Obstructed diffusion in phase-separated supported lipid bilayers: a combined atomic force microscopy and fluorescence recovery after photobleaching approach

Proteins and other macromolecules are believed to hinder molecular lateral diffusion in cellular membranes. We have constructed a well-characterized model system to better understand how obstacles in lipid bilayers obstruct diffusion. Fluorescence recovery after photobleaching was used to measure the lateral diffusion coefficient in single supported bilayers composed of mixtures of 1,2-dilauroylphosphotidylcholine (DLPC) and 1,2-distearoylphosphotidylcholine (DSPC). Because these lipids are immiscible and phase separate at room temperature, a novel quenching technique allowed us to construct fluid DLPC bilayers containing small disk-shaped gel-phase DSPC domains that acted as obstacles to lateral diffusion. Our experimental setup enabled us to analyze the same samples with atomic force microscopy and exactly characterize the size, shape, and number of gel-phase domains before measuring the obstacle-dependent diffusion coefficient. Lateral obstructed diffusion was found to be dependent on obstacle area fraction, size, and geometry. Analysis of our results using a free area diffusion model shows the possibility of unexpected long-range ordering of fluid-phase lipids around the gel-phase obstacles. This lipid ordering has implications for lipid-mediated protein interactions in cellular membranes.     

Dynamin self-assembly and the vesicle scission mechanism: how dynamin oligomers cleave the membrane neck of clathrin-coated pits during endocytosis

Recently, Gao et al. and Chappie et al. elucidated the crystal structures of the polytetrameric stalk domain of the dynamin-like virus resistance protein, MxA, and of the G-domain dimer of the large, membrane-deforming GTPase, dynamin, respectively. Combined, they provide a hypothetical oligomeric structure for the complete dynamin protein. Here, it is discussed how the oligomers are expected to form and how they participate in dynamin mediated vesicle fission during the process of endocytosis. The proposed oligomeric structure is compared with the novel mechanochemical model of dynamin function recently proposed by Bashkirov et al. and Pucadyil and Schmid. In conclusion, the new model of the dynamin oligomer has the potential to explain how short self-limiting fissogenic dynamin assemblies are formed and how concerted GTP hydrolysis is achieved. The oligomerisation of two other dynamin superfamily proteins, the guanylate binding proteins (GBPs) and the immunity-related GTPases (IRGs), is addressed briefly.     

Enzymatic hydrolysis of cod (Gadus morhua) by-products: Optimization of yield and properties of lipid and protein fractions

The main products of hydrolysis of fish by-products are hydrolysed protein and oil. The aim of this work was to study the effect of initial heat inactivation of endogenous enzymes, addition of water prior to hydrolysis, use of different commercial enzymes and combination of enzymes on the yield and purity of the protein and oil fractions after enzymatic hydrolysis of cod by-products. This study was designed to examine how all these factors were effective for destroying protein–lipid complexes in order to obtain pure oil and protein fractions and reduce the insoluble fraction.

Initial heating of raw material changed both raw material properties and inactivated endogenous enzymes thereby influencing the following hydrolysis. High amount of lipids in raw material combined with initial heating caused formation of protein–lipid complexes which was found in all protein containing fractions. The main constituents of the lipids in the complexes were phospholipids and other polar lipids. Insoluble protein–lipid complexes formed lead to increased amount of sludge, reduced FPH yield and high amount of lipids in FPH. The highest amount of separated oil was obtained in the experiments after initial heating without added water. These treatments also reduce amount of emulsion, which is not a desirable product after hydrolysis. Initial heating caused denaturation of protein, which decreased their emulsifying properties.

Results showed that it is not possible to obtain all desirable quality indicators such as: maximum oil and FPH yield, minimum emulsion and sludge yield and the highest protein recovery in FPH with the lowest amount of lipids in FPH fraction by using only one hydrolysis process. Therefore, the aim and requirements for the final products should be prioritised and defined very clearly before the process is designed taking into account the composition of raw material. Hydrolysis of unheated raw material with Alcalase and addition of water was the best compromise taking into account the mentioned quality indicators.     

Evidence for the hydrolysis of topical applied liposomal lipids in human stratum corneum

Liposomes were incubated with an extract of human plantar stratum corneum. The liposomal lipids were hydrolysed, if composed of soy-bean phosphatidylcholine or phosphatidylglycerol. Rigid lipids were not degraded. The temperature optimum of the hydrolysis was between 30-35degC. CaCl enhanced, while EDTA reduced the rate of hydrolysis, indicating that the hydrolysis is due to a phospholipase A(2).     

Enzyme-catalyzed hydrolysis of the supported phospholipid bilayers studied by atomic force microscopy

Atomic force microscopy (AFM) is employed to reveal the morphological changes of the supported phospholipid bilayers hydrolyzed by a phospholipase A₂ (PLA₂) enzyme in a buffer solution at room temperature. Based on the high catalytic selectivity of PLA₂ toward l-enantiomer phospholipids, five kinds of supported bilayers made of l- and d-dipalmitoylphosphatidylcholines (DPPC), including l-DPPC (upper leaflet adjacent to solution)/l-DPPC (bottom leaflet) (or l/l in short), l/d, d/l, d/d, and racemic ld/ld, were prepared on a mica surface in gel-phase, to explicate the kinetics and mechanism of the enzyme-induced hydrolysis reaction in detail. AFM observations for the l/l bilayer show that the hydrolysis rate for l-DPPC is significantly increased by PLA₂ and most of the hydrolysis products desorb from substrate surface in 40 min. As d-enantiomers are included in the bilayer, the hydrolysis rate is largely decreased in comparison with the l/l bilayer. The time used to hydrolyze the as-prepared bilayers by PLA₂ increases in the sequence of l/l, l/d, ld/ld, and d/l (d/d is inert to the enzyme action). d-enantiomers in the enantiomer hybrid bilayers remain on the mica surface at the end of the hydrolysis reaction. It was confirmed that the hydrolysis reaction catalyzed by PLA₂ preferentially occurs at the edges of pits or defects on the bilayer surface. The bilayer structures are preserved during the hydrolysis process. Based on these observations, a novel kinetics model is proposed to quantitatively account for the PLA₂-catalyzed hydrolysis of the supported phospholipid bilayers. The model simulation demonstrates that PLA₂ mainly binds with lipids at the perimeter of defects in the upper leaflet and leads to a hydrolysis reaction, yielding species soluble to the solution phase. The lipid molecules underneath subsequently flip up to the upper leaflet to maintain the hydrophilicity of the bilayer structure. Our analysis shows that d-enantiomers in the hybrid bilayers considerably reduce the hydrolysis rate by its ineffective binding with PLA₂.     

Hydrolysis of industrial substrates by an extremely stable thermolysin-like protease variant obtained by protein engineering

Various industrially used protein substrates were hydrolysed by a recently constructed, thermally stable, thermolysin-like protease variant (Boilysin; Van den Burg et al., Proc. Natl. Acad. Sci. 95: 2056–2060) and three industrial protease preparations. Hydrolysates were analysed by measuring the acid-soluble products and by SDS-PAGE of the breakdown products. The rate and extent of hydrolysis obtained by Boilysin was, in most cases, higher than those obtained with the three commercially available enzyme preperations tested. This suggests that protein hydrolysis with this new protease variant at elevated temperatures can result in improved substrate conversions.     

Selectivity in enzyme catalysed reaction: Preferential hydrolysis of saturated methyl ester over the corresponding unsaturated ester by pig liver esterase

Summary Saturated methyl ester is preferentially hydrolysed in presence of corresponding ,-unsaturated ester by Pig Liver Esterasse. This selectivity is known for aliphatic, aromatic and heterocyclic esters.Chemo, regio and stereoselective transformations are an important goal towards synthetic efficiency (Bartman and Trost, 1984). Among the various methods that are employed to achieve such reactions, enzymatic transformations are in the forefront of current research activities (Jones, 1986) because of several advantages over the chemical methods. These include extremely high degree of selectivity, easier isolation of products, specially with immobilized enzymes and their reusability. In the past decade this class of biocatalysts has provided a plathora of valuable selective transformations (Boland et.al, 1991). Pig Liver Esterase (PLE) is one such enzyme which has been thoroughly studied and used for asymmetric (Zhu and Tedford, 1990) and regioselective (Adachi et.al, 1986) hydrolysis of esters. Several chiral building blocks for the synthesis of complex natural products (Imori et.al., 1983) have been synthesized through PLE. In this communication, we report the preferential hydrolysis of a saturated ester over its unsaturated counterpart. The preference is total and works well for aliphatic, aromatic as well as heterocyclic systems.     

A lipid-phase separation model of low-temperature damage to biological membranes

An hypothesis is proposed to explain the damage caused to biological membranes exposed to low temperatures. The thesis rests on the general observation that the lipid components of most membranes are heterogeneous and undergo phase transitions from gel-phase lamellae to liquid-crystalline lamellae and some to a non-lamellar, hexagonal-II phase over a wide range of temperatures. As a consequence of these phase transitions the lateral distribution of the lipids characteristic of the growth temperature is disturbed and redistribution takes place on the basis of the temperature at which phase transitions occur. When membranes are cooled, first the non-lamellar forming lipids pass through a transition to a fluid lamellar phase and are miscible with bilayer-forming lipids into which they diffuse. On further cooling the high-melting-point lipids begin to crystallize and separate into a lamellar gel phase, in the process excluding the low-melting point lipids and intrinsic proteins. The lipids in these remaining regions form a gel phase at the lowest temperature. It is suggested that, because the non-lamellar lipids tend to undergo a liquid-crystalline to gel-phase transition at higher temperatures than lamellar-forming lipids, these will tend to phase separate into a gel phase domain rich in these lipids. Damage results when the membrane is reheated, whereupon the hexagonal-II-forming lipids give rise to non-lamellar structures. These probably take the form of inverted micelles sandwiched within the lipid bilayer and they completely destroy the permeability barrier properties of the membrane. The model is consistent with the phase behavior of membrane lipids and the action of cryoprotective agents in modifying lipid phase properties.     

Does the anthropometric model influence whole-body center of mass calculations in gait?

Examining whole-body center of mass (COM) motion is one of method being used to quantify dynamic balance and energy during gait. One common method for estimating the COM position is to apply an anthropometric model to a marker set and calculate the weighted sum from known segmental COM positions. Several anthropometric models are available to perform such a calculation. However, to date there has been no study of how the anthropometric model affects whole-body COM calculations during gait. This information is pertinent to researchers because the choice of anthropometric model may influence gait research findings and currently the trend is to consistently use a single model. In this study we analyzed a single stride of gait data from 103 young adult participants. We compared the whole-body COM motion calculated from 4 different anthropometric models (Plagenhoef et al., 1983; Winter, 1990; de Leva, 1996; Pavol et al., 2002). We found that anterior-posterior motion calculations are relatively unaffected by the anthropometric model. However, medial-lateral and vertical motions are significantly affected by the use of different anthropometric models. Our findings suggest that the researcher carefully choose an anthropometric model to fit their study populations when interested in medial-lateral or vertical motions of the COM. Our data can provide researchers a priori information on the model determination depending on the particular variable and how conservative they may want to be with COM comparisons between groups.     

Interpretation and predictions of the Emergent neutrality model: a reply to Barabás et al.

Formulated in 2006, Scheffer and van Nes’ Emergent neutrality model predicts that competing species might self‐organize into groups of species similar in their traits. Recently, Vergnon et al. showed that the model consistently generates multimodal species abundance distributions, in accordance with empirical data. Barabás et al. argue that Emergent neutrality model relies on unmodeled, ‘hidden’ species differences. They also suggest that an Emergent neutrality model explicitly integrating such differences may fail to generate multimodal species abundance distributions, while other models can robustly produce those patterns. Here we demonstrate that density dependence – the process deemed problematic by Barabás et al. – may permanently maintain groups of similar species without need for additional species differences. More broadly, we make it clear that density dependence is not the only likely mechanism that could allow the permanent coexistence of similar species in the Emergent neutrality framework. We welcome the finding that models other than Emergent neutrality can generate multimodal abundance distributions and we briefly discuss their novelty and relevance.     

Merocyanine 540, a fluorescent probe sensitive to lipid packing

Binding of the lipophilic probe merocyanine 540 to artificial bilayers was assessed by measuring the enhancement of fluorescence which results when dye enters the hydrophobic environment of the membrane. Titration of a constant amount of dye with increasing amounts of vesicles revealed that much more dye binds to multilamellar and 1000-Å, unilamellar vesicles which are in the fluid-phase state than to comparable vesicles which are in the gel-phase state. Incorporation of cholesterol into fluid-phase vesicles at levels of greater than 20 mol% reduced dye binding, whereas cholesterol had no effect at any concentration when incorporated into gel-phase vesicles. Sonicated 200–300-Å unilamellar gel-phase vesicles, which because of their reduced radius of curvature resemble fluid-phase bilayers in their more widely spaced exterior leaflet lipids, bound more dye than 1000-Å unilameilar gel-phase vesicles constructed from the same lipid. These results suggest that merocyanine 540 is able to sense the degree of lipid packing of bilayers and inserts preferentially into bilayers whose lipids are more widely spaced.     

Active flows cluster cell surface proteins

The plasma membrane of cells is a dynamic mixture of different lipids, proteins, and sugars. In a recent issue of Cell, Gowrishankar et?al. (2012) propose a model for how the actin cortex may generate and regulate lateral heterogeneity in the plasma membrane by actively clustering cell surface molecules.     

Nonlinear electron paramagnetic resonance studies of the interaction of cytochrome c oxidase with spin-labeled lipids in gel-phase membranes

The interaction of lipids, spin-labeled at different positions in the sn-2 chain, with cytochrome c oxidase reconstituted in gel-phase membranes of dimyristoylphosphatidylglycerol has been studied by electron paramagnetic resonance (EPR) spectroscopy. Nonlinear EPR methods, both saturation transfer EPR and progressive saturation EPR, were used. Interaction with the protein largely removes the flexibility gradient of the lipid chains in gel-phase membranes. The rotational mobility of the chain segments is reduced, relative to that for gel-phase lipids, by the intramembranous interaction with cytochrome c oxidase. This holds for all positions of chain labeling, but the relative effect is greater for chain segments closer to the terminal methyl ends. Modification of the paramagnetic metal-ion centers in the protein by binding azide has a pronounced effect on the spin-lattice relaxation of the lipid spin labels. This demonstrates that the centers modified are sufficiently close to the first-shell lipids to give appreciable dipolar interactions and that their vertical location in the membrane is closer to the 5-position than to the 14-position of the lipid chains.     

Incorporation of exogenous lipids modulates insulin signaling in the hepatoma cell line, HepG2.

The lipid content of cultured cells can be experimentally modified by supplementing the culture medium with specific lipids or by the use of phospholipases. In the case of the insulin receptor, these methods have contributed to a better understanding of lipid disorder-related diseases. Previously, our laboratory demonstrated that experimental modification of the cellular lipid composition of an insulin-sensitive rat hepatoma cell line (ZHC) resulted in an alteration in insulin receptor binding and biological action (Bruneau et al., Biochim. Biophys. Acta 928 (1987) 287-296/297-304). In this paper, we have examined the effects of lipid modification in another hepatoma cell line, HepG2. Exogenous linoleic acid (LA, n-6), eicosapentaenoic acid (EPA, n-3) or hemisuccinate of cholesterol (CHS) was added to HepG2 cells, to create a cellular model in which membrane composition was modified. In this model, we have shown that: (1) lipids were incorporated in treated HepG2 cells, but redistributed differently when compared to treated ZHC cells; (2) that insulin signaling events, such as insulin receptor autophosphorylation and the phosphorylation of the major insulin receptor substrate (IRS-1) were altered in response to the addition of membrane lipids or cholesterol derived components; and (3) different lipids affected insulin receptor signaling differently. We have also shown that the loss of insulin receptor autophosphorylation in CHS-treated cells can be correlated with a decreased sensitivity to insulin. Overall, the results suggest that the lipid environment of the insulin receptor may play an important role in insulin signal transduction.     

Microbial carboxylic ester hydrolases (EC 3.1.1) in food biotechnology

Ester linkages between carboxylic acid groups and hydroxyl groups are basic to the structure of carboxyesters and lipids, and occur commonly as modifications of polysaccharide molecules. Microorganisms produce enzymes which hydrolyse the carboxylic ester linkages in substrates which are being utilized for growth. Such esterase reactions are frequently easily reversible, depending on the concentration of reactants or availability of water. The importance of esters as flavour compounds has resulted in the selection of yeast strains which produce esters in beverage fermentation. The synthetic potential of triacylglycerol lipases (EC 3.1.1.3) has been exploited by use of the purified enzymes in environments of low water activity. The techniques of molecular biology facilitate analysis of the homology between carboxylesterase enzymes and a detailed knowledge of structure and specificity provides the opportunity to modify enzymes to suit particular applications in biotechnology.
Esterase activity can be assayed conveniently by using synthetic chromogenic esters of naphthol or nitrophenol. Naphthyl and nitrophenyl acetates are readily hydrolysed by a wide range of enzymes including lipases (Hofelmann et al. 1985; Brahimi-Horn et al. 1990; Gilbert et al. 1991), serine protease (Klapper et al. 1973) and acetylxylan esterase (Lee et al. 1987). Electrophoretic separation followed by detection using chromogenic esters has demonstrated polymorphism of esterase enzymes and has been used to type strains of bacteria (Goullet & Picard 1990, 1991; Picard & Goullet 1990) but was less discriminating with yeasts (Campbell et al. 1972) and edible mushrooms (Itavaara 1988).     

Are Accuracy and Reaction Time Affected via Different Processes?

A recent study by van Ede et al. (2012) shows that the accuracy and reaction time in humans of tactile perceptual decisions are affected by an attentional cue via distinct cognitive and neural processes. These results are controversial as they undermine the notion that accuracy and reaction time are influenced by the same latent process that underlie the decision process. Typically, accumulation-to-bound models (like the drift diffusion model) can explain variability in both accuracy and reaction time by a change of a single parameter. To elaborate the findings of van Ede et al., we fitted the drift diffusion model to their behavioral data. Results show that both changes in accuracy and reaction time can be partly explained by an increase in the accumulation of sensory evidence (drift rate). In addition, a change in non-decision time is necessary to account for reaction time changes as well. These results provide a subtle explanation of how the underlying dynamics of the decision process might give rise to differences in both the speed and accuracy of perceptual tactile decisions. Furthermore, our analyses highlight the importance of applying a model-based approach, as the observed changes in the model parameters might be ecologically more valid, since they have an intuitive relationship with the neuronal processes underlying perceptual decision making.     

Domains in binary SOPC/POPE lipid mixtures studied by pulsed field gradient 1H MAS NMR

We studied domain formation in mixtures of the monounsaturated lipids SOPC and POPE as a function of temperature and composition by NMR. Magic angle spinning at kHz frequencies restored resolution of (1)H NMR lipid resonances in the fluid phase, whereas the linewidth of gel-phase lipids remained rather broad and spinning frequency dependent. In regions of fluid- and gel-phase coexistence, spectra are a superposition of resonances from fluid and gel domains, as indicated by the existence of isosbestic points. Quantitative determination of the amount of lipid in the coexisting phases is straightforward and permitted construction of a binary phase diagram. Lateral rates of lipid diffusion were determined by (1)H MAS NMR with pulsed field gradients. At the onset of the phase transition near 25 degrees C apparent diffusion rates became diffusion time dependent, indicating that lipid movement is obstructed by the formation of gel-phase domains. A percolation threshold at which diffusion of fluid-phase lipid becomes confined to micrometer-size domains was observed when approximately 40% of total lipid had entered the gel phase. The results indicate that common phosphatidylethanolamines may trigger domain formation in membranes within a physiologically relevant temperature range. This novel NMR approach may aid the study of lipid rafts.     

AFM study of the thermotropic behaviour of supported DPPC bilayers with and without the model peptide WALP23

Temperature-controlled Atomic Force Microscopy (TC-AFM) in Contact Mode is used here to directly image the mechanisms by which melting and crystallization of supported, hydrated DPPC bilayers proceed in the presence and absence of the model peptide WALP23. Melting from the gel L_β′ to the liquid-crystalline L_α phase starts at pre-existing line-type packing defects (grain boundaries) in absence of the peptide. The exact transition temperature is shown to be influenced by the magnitude of the force exerted by the AFM probe on the bilayer, but is higher than the main transition temperature of non-supported DPPC vesicles in all cases due to bilayer–substrate interactions. Cooling of the fluid L_α bilayer shows the formation of the line-type defects at the borders between different gel-phase regions that originate from different nuclei. The number of these defects depends directly on the rate of cooling through the transition, as predicted by classical nucleation theory.The presence of the transmembrane, synthetic model peptide WALP23 is known to give rise to heterogeneity in the bilayer as microdomains with a striped appearance are formed in the DPPC bilayer. This striated phase consists of alternating lines of lipids and peptide. It is shown here that melting starts with the peptide-associated lipids in the domains, whose melting temperature is lowered by 0.8–2.0 °C compared to the remaining, peptide-free parts of the bilayer. The stabilization of the fluid phase is ascribed to adaptations of the lipids to the shorter peptide. The lipids not associated with the peptide melt at the same temperature as those in the pure DPPC supported bilayer.