products and engineering applications has been growing exponentially. As a result, MWCNT pollution into environmental compartments has been increasing. We here demonstrate that the exposure to MWCNTs can affect bacterial plasmid transfer rates in aquatic environments, an important process connected to the spread of antimicrobial resistance genes in microbial communities. This is mechanistically explained by the ability of MWCNTs to form bigger agglomerates, hence providing novel surfaces for bacterial interactions. Consequently, increasing pollution with MWCNTs has the potential to elevate the ongoing spread of antimicrobial resistance, a major threat to human health in the 21st century.

A workflow to investigate the impacts of weathered multi-walled carbon nanotubes to the mud snail Lymnaea stagnalis.

Although the development and application of nanomaterials is a growing industry, little data is available on the ecotoxicological effects on aquatic organisms. Therefore, we set up a workflow to address the potential uptake of weathered multi-walled carbon nanotubes (wMWCNTs) by a model organism, the pulmonary mud snail Lymnaea stagnalis (L. stagnalis), which plays an important role in the food web. It represents a suitable organism for this approach because as a grazer it potentially ingests large amounts of sedimented wMWCNTs. As food source for L. stagnalis, benthic biofilm was investigated by the use of a transmission electron microscope (TEM) and a scanning electron microscope (SEM) after exposure with wMWCNTs. In addition, isotopic labeling was applied with 14C-wMWCNTs (0.1 mg/L) to quantify fate, behavior, and enrichment of 14C-wMWCNTs in benthic biofilm and in L. stagnalis. Enrichment in benthic biofilm amounted to 529.0 µg wMWCNTs/g dry weight and in L. stagnalis to 79.6 µg wMWCNTs/g dry weight. A bioconcentration factor (BCF) for L. stagnalis was calculated (3500 L/kg). We demonstrate the accumulation of wMWCNTs (10 mg/L) in the digestive tract of L. stagnalis in an effect study. Moreover, the physiological markers glycogen and triglycerides as indicators for the physiological state, as well as the RNA/DNA ratio as growth indicator, were examined. No significant differences between exposed and control animals were analyzed for glycogen and triglycerides after 24 days of exposure, but a decreasing trend is recognizable for triglycerides. In contrast, the significant reduction in the RNA/DNA ratio of L. stagnalis indicated an inhibition of growth with a following recovery after depuration. The described workflow enables a comprehensive determination of the fate and the behavior of wMWCNTs specifically and in general all kinds of CNTs in the aquatic environment and therefore contributes to a holistic risk assessment of wMWCNTs.

Lipid Phase Control and Secondary Structure of Viral Fusion Peptides Anchored in Monoolein Membranes.

The fusion of lipid membranes involves major changes of the membrane curvatures and is mediated by fusion proteins that bind to the lipid membranes. For a better understanding of the way fusion proteins steer this process, we have studied the interaction of two different viral fusion peptides, HA2-FP and TBEV-FP, with monoolein mesophases as a function of temperature and pressure at limited hydration. The fusion peptides are derived from the influenza virus hemagglutinin fusion protein (HA2-FP) and from the tick-borne encephalitis virus envelope glycoprotein E (TBEV-FP). By using synchrotron X-ray diffraction, the changes of the monoolein phase behavior upon binding the peptides have been determined and the concomitant secondary structures of the peptides have been analyzed by FTIR spectroscopy. As main results we have found that the fusion peptides interact differently with monoolein and change the pressure and temperature dependent lipid phase behavior to different extents. However, they both destabilize the fluid lamellar phase and favor phases with negative curvature, i.e. inverse bicontinuous cubic and inverse hexagonal phases. These peptide-induced phase changes can partially be reversed by the application of high pressure, demonstrating that the promotion of negative curvature is achieved by a less dense packing of the monoolein membranes by the fusion peptides. Pressure jumps across the cubic-lamellar phase transition reveal that HA2-FP has a negligible effect on the rates of the cubic and the lamellar phase formation. Interestingly, the secondary structures of the fusion peptides appear unaffected by monoolein fluid-fluid phase transitions, suggesting that the fusion peptides are the structure dominant species in the fusion process of lipid membranes.

Phosphorylation Weakens but Does Not Inhibit Membrane Binding and Clustering of K-Ras4B.

K-Ras4B is one of the most frequently mutated Ras isoforms in cancer. The signaling activity of K-Ras4B depends on its localization to the plasma membrane (PM), which is mainly mediated by its polybasic farnesylated C-terminus. On top of the constitutive cycles that maintain the PM enrichment of K-Ras4B, conditional phosphorylation at Ser181 located within this motif has been found to be involved in regulating K-Ras4B's cell distribution and signaling activity. However, discordant observations have undermined our understanding of the role this phosphorylation plays. Here, we report an efficient strategy for producing K-Ras4B simultaneously bearing phosphate, farnesyl, and methyl modifications on a preparative scale, a very useful in vitro system when used in concert with model biomembranes. By using this system, we determined that phosphorylation at Ser181 does not fully inhibit membrane binding and clustering of K-Ras4B but reduces its membrane binding affinity, depending on membrane fluidity. In addition, phosphorylated K-Ras4B maintains tight association with its cytosolic shuttle protein PDEδ. After delivering K-Ras4B containing nonhydrolyzable phosphoserine mimetic into cells, the protein displayed a decreasing PM distribution compared with nonphosphorylable K-Ras4B, implying that phosphorylation might facilitate the dissociation of K-Ras4B from the PM. In addition, phosphorylation does not alter the localization of K-Ras4B in the liquid-disordered lipid subdomains of the membrane but slightly alters the thermotropic properties of K-Ras4B-incorporated membranes probably due to minor differences in membrane partitioning and dynamics. These results provide novel mechanistic insights into the role that phosphorylation at Ser181 plays in regulating K-Ras4B's distribution and activity.

Influence of isoform-specific Ras lipidation motifs on protein partitioning and dynamics in model membrane systems of various complexity.

The partitioning of the lipidated signaling proteins N-Ras and K-Ras4B into various membrane systems, ranging from single-component fluid bilayers, binary fluid mixtures, heterogeneous raft model membranes up to complex native-like lipid mixtures (GPMVs) in the absence and presence of integral membrane proteins have been explored in the last decade in a combined chemical-biological and biophysical approach. These studies have revealed pronounced isoform-specific differences regarding the lateral distribution in membranes and formation of protein-rich membrane domains. In this context, we will also discuss the effects of lipid head group structure and charge density on the partitioning behavior of the lipoproteins. Moreover, the dynamic properties of N-Ras and K-Ras4B have been studied in different model membrane systems and native-like crowded milieus. Addition of crowding agents such as Ficoll and its monomeric unit, sucrose, gradually favors clustering of Ras proteins in forming small oligomers in the bulk; only at very high crowder concentrations association is disfavored.

Regulation of K-Ras4B Membrane Binding by Calmodulin.

K-Ras4B is a membrane-bound small GTPase with a prominent role in cancer development. It contains a polybasic farnesylated C-terminus that is required for the correct localization and clustering of K-Ras4B in distinct membrane domains. PDEδ and the Ca(2+)-binding protein calmodulin (CaM) are known to function as potential binding partners for farnesylated Ras proteins. However, they differ in the number of interaction sites with K-Ras4B, leading to different modes of interaction, and thus affect the subcellular distribution of K-Ras4B in different ways. Although it is clear that Ca(2+)-bound CaM can play a role in the dynamic spatial cycle of K-Ras4B in the cell, the exact molecular mechanism is only partially understood. In this biophysical study, we investigated the effect of Ca(2+)/CaM on the interaction of GDP- and GTP-loaded K-Ras4B with heterogeneous model biomembranes by using a combination of different spectroscopic and imaging techniques. The results show that Ca(2+)/CaM is able to extract K-Ras4B from negatively charged membranes in a nucleotide-independent manner. Moreover, the data demonstrate that the complex of Ca(2+)/CaM and K-Ras4B is stable in the presence of anionic membranes and shows no membrane binding. Finally, the influence of Ca(2+)/CaM on the interaction of K-Ras4B with membranes is compared with that of PDEδ, which was investigated in a previous study. Although both CaM and PDEδ exhibit a hydrophobic binding pocket for farnesyl, they have different effects on membrane binding of K-Ras4B and hence should be capable of regulating K-Ras4B plasma membrane localization in the cell.

Lipoprotein insertion into membranes of various complexity: lipid sorting, interfacial adsorption and protein clustering.

In a combined chemical-biological and biophysical approach we explored the membrane partitioning of the lipidated signaling proteins N-Ras and K-Ras4B into membrane systems of different complexity, ranging from one-component lipid bilayers and anionic binary and ternary heterogeneous membrane systems even up to partitioning studies on protein-free and protein-containing giant plasma membrane vesicles (GPMVs). To yield a pictorial view of the localization process, imaging using confocal laser scanning and atomic force microscopy was performed. The results reveal pronounced isoform-specific differences regarding the lateral distribution and formation of protein-rich membrane domains. Line tension is one of the key parameters controlling not only the size and dynamic properties of segregated lipid domains but also the partitioning process of N-Ras that acts as a lineactant. The formation of N-Ras protein clusters is even recorded for almost vanishing hydrophobic mismatch. Conversely, for K-Ras4B, selective localization and clustering are electrostatically mediated by its polybasic farnesylated C-terminus. The formation of K-Ras4B clusters is also observed for the multi-component GPMV membrane, i.e., it seems to be a general phenomenon, largely independent of the details of the membrane composition, including the anionic charge density of lipid headgroups. Our data indicate that unspecific and entropy-driven membrane-mediated interactions play a major role in the partitioning behavior, thus relaxing the need for a multitude of fine-tuned interactions. Such a scenario seems also to be reasonable recalling the high dynamic nature of cellular membranes. Finally, we note that even relatively simple models of heterogeneous membranes are able to reproduce many of the properties of much more complex biological membranes.

Effect of the N-Terminal Helix and Nucleotide Loading on the Membrane and Effector Binding of Arl2/3.

The small GTP-binding proteins Arl2 and Arl3, which are close homologs, share a number of interacting partners and act as displacement factors for prenylated and myristoylated cargo. Nevertheless, both proteins have distinct biological functions. Whereas Arl3 is considered a ciliary protein, Arl2 has been reported to be involved in tubulin folding, mitochondrial function, and Ras signaling. How these different roles are attained by the two homolog proteins is not fully understood. Recently, we showed that the N-terminal amphipathic helix of Arl3, but not that of Arl2, regulates the release of myristoylated ciliary proteins from the GDI-like solubilizing factor UNC119a/b. In the biophysical study presented here, both proteins are shown to exhibit a preferential localization and clustering in liquid-disordered domains of phase-separated membranes. However, the membrane interaction behavior differs significantly between both proteins with regard to their nucleotide loading. Whereas Arl3 and other Arf proteins with an N-terminal amphipathic helix require GTP loading for the interaction with membranes, Arl2 binds to membranes in a nucleotide-independent manner. In contrast to Arl2, the N-terminal helix of Arl3 increases the binding affinity to UNC119a. Furthermore, UNC119a impedes membrane binding of Arl3, but not of Arl2. Taken together, these results suggest an interplay among the nucleotide status of Arl3, the location of the N-terminal helix, membrane fluidity and binding, and the release of lipid modified cargos from carriers such as UNC119a. Since a specific Arl3-GEF is postulated to reside inside cilia, the N-terminal helix of Arl3•GTP would be available for allosteric regulation of UNC119a cargo release only inside cilia.

Exploring the structure and phase behavior of plasma membrane vesicles under extreme environmental conditions.

Not only drastic temperature- but also pressure-induced perturbations of membrane organization pose a serious challenge to the biological cell. Although high hydrostatic pressure significantly influences the structural properties and thus functional characteristics of cells, this has not prevented life from invading the high pressure habitats of marine depths where pressures up to the 100 MPa level are encountered. Here, the temperature- and pressure-dependent structure and phase behavior of giant plasma membrane vesicles have been explored in the absence and presence of membrane proteins using a combined spectroscopic and microscopic approach. Demixing into extended liquid-ordered and liquid-disordered domains is observed over a wide range of temperatures and pressures. Only at pressures beyond 200 MPa a physiologically unfavorable all gel-like ordered lipid phase is reached at ambient temperature. This is in fact the pressure range where the membrane-protein function has generally been observed to cease, thereby shedding new light on the possible origin of this observation.

Interaction of the human N-Ras protein with lipid raft model membranes of varying degrees of complexity.

Ternary lipid mixtures composed of cholesterol, saturated (frequently with sphingosine backbone), and unsaturated phospholipids show stable phase separation and are often used as model systems of lipid rafts. Yet, their ability to reproduce raft properties and function is still debated. We investigated the properties and functional aspects of three lipid raft model systems of varying degrees of biological relevance--PSM/POPC/Chol, DPPC/POPC/Chol, and DPPC/DOPC/Chol--using 2H solid-state nuclear magnetic resonance (NMR) spectroscopy, fluorescence microscopy, and atomic force microscopy. While some minor differences were observed, the general behavior and properties of all three model mixtures were similar to previously investigated influenza envelope lipid membranes, which closely mimic the lipid composition of biological membranes. For the investigation of the functional aspects, we employed the human N-Ras protein, which is posttranslationally modified by two lipid modifications that anchor the protein to the membrane. It was previously shown that N-Ras preferentially resides in liquid-disordered domains and exhibits a time-dependent accumulation in the domain boundaries of influenza envelope lipid membranes. For all three model mixtures, we observed the same membrane partitioning behavior for N-Ras. Therefore, we conclude that even relatively simple models of raft membranes are able to reproduce many of their specific properties and functions.

Discrete and polymeric heteronuclear constructs derived from triangular 2,2'-bipyrazine complexes of cis-a2Pt(II) (with a = NH3 or a2 = en).

cis-[Pta2(H2O)2](2+) (with a = NH3 or a2 = en) and 2,2'-bipyrazine (2,2'-bpz) react in water preferentially to cyclic, triangular complexes of composition [{cis-Pta2(2,2'-bpz-N4,N4')}3](6+) (a = NH3, 1a; a2 = en, 1b). In their C3-symmetric conformation (all bridging pyrazine rings adopt cisoid orientations with respect to the central C2-C2' bond), 1a and 1b provide three pairs of N1,N1' donor sites each, which are capable of chelating additional (hetero)metal ions. The latter can in turn bridge to other N1,N1' sites in an intermolecular fashion or simply complete their coordination spheres with other ligands (water, anions). Four previously not observed structural variants of heteronuclear (Pt(II), Cu(II); Pt(II), Ag(I); Pt(II), Cd(II)) constructs have now been isolated by us and are reported here: a decanuclear Pt6Cu4 complex, [{(en)Pt(2,2'-bpz)}3]2Cu4(H2O)6(NO3)20·11H2O (2) with the appearance of a paddle-wheel, a dodecanuclear Pt6Cu6 capsule with a Cu(2+) ion in its interior and additional Cu(2+) ions in its periphery giving a total composition of [{(en)Pt(2,2'-bpz)}3]2Cu11(NO3)34(H2O)18·3H2O (3), as well as two coordination polymers, {[{(en)Pt(2,2'-bpz)}3]Cd2(H2O)7}(SO4)5·{[Cd(H2O)6](SO4)}·15H2O (4) and [{cis-(NH3)2Pt(2,2'-bpz)}3]Ag(SiF6)3(BF4)·7H2O (5). The structures of these four compounds are dominated by host-guest interactions between the triangular metal vases of 1a and 1b and anions, respectively, as well as hydrogen bonding involving anions and water molecules and electrostatics. In aqueous solution considerable dissociation of the heteronuclear compounds 2-5 into the triangular Pt complexes 1 and the heterometal ions takes place. Preliminary AFM (atomic force microscopy) studies reveal that the +6 cations of 1a have a strong tendency to interact with double-stranded DNA with formation of condensed DNA states.

Gibbs energy determinants of lipoprotein insertion into lipid membranes: the case study of Ras proteins.

In a combined chemical-biological and biophysical approach we explored the Gibbs (free) energy contributions to the membrane partitioning of lipidated proteins, and compared the theoretical predictions with recent experimental data on the membrane insertion of Ras proteins of various anchor systems into rationally designed model biomembrane systems. Various factors fostering or reducing the membrane partitioning properties are discussed, including hydrophobic effects, lipid chain mismatch, electrostatic interactions, membrane-mediated protein-protein interactions, and terms that account for line tension effects between coexisting lipid domains, lipid sorting, and changes in the lateral organization of the lipid bilayer system. From these data, it is apparent that two membrane anchoring motifs are needed to facilitate firm membrane binding. For heterogeneous membranes, localization and sequestration at domain boundaries as well as formation of protein clusters and collective lateral organization via an effective lipid sorting mechanism provide complementary ways of inducing membrane nanodomains that could potentially operate as effective, high fidelity signalling platforms.

The role of G-domain orientation and nucleotide state on the Ras isoform-specific membrane interaction.

Ras proteins are proto-oncogenes that function as molecular switches linking extracellular stimuli with an overlapping but distinctive range of biological outcomes. Although modulatable interactions between the membrane and the Ras C-terminal hypervariable region (HVR) harbouring the membrane anchor motifs enable signalling specificity to be determined by their location, it is becoming clear that the spatial orientation of different Ras proteins is also crucial for their functions. To reveal the orientation of the G-domain at membranes, we conducted an extensive study on different Ras isoforms anchored to model raft membranes. The results show that the G-domain mediates the Ras-membrane interaction by inducing different sets of preferred orientations in the active and inactive states with largely parallel orientation relative to the membrane of most of the helices. The distinct locations of the different isoforms, exposing them to different effectors and regulators, coupled with different G-domain-membrane orientation, suggests synergy between this type of recognition motif and the specificity conferred by the HVR, thereby validating the concept of isoform specificity in Ras.

Dissociation of the K-Ras4B/PDEδ complex upon contact with lipid membranes: membrane delivery instead of extraction.

K-Ras4B is a small GTPase whose selective membrane localization and clustering into microdomains are mediated by its polybasic farnesylated C-terminus. The importance of the subcellular distribution for the signaling activity of K-Ras4B became apparent from recent in vivo studies, showing that the delta subunit of cGMP phosphodiesterase (PDEδ), which possesses a hydrophobic prenyl-binding pocket, is able to function as a potential binding partner for farnesylated proteins, thereby leading to a modulation of the spatiotemporal organization of K-Ras. Even though PDEδ has been suggested to serve as a cytosolic carrier for Ras, the functional transport mechanism still remains largely elusive. In this study, the effect of PDEδ on the interaction of GDP- and GTP-loaded K-Ras4B with neutral and anionic model biomembranes has been investigated by a combination of different spectroscopic and imaging techniques. The results show that PDEδ is not able to extract K-Ras4B from membranes. Rather, the K-Ras4B/PDEδ complex formed in bulk solution turned out to be unstable in the presence of heterogeneous membranes, resulting in a release of farnesylated K-Ras4B upon membrane contact. With the additional observation of enhanced membrane affinity for the K-Ras4B/PDEδ complex, a molecular mechanism for the PDEδ-K-Ras4B-membrane interaction could be proposed. This includes an effective delivery of PDEδ-solubilized K-Ras4B to the plasma membrane, probably through cytoplasmic diffusion, the dissociation of the K-Ras4B/PDEδ complex upon plasma membrane contact, and finally the membrane binding of released farnesylated K-Ras4B that leads to K-Ras4B-enriched microdomain formation.

The effect of Aß on IAPP aggregation in the presence of an isolated ß-cell membrane.

Fibrillar aggregates of the islet amyloid polypeptide (IAPP) and amyloid-ß (Aß) are known to deposit at pancreatic ß-cells and neuronal cells and are associated with the cell degenerative diseases type-2 diabetes mellitus (T2DM) and Alzheimer's disease (AD), respectively. Since IAPP is secreted by ß-cells and a membrane-damaging effect of IAPP has been discussed as a reason for ß-cell dysfunction and the development of T2DM, studies of the interaction of IAPP with the ß-cell membrane are of high relevance for gaining a molecular-level understanding of the underlying mechanism. Recently, it has also been shown that patients suffering from T2DM exhibit an increased risk to develop AD and vice versa, and a molecular link between AD and T2DM has been suggested. In this study, membrane lipids from the rat insulinoma-derived INS-1E ß-cell line were isolated, and their interaction with the amyloidogenic peptides IAPP and Aß and a mixture of both peptides has been studied. To yield insight into the associated peptides' conformational changes and their effect on the membrane integrity during aggregation, we have carried out attenuated total reflection Fourier transform infrared spectroscopy, fluorescence microscopy, and atomic force microscopy experiments. The IAPP-Aß heterocomplexes formed were shown to adsorb, aggregate, and permeabilize the isolated ß-cell membrane significantly slower than pure IAPP, however, at a rate that is much faster than that of pure Aß. In addition, it could be shown that isolated ß-cell membranes cause similar effects on the kinetics of IAPP and IAPP-Aß fibril formation as anionic heterogeneous model membranes.

Temperature-pressure phase diagram of a heterogeneous anionic model biomembrane system: results from a combined calorimetry, spectroscopy and microscopy study.

By using Fourier transform infrared (FT-IR) spectroscopy in combination with differential scanning calorimetry (DSC) coupled with pressure perturbation calorimetry (PPC), ultrasound velocimetry, Laurdan fluorescence spectroscopy, fluorescence microscopy and atomic force microscopy (AFM), the temperature and pressure dependent phase behavior of the five-component anionic model raft lipid mixture DOPC/DOPG/DPPC/DPPG/cholesterol (20:5:45:5:25 mol%) was investigated. A temperature range from 5 to 65 °C and a pressure range up to 16 kbar were covered to establish the temperature-pressure phase diagram of this heterogeneous model biomembrane system. Incorporation of 10-20 mol% PG still leads to liquid-ordered (l(o))-liquid-disordered (l(d)) phase coexistence regions over a wide range of temperatures and pressures. Compared to the corresponding neutral model raft mixture (DOPC/DPPC/Chol 25:50:25 mol%), the p,T-phase diagram is - as expected and in accordance with the Gibbs phase rule - more complex, the phase sequence as a function of temperature and pressure is largely similar, however. This anionic heterogeneous model membrane system will serve as a more realistic model biomembrane system to study protein interactions with anionic lipid bilayers displaying liquid-disordered/liquid-ordered domain coexistence over a wide range of the temperature-pressure plane, thus allowing also studies of biologically relevant systems encountered under extreme environmental conditions.

Membrane-mediated induction and sorting of K-Ras microdomain signaling platforms.

The K-Ras4B GTPase is a major oncoprotein whose signaling activity depends on its correct localization to negatively charged subcellular membranes and nanoclustering in membrane microdomains. Selective localization and clustering are mediated by the polybasic farnesylated C-terminus of K-Ras4B, but the mechanisms and molecular determinants involved are largely unknown. In a combined chemical biological and biophysical approach we investigated the partitioning of semisynthetic fully functional lipidated K-Ras4B proteins into heterogeneous anionic model membranes and membranes composed of viral lipid extracts. Independent of GDP/GTP-loading, K-Ras4B is preferentially localized in liquid-disordered (l(d)) lipid domains and forms new protein-containing fluid domains that are recruiting multivalent acidic lipids by an effective, electrostatic lipid sorting mechanism. In addition, GDP-GTP exchange and, thereby, Ras activation results in a higher concentration of activated K-Ras4B in the nanoscale signaling platforms. Conversely, palmitoylated and farnesylated N-Ras proteins partition into the l(d) phase and concentrate at the l(d)/l(o) phase boundary of heterogeneous membranes. Next to the lipid anchor system, the results reveal an involvement of the G-domain in the membrane interaction process by determining minor but yet significant structural reorientations of the GDP/GTP-K-Ras4B proteins at lipid interfaces. A molecular mechanism for isoform-specific Ras signaling from separate membrane microdomains is postulated from the results of this study.