Advanced Imaging Approaches to Reveal Molecular Mechanisms Governing Neuroendocrine Secretion.

Identification of the molecular mechanisms governing neuroendocrine secretion and resulting intercellular communication is one of the great challenges of cell biology to better understand organism physiology and neurosecretion disruption-related pathologies such as hypertension, neurodegenerative, or metabolic diseases. To visualize molecule distribution and dynamics at the nanoscale, many imaging approaches have been developed and are still emerging. In this review, we provide an overview of the pioneering studies using transmission electron microscopy, atomic force microscopy, total internal reflection microscopy, and super-resolution microscopy in neuroendocrine cells to visualize molecular mechanisms driving neurosecretion processes, including exocytosis and associated fusion pores, endocytosis and associated recycling vesicles, and protein-protein or protein-lipid interactions. Furthermore, the potential and the challenges of these different advanced imaging approaches for application in the study of neuroendocrine cell biology are discussed, aiming to guide researchers to select the best approach for their specific purpose around the crucial but not yet fully understood neurosecretion process.

Impact of Carbon Source Supplementations on Pseudomonas aeruginosa Physiology.

Pseudomonas aeruginosa is an opportunistic pathogen highly resistant to a wide range of antimicrobial agents, making its infections very difficult to treat. Since microorganisms need to perpetually adapt to their surrounding environment, understanding the effect of carbon sources on P. aeruginosa physiology is therefore essential to avoid increasing drug-resistance and better fight this pathogen. By a global proteomic approach and phenotypic assays, we investigated the impact of various carbon source supplementations (glucose, glutamate, succinate, and citrate) on the physiology of the P. aeruginosa PA14 strain. A total of 581 proteins were identified as differentially expressed in the 4 conditions. Most of them were more abundant in citrate supplementation and were involved in virulence, motility, biofilm development, and antibiotic resistance. Phenotypic assays were performed to check these hypotheses. By coupling all this data, we highlight the importance of the environment in which the bacterium evolves on its metabolism, and thus the necessity to better understand the metabolic pathways implied in its adaptative response according to the nutrient availability.

Membrane phospholipid composition of Pseudomonas aeruginosa grown in a cystic fibrosis mucus-mimicking medium.

BACKGROUND: Pseudomonas aeruginosa is a bacterium able to induce serious pulmonary infections in cystic fibrosis (CF) patients. This bacterium is very often antibiotic resistant, partly because of its membrane impermeability, which is linked to the membrane lipid composition. This work aims to study the membrane phospholipids of P. aeruginosa grown in CF sputum-like media. METHODS: Three media were used: Mueller Hilton broth (MHB), synthetic cystic fibrosis medium (SCFM) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) complemented SCFM (SCFM-PC). Lipids were extracted and LC-MS/MS analyses were performed. Growth curves, atomic force microscopy images and minimal inhibitory concentration determination were performed in order to compare the growth and potentially link lipid modifications to antibiotic resistance. RESULTS: Semi-quantification showed phospholipid quantity variation depending on the growth medium. Phosphatidylcholines were detected in traces in SCFM. MS/MS experiments showed an increase of phospholipids derived from DOPC in SCFM-PC. We observed no influence of the medium on the bacterial growth and a minor influence on the bacterial shape. MIC values were generally higher in SCFM and SCFM-PC than in MHB. CONCLUSIONS: We defined a CF sputum-like media which can be used for the membrane lipid extraction of P. aeruginosa. We also showed that the growth medium does have an influence on its membrane lipid composition and antibiotic resistance, especially for SCFM-PC in which P. aeruginosa uses DOPC, in order to make its own membrane. GENERAL SIGNIFICANCE: Our results show that considerable caution must be taken when choosing a medium for lipid identification and antibiotic testing -especially for phospholipids-enriched media.

Deleterious Effects of an Air Pollutant (NO2) on a Selection of Commensal Skin Bacterial Strains, Potential Contributor to Dysbiosis?

The skin constitutes with its microbiota the first line of body defense against exogenous stress including air pollution. Especially in urban or sub-urban areas, it is continuously exposed to many environmental pollutants including gaseous nitrogen dioxide (gNO2). Nowadays, it is well established that air pollution has major effects on the human skin, inducing various diseases often associated with microbial dysbiosis. However, very few is known about the impact of pollutants on skin microbiota. In this study, a new approach was adopted, by considering the alteration of the cutaneous microbiota by air pollutants as an indirect action of the harmful molecules on the skin. The effects of gNO2 on this bacterial skin microbiota was investigated using a device developed to mimic the real-life contact of the gNO2 with bacteria on the surface of the skin. Five strains of human skin commensal bacteria were considered, namely Staphylococcus aureus MFP03, Staphylococcus epidermidis MFP04, Staphylococcus capitis MFP08, Pseudomonas fluorescens MFP05, and Corynebacterium tuberculostearicum CIP102622. Bacteria were exposed to high concentration of gNO2 (10 or 80 ppm) over a short period of 2 h inside the gas exposure device. The physiological, morphological, and molecular responses of the bacteria after the gas exposure were assessed and compared between the different strains and the two gNO2 concentrations. A highly significant deleterious effect of gNO2 was highlighted, particularly for S. capitis MFP08 and C. tuberculostearicum CIP102622, while S. aureus MFP03 seems to be the less sensitive strain. It appeared that the impact of this nitrosative stress differs according to the bacterial species and the gNO2 concentration. Thus the exposition to gNO2 as an air pollutant could contribute to dysbiosis, which would affect skin homeostasis. The response of the microbiota to the nitrosative stress could be involved in some pathologies such as atopic dermatitis.

Chromogranin A preferential interaction with Golgi phosphatidic acid induces membrane deformation and contributes to secretory granule biogenesis.

Chromogranin A (CgA) is a key luminal actor of secretory granule biogenesis at the trans-Golgi network (TGN) level but the molecular mechanisms involved remain obscure. Here, we investigated the possibility that CgA acts synergistically with specific membrane lipids to trigger secretory granule formation. We show that CgA preferentially interacts with the anionic glycerophospholipid phosphatidic acid (PA). In accordance, bioinformatic analysis predicted a PA-binding domain (PABD) in CgA sequence that effectively bound PA (36:1) or PA (40:6) in membrane models. We identified PA (36:1) and PA (40:6) as predominant species in Golgi and granule membranes of secretory cells, and we found that CgA interaction with these PA species promotes artificial membrane deformation and remodeling. Furthermore, we demonstrated that disruption of either CgA PABD or phospholipase D (PLD) activity significantly alters secretory granule formation in secretory cells. Our findings show for the first time the ability of CgA to interact with PLD-generated PA, which allows membrane remodeling and curvature, key processes necessary to initiate secretory granule budding.

Determination of the collision cross sections of cardiolipins and phospholipids from Pseudomonas aeruginosa by traveling wave ion mobility spectrometry-mass spectrometry using a novel correction strategy.

Collision cross section (CCS) values are descriptors of the 3D structure of ions which can be determined by ion mobility spectrometry (IMS). Currently, most lipidomic studies involving CCS value determination concern eukaryote samples (e.g. human, bovine) and to a lower extent prokaryote samples (e.g. bacteria). Here, we report CCS values obtained from traveling wave ion mobility spectrometry (TWCCSN2) measurements from the bacterial membrane of Pseudomonas aeruginosa-a bacterium ranked as priority 1 for the R&D of new antibiotics by the World Health Organization. In order to cover the lack of reference compounds which could cover the m/z and CCS ranges of the membrane lipids of P. aeruginosa, three calibrants (polyalanine, dextran and phospholipids) were used for the TWCCSN2 calibration. A shift from the published lipid CCS values was systematically observed (ΔCCS% up to 9%); thus, we proposed a CCS correction strategy. This correction strategy allowed a reduction in the shift (ΔCCS%) between our measurements and published values to less than 2%. This correction was then applied to determine the CCS values of Pseudomonas aeruginosa lipids which have not been published yet. As a result, 32 TWCCSN2 values for [M+H]+ ions and 24 TWCCSN2 values for [M-H]- ions were obtained for four classes of phospholipids (phosphatidylethanolamines (PE), phosphatidylcholines (PC), phosphatidylglycerols (PG) and diphosphatidylglycerols-known as cardiolipins (CL)). Graphical abstract.

Identification by mass spectrometry of glucosaminylphosphatidylglycerol, a phosphatidylglycerol derivative, produced by Pseudomonas aeruginosa.

RATIONALE: Pseudomonas aeruginosa is an opportunistic pathogen bacterium widely considered to be an excellent research model in several areas of molecular studies, namely genomics and proteomics. However, its lipid metabolism is still not totally decrypted. While it is known that this bacterium has the particularity to produce phosphatidylcholine, a lipid mainly found in eukaryotes, other singularities are still to be discovered. METHODS: P. aeruginosa was grown as planktonic cultures to the stationary state. Membrane pellets were collected and lipids were extracted using the Bligh and Dyer protocol. Lipid extracts were analyzed by Electrospray Ionization Mass Spectrometry (ESI-MS) using high-resolution mass spectrometer (LTQ Orbitrap Elite, Thermo Scientific) in the negative mode. MSn spectra were recorded both in the Orbitrap and in the ion trap analyzer (collision-induced dissociation (CID) or higher energy collision-induced dissociation (HCD) mode). RESULTS: We observed by mass spectrometry and thin layer chromatography that P. aeruginosa produced an unreferenced lipid in classical growth conditions. MS2 analysis of the unknown ion indicates that it is a phosphatidylglycerol derivative. The exact mass shift corresponds to glucosamine which is largely found in the metabolism of this bacterium. MS3 analysis of secondary ions allowed us to conclude that this lipid is a glucosaminylphosphatidylglycerol, a phosphatidylglycerol derivative containing a glucosamine substituted at C4. CONCLUSIONS: We show here that P. aeruginosa is able to produce glucosaminylphosphatidylglycerols via a probable esterification of phosphatidylglycerols by glucosamine.

Unsaturated Fatty Acids Affect Quorum Sensing Communication System and Inhibit Motility and Biofilm Formation of Acinetobacter baumannii.

The increasing threat of Acinetobacter baumannii as a nosocomial pathogen is mainly due to the occurrence of multidrug-resistant strains that are associated with the real problem of its eradication from hospital wards. The particular ability of this pathogen to form biofilms contributes to its persistence, increases antibiotic resistance, and promotes persistent/device-related infections. We previously demonstrated that virstatin, which is a small organic compound known to decrease virulence of Vibrio cholera via an inhibition of T4-pili expression, displayed very promising activity to prevent A. baumannii biofilm development. Here, we examined the antibiofilm activity of mono-unsaturated chain fatty acids, palmitoleic (PoA), and myristoleic (MoA) acids, presenting similar action on V. cholerae virulence. We demonstrated that PoA and MoA (at 0.02 mg/mL) were able to decrease A. baumannii ATCC 17978 biofilm formation up to 38% and 24%, respectively, presented a biofilm dispersing effect and drastically reduced motility. We highlighted that these fatty acids decreased the expression of the regulator abaR from the LuxIR-type quorum sensing (QS) communication system AbaIR and consequently reduced the N-acyl-homoserine lactone production (AHL). This effect can be countered by addition of exogenous AHLs. Besides, fatty acids may have additional non-targeted effects, independent from QS. Atomic force microscopy experiments probed indeed that PoA and MoA could also act on the initial adhesion process in modifying the material interface properties. Evaluation of fatty acids effect on 22 clinical isolates showed a strain-dependent antibiofilm activity, which was not correlated to hydrophobicity or pellicle formation ability of the tested strains, and suggested a real diversity in cell-to-cell communication systems involved in A. baumannii biofilm formation.

Pseudomonas aeruginosa produces phosphatidyltris(hydroxymethyl)aminomethane and derivatives when grown in Tris-buffered medium.

For optimal growth of a microorganism, the pH of the culture medium should be set at an optimum value. For that reason, growth media require buffering agents. We show in this study that, when grown in a medium supplemented with tris(hydroxymethyl)aminomethane (Tris), Pseudomonas aeruginosa is able to use this organic compound to produce new phospholipids. We thus pointed out that phosphatidyltris(hydroxymethyl)aminomethane as well as diphosphatidyltris(hydroxymethyl)aminomethane was detected in membrane lipid extracts of bacteria grown in Tris-buffered medium. Moreover, the amounts of lysoglycerophospholipids in the lipidome of P. aeruginosa grown in Tris-buffered medium increased leading to the presence of lysophosphatidylglycerol and lysophosphatidyltris(hydroxymethyl)aminomethane as well as other lysophospholipid derivatives. Finally, we investigated the effect of the presence of these exogenous phospholipids on the susceptibility of P. aeruginosa to some antibiotics. We observed a decrease of the minimal inhibitory concentrations of different antibiotic families, i.e., fluoroquinolones, aminoglycosides, ß-lactams and polymyxins, proving the importance of the buffer choice for growth medium and its impact on the lipidome.

Two methods for one-point anchoring of a linear polysaccharide on a gold surface.

Two strategies to achieve a one-point anchoring of a hydrolyzed pullulan (P9000) on a gold surface are compared. The first strategy consists of forming a self-assembled monolayer of a 6-amino-1-hexanethiol (AHT) and then achieving reductive amination on the surface between the aminated surface and the aldehyde of the polysaccharide reductive end sugar. The second consists of incorporating a thiol function at the extremity of the pullulan (via the same reductive amination), leading to P9000-AHT and then immobilizing it on gold by a spontaneous reaction between solid gold and thiol. The modified pullulan was characterized by NMR and size-exclusion chromatography coupled to a light-scattering detector. P9000-AHT appears to be in a disulfide dimer form in solution but recovers its unimer form with dithiothreitol (DTT) treatment. The comparison of the two strategies by contact angle and XPS revealed that the second strategy is more efficient for the pullulan one-point anchoring. P9000-AHT even in its dimer form is easily grafted onto the surface. The grafted polymer seems to be more in a coil conformation than in a rigid brush. Furthermore, QCM measurements highlighted that the second strategy leads to a grafting density of around 3.5 × 10(13) molecules·cm(-2) corresponding to a high surface coverage. The elaboration of a dense and oriented layer of polysaccharides covalently linked to a gold surface might enhance the use of such modified polysaccharides in various fields.

Characterisation of pellicles formed by Acinetobacter baumannii at the air-liquid interface.

The clinical importance of Acinetobacter baumannii is partly due to its natural ability to survive in the hospital environment. This persistence may be explained by its capacity to form biofilms and, interestingly, A. baumannii can form pellicles at the air-liquid interface more readily than other less pathogenic Acinetobacter species. Pellicles from twenty-six strains were morphologically classified into three groups: I) egg-shaped (27%); II) ball-shaped (50%); and III) irregular pellicles (23%). One strain representative of each group was further analysed by Brewster's Angle Microscopy to follow pellicle development, demonstrating that their formation did not require anchoring to a solid surface. Total carbohydrate analysis of the matrix showed three main components: Glucose, GlcNAc and Kdo. Dispersin B, an enzyme that hydrolyzes poly-N-acetylglucosamine (PNAG) polysaccharide, inhibited A. baumannii pellicle formation, suggesting that this exopolysaccharide contributes to pellicle formation. Also associated with the pellicle matrix were three subunits of pili assembled by chaperon-usher systems: the major CsuA/B, A1S_1510 (presented 45% of identity with the main pilin F17-A from enterotoxigenic Escherichia coli pili) and A1S_2091. The presence of both PNAG polysaccharide and pili systems in matrix of pellicles might contribute to the virulence of this emerging pathogen.

Characterization of membrane lipidome changes in Pseudomonas aeruginosa during biofilm growth on glass wool.

Bacteria cells within biofilms are physiologically distinct from their planktonic counterparts. In particular they are more resistant to detrimental environmental conditions. In this study, we monitored the evolution of the phospholipid composition of the inner and outer membranes of P. aeruginosa during the biofilm formation (i.e., from 1-, 2-, to 6-day-old biofilm). Lipidome analyses were performed by electrospray ionization mass spectrometry. In addition to the lipidomic analysis, the fatty acid composition was analysed by gas chromatography/mass spectrometry. We found that the lipidome alterations of the inner and the outer membranes varied with the biofilm age. These alterations in phospholipid compositions reflect a higher diversity in sessile organisms than in planktonic counterparts. The diversity is characterized by the presence of PE 30∶1, PE 31∶0 and PG 31∶0 for the lower masses as well as PE 38∶1, 38∶2, 39∶1, 39∶2 and PG 38∶0, 38∶1, 38∶2, 39∶1, 39∶2 for the higher masses. However, this lipidomic feature tends to disappear with the biofilm age, in particular the high mass phospholipids tend to disappear. The amount of branched chains phospholipids mainly located in the outer membrane decreased with the biofilm age, whereas the proportion of cyclopropylated phospholipids increased in both membranes. In bacteria present in oldest biofilms, i.e., 6-day-old, the phospholipid distribution moved closer to that of planktonic bacteria.

Virstatin inhibits biofilm formation and motility of Acinetobacter baumannii.

BACKGROUND: Acinetobacter baumannii has emerged as an opportunistic nosocomial pathogen causing infections worldwide. One reason for this emergence is due to its natural ability to survive in the hospital environment, which may be explained by its capacity to form biofilms. Cell surface appendages are important determinants of the A. baumannii biofilm formation and as such constitute interesting targets to prevent the development of biofilm-related infections. A chemical agent called virstatin was recently described to impair the virulence of Vibrio cholerae by preventing the expression of its virulence factor, the toxin coregulated pilus (type IV pilus). The objective of this work was to investigate the potential effect of virstatin on A. baumannii biofilms. RESULTS: After a dose-response experiment, we determined that 100 µM virstatin led to an important decrease (38%) of biofilms formed by A. baumannii ATCC17978 grown under static mode. We demonstrated that the production of biofilms grown under dynamic mode was also delayed and reduced. The biofilm susceptibility to virstatin was then tested for 40 clinical and reference A. baumannii strains. 70% of the strains were susceptible to virstatin (with a decrease of 10 to 65%) when biofilms grew in static mode, whereas 60% of strains respond to the treatment when their biofilms grew in dynamic mode. As expected, motility and atomic force microscopy experiments showed that virstatin acts on the A. baumannii pili biogenesis. CONCLUSIONS: By its action on pili biogenesis, virstatin demonstrated a very promising antibiofilm activity affecting more than 70% of the A. baumannii clinical isolates.

Organization of "Pullulan"-block-polyether copolymers at the aqueous solution/air interface.

Interfacial behavior of a thermosensitive homologous series of linear copolymers based on polyetheramine (commercially named Jeffamine® M2005) and on pullulan is investigated. The influence of the polysaccharide block length has been significantly highlighted on the copolymer structuring at the aqueous solution/air interface. Indeed, these systems induce a decrease in the surface tension depending on the pullulan length. To highlight the impact of the pullulan length in such a series at the aqueous solution/air interface, the interfacial rheological measurement and the transferred Gibbs films of these systems have been studied. The storage modulus of the adsorption layers and the Gibbs film thickness increase along with the length of pullulan in the block, probably because of a structuring effect in the subphase and to an entanglement effect of the pullulan chains under the interface. Moreover, the results obtained through the transferred Gibbs films backup those obtained by the surface tension and the interfacial rheological measurements.

Competition for space between a protein and lipid monolayers.

Competitive adsorption is a general problem both in polymer and in biological systems. The equilibrium composition at a surface in contact either with polymer solutions or biological fluids depends on the competition between all the surface active material present in the medium. Such competition is particularly important in cell membranes where membrane proteins generated on ribosomes have to incorporate in the cell. Here we use fluovideo microscopy to study the competition for adsorption at the air/water interface between the enzyme glucose oxidase (GOx) and fluid monolayers of pentadecanoic acid (PDA). Although water soluble, GOx has a strong affinity for the air/water interface. We show that under certain conditions it inserts in the monolayer and causes a contraction of the Langmuir film and the formation of condensed domains. When exposed to a heterogeneous surface it is inserted in the less dense regions. Its crystallization leads to the deformation of the condensed domains followed by the destruction of their initial shape. By compressing the layer the protein is not removed from the interface where it eventually forms three-dimensional structures.

Growth of Acinetobacter baumannii in pellicle enhanced the expression of potential virulence factors.

BACKGROUND: Interestingly, Acinetobacter baumannii presents an enhanced capacity to form biofilms (also named pellicles) at the air-liquid interface as compared to the other Acinetobacter species. This characteristic questions the contribution of this phenotype to an increased risk of clinical infections by this pathogen. METHODOLOGY/PRINCIPAL FINDINGS: By a proteomic approach using 2-D gel electrophoresis-LC-MS/MS mass spectrometry, we compared the membrane protein patterns of A. baumannii 77, a pellicle-forming clinical isolate, grown in planktonic and in sessile modes. We identified 52 proteins with a differential expression, including 32 up-regulated and 20 down-regulated in the pellicle state. Several proteins, differentially expressed during pellicle development, were of particular interest. We determined the over-expression of four siderophore iron uptake systems including the acinetobactin and enterobactin receptors and confirmed that the development of this type of biofilm is promoted by ferric ions. Two over-expressed proteins, CarO and an OprD-homologue, putative carbapenem-resistance associated porins, would be involved in the transport of specific compounds, like ornithine, a biosynthesis precursor of a siderophore from the hydroxamate family. We evidenced the overexpression of a lipase and a transporter of LCFA that may be involved in the recycling of lipids inside the pellicle matrix. Finally, we demonstrated both by proteomic and by AFM studies that this particular type of biofilm required multiple pili systems to maintain this cohesive structure at the air-liquid interface; two of these systems have never been described in A. baumannii. CONCLUSIONS/SIGNIFICANCE: Our study demonstrated that several proteins, overexpressed at a late state of pellicle development, could be potentially involved in virulence processes. Therefore, regarding the number of potential virulence factors that are over-expressed in this growth mode, the pellicle-forming clinical isolates should be kept under survey.

Impact of the biofilm mode of growth on the inner membrane phospholipid composition and lipid domains in Pseudomonas aeruginosa.

Many studies using genetic and proteomic approaches have revealed phenotypic differences between planktonic and sessile bacteria but the mechanisms of biofilm formation and the switch between the two growth modes are not well understood yet. In this study, we focused on inner membrane lipidome modifications when Pseudomonas aeruginosa cells were grown as biofilm. Lipid analyses were performed by Electrospray Ionization Mass Spectrometry. Results showed a drastic decrease of the uneven-numbered chain phospholipids and a slight increase of long chain PEs in sessile organisms as compared with planktonic counterparts, suggesting a better lipid stability in the bilayer and a decrease in membrane fluidity. The impact of sessile growth on lipid domains was then investigated by Brewster Angle Microscopy (BAM) and Atomic Force Microscopy (AFM). Observations showed that inner membrane lipids of P. aeruginosa formed domains when the pressure was close to physiological conditions and that these domains were larger for lipids extracted from biofilm bacteria. This is coherent with the mass spectrometry analyses.

Inner membrane lipids of Escherichia coli form domains.

In prokaryotic cells, the hypothesis of the existence of lipid domains was considered. In order to test this hypothesis and study the organization of lipids in the inner membrane of Escherichia coli, we elaborated Langmuir films mimicking the inner leaflet of this membrane by considering lipids extracted from the inner membrane of E coli by Folch protocol. Lipid monolayers were elaborated by using these extracts (Langmuir technique); the organization of the resulting films was studied at the air-water interface by Brewster angle microscopy and after transfer onto muscovite by atomic force microscopy. The existence of domains was demonstrated for different interfacial pressures of biological interest, and their stability was studied.

Behaviour of bacterial division protein FtsZ under a monolayer with phospholipid domains.

Assembly of the tubulin-like protein FtsZ at or near the cytoplasmic membrane is one of the earliest steps in division of bacteria such as Escherichia coli. Exactly what constitutes the site at which FtsZ acts is less clear. To investigate the influence of the membrane phospholipids on FtsZ localization and assembly, we have elaborated with the Langmuir technique a two-lipid monolayer made of dilauryl-phosphatidylethanolamine (DLPE) and dipalmitoyl-phosphatidylglycerol (DPPG). This monolayer comprised stable condensed domains in an expanded continuous phase. In the presence of GTP, FtsZ assembly disrupts the condensed domains within 5 min. After several hours, with or without GTP, FtsZ assembled into large aggregates at the domain interface. We suggest that the GTP-induced polymerization of FtsZ is coupled to the association of FtsZ protofilaments with domain interfaces.

Biological processes in organised media.

Embedding a simple Michaelis-Menten enzyme in a gel slice may allow the catalysis of not only scalar processes but also vectorial ones, including uphill transport of a substrate between two compartments, and may make it seem as if two enzymes or transporters are present or as if an allosterically controlled enzyme/transporter is operating. The values of kinetic parameters of an enzyme in a partially hydrophobic environment are usually different from those actually measured in a homogeneous aqueous solution. This implies that fitting kinetic data (expressed in reciprocal co-ordinates) from in vivo studies of enzymes or transporters to two straight lines or a sigmoidal curve does not prove the existence of two different membrane mechanisms or allosteric control. In the artificial transport systems described here, a functional asymmetry was sufficient to induce uphill transport, therefore, although the active transport systems characterised so far correspond to proteins asymmetrically anchored in a membrane, the past or present existence of structurally symmetrical systems of transport in vivo cannot be excluded. The fact that oscillations can be induced in studies of the maintenance of the electrical potential of frog skin by addition of lithium allowed evaluation of several parameters fundamental to the functioning of the system in vivo (e.g., relative volumes of internal compartments, characteristic times of ionic exchanges between compartments). Hence, under conditions that approach real biological complexity, increasing the complexity of the behaviour of the system may provide information that cannot be obtained by a conventional, reductionist approach.