A kinetic study of the growth of fatty acid vesicles

Membrane vesicles composed of fatty acids can be made to grow and divide under laboratory conditions, and thus provide a model system relevant to the emergence of cellular life. Fatty acid vesicles grow spontaneously when alkaline micelles are added to buffered vesicles. To investigate the mechanism of this process, we used stopped-flow kinetics to analyze the dilution of non-exchanging FRET probes incorporated into preformed vesicles during growth. Oleate vesicle growth occurs in two phases (fast and slow), indicating two pathways for the incorporation of fatty acid into preformed vesicles. We propose that the fast phase, which is stoichiometrically limited by the preformed vesicles, results from the formation of a "shell" of fatty acid around a vesicle, followed by rapid transfer of this fatty acid into the preformed vesicle. The slower phase may result from incorporation of fatty acid which had been trapped in an intermediate state. We provide independent evidence for the rapid transformation of micelles into an aggregated intermediate form after transfer from high to low pH. Our results show that the most efficient incorporation of added oleate into oleic acid/oleate vesicles occurs under conditions that avoid a large transient increase in the micelle/vesicle ratio.     

OmpA can form folded and unfolded oligomers

The monomeric outer membrane protein OmpA from Escherichia coli has long served as a model protein for studying the folding and membrane insertion of β-barrel membrane proteins. Here we report that when OmpA is refolded in limiting amounts of surfactant (close to the cmc), it has a high propensity to form folded and unfolded oligomers. The oligomers exist both in a folded and (partially) unfolded form which both dissociate under denaturing conditions. Oligomerization does not require the involvement of the periplasmic domain and is not strongly affected by ionic strength. The folded dimers can be isolated and show native-like secondary structure; they are resistant to proteolytic attack and do not dissociate in high surfactant concentrations, indicating high kinetic stability once formed. Remarkably, OmpA also forms significant amounts of higher order structures when refolding in the presence of lipid vesicles. We suggest that oligomerization occurs by domain swapping favored by the high local concentration of OmpA molecules congregating on the same micelle or vesicle. In this model, the unfolded oligomer is stabilized by a small number of intermolecular β-strand contacts and subsequently folds to a more stable state where these intermolecular contacts are consolidated in a native-like fashion by contacts between complementary β-strands from different molecules. Our model is supported by the ability of complementary fragments to associate with each other in vitro. Oligomerization is probably avoided in the cell by the presence of cellular chaperones which maintain the protein in a monomeric state.     

Automated formation of multicomponent-encapuslating vesosomes using continuous flow microcentrifugation

Vesosomes – hierarchical assemblies consisting of membrane-bound vesicles of various scales – are potentially powerful models of cellular compartmentalization. Current methods of vesosome fabrication are labor intensive, and offer little control over the size and uniformity of the final product. In this article, we report the development of an automated vesosome formation platform using a microfluidic device and a continuous flow microcentrifuge. In the microfluidic device, water-in-oil droplets containing nanoscale vesicles in the water phase were formed using T-junction geometry, in which a lipid monolayer is formed at the oil/water interface. These water-in-oil droplets were then immediately transferred to the continuous flow microcentrifuge. When a water-in-oil droplet passed through a second lipid monolayer formed in the continuous flow microcentrifuge, a bilayer-encapsulated vesosome was created, which contained all of the contents of the aqueous phase encapsulated within the vesosome. Encapsulation of nanoscale liposomes within the outer vesosome membrane was confirmed by fluorescence microscopy. Laser diffraction analysis showed that the vesosomes we fabricated were uniform (coefficient of variation of 0.029). The yield of the continuous flow microcentrifuge is high, with over 60% of impinging water droplets being converted to vesosomes. Our system provides a fully automatable route for the generation of vesosomes encapsulating arbitrary contents. The method employed in this work is simple and can be readily applied to a variety of systems, providing a facile platform for fabricating multicomponent carriers and model cells.     

Probing the dynamics of intact cells and nuclear envelope precursor membrane vesicles by deuterium solid state NMR spectroscopy

Membrane dynamics is an essential part of many cellular mechanisms such as intracellular trafficking, membrane fusion/fission and mitotic organelle reconstitution. The dynamics of membranes is dependent primarily on their phospholipid and cholesterol composition and how these molecules are ordered in relation to one another. To determine the physical status of membranes in whole cells or purified membranes of subcellular compartments we have developed a novel application exploiting solid-state ²H-NMR spectroscopy. We utilise this method to probe the dynamics of intact sperm and nuclear envelope precursor membranes. We show, using mass spectrometry, that either multilamellar or small unilamellar vesicles of deuterium-labelled palmitoyl-oleoylphosphatidylcholine can be used to probe the dynamics of sperm cells or nuclear envelope precursor membrane vesicles, respectively. Using ²H-NMR we determine the order parameters of sperm cells and nuclear envelope precursor membrane vesicles. We demonstrate that whole sperm membranes are more dynamic than nuclear envelope precursor membranes due to the higher cholesterol levels of the latter. Our new application can be exploited as a generic method for monitoring membrane dynamics in whole cells, various subcellular membrane compartments and membrane domains in subcellular compartments.     

Autotrophy versus heterotrophy: The origin of carbon determines its fate in a symbiotic sea anemone

Cnidarians - corals and their relatives - dominate the shallow, illuminated sea-floor in tropical nutrient-poor seas, mainly because their food sources are both heterotrophic, by predation, and autotrophic, from their intracellular symbiotic microalgae, the zooxanthellae. This trophic flexibility is rare in the animal kingdom, and the cellular usage and biological lifetime of the organic food molecules derived from both sources have never been investigated. Here we show that autotrophically-derived carbon is stored mostly in fatty tissue and is quickly consumed by the organism, while predation-derived carbon is incorporated throughout the animal’s body and remains there for a much longer time. We hypothesize that the simple photosynthate molecules are used primarily for immediate energetic processes like respiration, whereas the more complex heterotrophic organic molecules are utilized to build cellular structures such as proteins and membranes. Our study reveals, for the first time, the different fates and roles of autotrophy and heterothrophy in zooxanthellate coelenterates.     

Reconstitution of bacteriorhodopsin into cyclic lipid vesicles

Bacteriorhodopsin (bR), a membrane protein that can generate a light-driven proton pump, was successfully reconstituted into vesicles composed of an artificial cyclic lipid that mimics archaeal membrane lipids. Unlike reconstituted bR in 1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicles, the net topology and structure of bR molecules in cyclic lipid vesicles are identical to those in the native purple membrane of Halobacterium salinarum.     

Protein-mediated Selective Enclosure of Early Replicators Inside of Membranous Vesicles: First Step Towards Cell Membranes

Containment in cell membranes is essential for all contemporary life, and apparently even the earliest life forms had to be somehow contained. It has been postulated that random enclosure of replicating molecules inside of spontaneously assembled vesicles would have formed the initial cellular ancestors. However, completely random re-formation or division of such primitive vesicles would have abolished the heritability of their contents, nullifying any selective advantage to them. We propose that the containment of the early replicators in membranous vesicles was adopted only after the invention of genetically encoded proteins, and that selective enclosure of target molecules was mediated by specific proteins. A similar containment process is still utilised by various RNA- and retroviruses to isolate their replication complexes from the host’s intracellular environment. Such selective encapsulation would have protected the replicators against competitor and parasitic sequences, and provided a strong positive selection within the replicator communities.     

In vitro formation of the endoplasmic reticulum occurs independently of microtubules by a controlled fusion reaction

We have established an in vitro system for the formation of the endoplasmic reticulum (ER). Starting from small membrane vesicles prepared from Xenopus laevis eggs, an elaborate network of membrane tubules is formed in the presence of cytosol. In the absence of cytosol, the vesicles only fuse to form large spheres. Network formation requires a ubiquitous cytosolic protein and nucleoside triphosphates, is sensitive to N-ethylmaleimide and high cytosolic Ca(2+) concentrations, and proceeds via an intermediate stage in which vesicles appear to be clustered. Microtubules are not required for membrane tubule and network formation. Formation of the ER network shares significant similarities with formation of the nuclear envelope. Our results suggest that the ER network forms in a process in which cytosolic factors modify and regulate a basic reaction of membrane vesicle fusion.     

Profiling and functional classification of esterases in olive (Olea europaea) pollen during germination

Background and Aims

A pollen grain contains a number of esterases, many of which are released upon contact with the stigma surface. However, the identity and function of most of these esterases remain unknown. In this work, esterases from olive pollen during its germination were identifided and functionally characterized.

Methods

The esterolytic capacity of olive (Olea europaea) pollen was examined using in vitro and in-gel enzymatic assays with different enzyme substrates. The functional analysis of pollen esterases was achieved by inhibition assays by using specific inhibitors. The cellular localization of esterase activities was performed using histochemical methods.

Key Results

Olive pollen showed high levels of non-specific esterase activity, which remained steady after hydration and germination. Up to 20 esterolytic bands were identified on polyacrylamide gels. All the inhibitors decreased pollen germinability, but only diisopropyl fluorophosphate (DIFP) hampered pollen tube growth. Non-specific esterase activity is localized on the surface of oil bodies (OBs) and small vesicles, in the pollen intine and in the callose layer of the pollen tube wall. Acetylcholinesterase (AChE) activity was mostly observed in the apertures, exine and pollen coat, and attached to the pollen tube wall surface and to small cytoplasmic vesicles.

Conclusions

In this work, for the first time a systematic functional characterization of esterase enzymes in pollen from a plant species with wet stigma has been carried out. Olive pollen esterases belong to four different functional groups: carboxylesterases, acetylesterases, AChEs and lipases. The cellular localization of esterase activity indicates that the intine is a putative storage site for esterolytic enzymes in olive pollen. Based on inhibition assays and cellular localization of enzymatic activities, it can be concluded that these enzymes are likely to be involved in pollen germination, and pollen tube growth and penetration of the stigma.     

Two interdependent mechanisms of antimicrobial activity allow for efficient killing in nylon-3-based polymeric mimics of innate immunity peptides

Novel synthetic mimics of antimicrobial peptides have been developed to exhibit structural properties and antimicrobial activity similar to those of natural antimicrobial peptides (AMPs) of the innate immune system. These molecules have a number of potential advantages over conventional antibiotics, including reduced bacterial resistance, cost-effective preparation, and customizable designs. In this study, we investigate a family of nylon-3 polymer-based antimicrobials. By combining vesicle dye leakage, bacterial permeation, and bactericidal assays with small-angle X-ray scattering (SAXS), we find that these polymers are capable of two interdependent mechanisms of action: permeation of bacterial membranes and binding to intracellular targets such as DNA, with the latter necessarily dependent on the former. We systemically examine polymer-induced membrane deformation modes across a range of lipid compositions that mimic both bacteria and mammalian cell membranes. The results show that the polymers' ability to generate negative Gaussian curvature (NGC), a topological requirement for membrane permeation and cellular entry, in model Escherichia coli membranes correlates with their ability to permeate membranes without complete membrane disruption and kill E. coli cells. Our findings suggest that these polymers operate with a concentration-dependent mechanism of action: at low concentrations permeation and DNA binding occur without membrane disruption, while at high concentrations complete disruption of the membrane occurs. This article is part of a Special Issue entitled: Interfacially Active Peptides and Proteins. Guest Editors: William C. Wimley and Kalina Hristova.     

Glutamine Flux Imaging Using Genetically Encoded Sensors

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for biological molecules became available in the past 15 years, some of which became indispensable tools that are used routinely in many laboratories.One of the exciting applications of genetically encoded sensors is the use of these sensors in investigating cellular transport processes. Properties of transporters such as kinetics and substrate specificities can be investigated at a cellular level, providing possibilities for cell-type specific analyses of transport activities. In this article, we will demonstrate how transporter dynamics can be observed using genetically encoded glutamine sensor as an example. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed.     

The Role of the Lipid Bilayer in Tau Aggregation

Tau is a microtubule associated protein whose aggregation is implicated in a number of neurodegenerative diseases. We investigate the mechanism by which anionic lipid vesicles induce aggregation of tau in vitro using K18, a fragment of tau corresponding to the four repeats of the microtubule binding domain. Our results show that aggregation occurs when the amount of K18 bound to the lipid bilayer exceeds a critical surface density. The ratio of protein/lipid at the critical aggregation concentration is pH-dependent, as is the binding affinity. At low pH, where the protein binds with high affinity, the critical surface density is independent both of total lipid concentration as well as the fraction of anionic lipid present in the bilayer. Furthermore, the aggregates consist of both protein and vesicles and bind the β-sheet specific dye, Thioflavin T, in the manner characteristic of pathological aggregates. Our results suggest that the lipid bilayer facilitates protein-protein interactions both by screening charges on the protein and by increasing the local protein concentration, resulting in rapid aggregation. Because anionic lipids are abundant in cellular membranes, these findings contribute to understanding tau-lipid bilayer interactions that may be relevant to disease pathology.     

Amino acid sequence preferences to control cell‐specific organization of endothelial cells,smooth muscle cells,and fibroblasts

Effective surface modification with biocompatible molecules is known to be effective in reducing the life‐threatening risks related to artificial cardiovascular implants. In recent strategies in regenerative medicine, the enhancement and support of natural repair systems at the site of injury by designed biocompatible molecules have succeeded in rapid and effective injury repair. Therefore, such a strategy could also be effective for rapid endothelialization of cardiovascular implants to lower the risk of thrombosis and stenosis. To achieve this enhancement of the natural repair system, a biomimetic molecule that mimics proper cellular organization at the implant location is required. In spite of the fact that many reported peptides have cell‐attracting properties on material surfaces, there have been few peptides that could control cell‐specific adhesion. For the advanced cardiovascular implants, peptides that can mimic the natural mechanism that controls cell‐specific organization have been strongly anticipated. To obtain such peptides, we hypothesized the cellular bias toward certain varieties of amino acids and examined the cell preference (in terms of adhesion, proliferation, and protein attraction) of varieties and of repeat length on SPOT peptide arrays. To investigate the role of specific peptides in controlling the organization of various cardiovascular‐related cells, we compared endothelial cells (ECs), smooth muscle cells (SMCs), and fibroblasts (FBs). A clear, cell‐specific preference was found for amino acids (longer than 5‐mer) using three types of cells, and the combinational effect of the physicochemical properties of the residues was analyzed to interpret the mechanism. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Experimental approaches for addressing fundamental biological questions in living, functioning cells with single molecule precision

In recent years, single molecule experimentation has allowed researchers to observe biological processes at the sensitivity level of single molecules in actual functioning, living cells, thereby allowing us to observe the molecular basis of the key mechanistic processes in question in a very direct way, rather than inferring these from ensemble average data gained from traditional molecular and biochemical techniques. In this short review, we demonstrate the impact that the application of single molecule bioscience experimentation has had on our understanding of various cellular systems and processes, and the potential that this approach has for the future to really address very challenging and fundamental questions in the life sciences.     

A protective function for aggressive mimicry?

Mimicry often involves a protective element, whereby the risk of predation on mimics is reduced owing to their resemblance to unpalatable models. However, protection from predation has so far seemed unimportant in aggressive mimicry, where mimics are usually predators rather than prey. Here, we demonstrate that bluestriped fangblennies (Plagiotremus rhinorhynchos), which are aggressive mimics of juvenile bluestreak cleaner wrasse (Labroides dimidiatus), derive significant protection benefits from their resemblance to cleaner fish. Field observations revealed that mimetic fangblennies were chased by potential victims less often than individuals of a closely related, ecologically and behaviourally similar but non-mimetic species (Plagiotremus tapeinosoma). After attacks, proximity to models protected mimics from retaliation by victims, but the effect of colour similarity was less clear. Both colour resemblance and physical proximity to models thus appear to protect cleaner-fish mimics from aggression by potential and actual victims of their attacks. Our results suggest that the mimicry types observed in nature, which are usually distinguished on the basis of the benefits accrued to mimics, may in fact overlap greatly in the benefits provided.     

The chemical logic of a minimum protocell

Traditional schemes for the origin of cellular life on earth generally suppose that the chance assembly of polymer synthesis systems was the initial event, followed by incorporation into a membrane-enclosed volume to form the earliest cells. Here we discuss an alternative system consisting of replicating membrane vesicles, which we define as minimum protocells. These consist of vesicular bilayer membranes that self-assemble from relatively rare organic amphiphiles present in the prebiotic environment. If some of the amphiphiles are primitive pigment molecules asymmetrically oriented in the bilayer, light energy can be captured in the form of electrochemical ion gradients. This energy could then be used to convert relatively common precursor molecules into membrane amphiphiles, thereby providing an initial photosynthetic growth process, as well as an appropriate microenvironment for incorporation and evolution of polymer synthesis systems.     

Cleaner wrasse mimics inflict higher costs on their models when they are more aggressive towards signal receivers

Aggressive mimics are predatory species that resemble a 'model' species to gain access to food, mating opportunities or transportation at the expense of a signal receiver. Costs to the model may be variable, depending on the strength of the interaction between mimics and signal receivers. In the Indopacific, the bluestriped fangblenny Plagiotremus rhinorhynchos mimics juvenile cleaner wrasse Labroides dimidiatus. Instead of removing ectoparasites from larger coral reef fish, fangblennies attack fish to feed on scales and body tissue. In this study, juvenile cleaner wrasse suffered significant costs when associated with P. rhinorhynchos mimics in terms of reduced cleaning activity. Furthermore, the costs incurred by the model increased with heightened aggression by mimics towards signal receivers. This was apparently because of behavioural changes in signal receivers, as cleaning stations with mimics that attacked frequently were visited less. Variation in the costs incurred by the model may influence mimicry accuracy and avoidance learning by the signal receiver and thus affect the overall success and maintenance of the mimicry system.