Transfer of insulin receptors and of glucose transport-inducing proteins onto phospholipid vesicles

Insulin receptors and glucose transport-inducing proteins have been extracted from rat liver membranes onto positively charged lipid bilayer vesicles. The extraction was carried out during the incubation of the vesicles with lipid vesicles caused an overall enhancement of specific insulin binding and of glucose transport inducement. The latter has been inferred from the oxidation rate of transported glucose through a spherical bilayer membrane entrapping the oxidizing glucose oxidase. Glucose transport is not enhanced by insulin binding, indicating that the two functions become dissociated when the proteins are transferred from the plasma membrane onto the bilayer vesicles.     

Protein diffusion on charged membranes: a dynamic mean-field model describes time evolution and lipid reorganization

As charged macromolecules adsorb and diffuse on cell membranes in a large variety of cell signaling processes, they can attract or repel oppositely charged lipids. This results in lateral membrane rearrangement and affects the dynamics of protein function. To address such processes quantitatively we introduce a dynamic mean-field scheme that allows self-consistent calculations of the equilibrium state of membrane-protein complexes after such lateral reorganization of the membrane components, and serves to probe kinetic details of the process. Applicable to membranes with heterogeneous compositions containing several types of lipids, this comprehensive method accounts for mobile salt ions and charged macromolecules in three dimensions, as well as for lateral demixing of charged and net-neutral lipids in the membrane plane. In our model, the mobility of membrane components is governed by the diffusion-like Cahn-Hilliard equation, while the local electrochemical potential is based on nonlinear Poisson-Boltzmann theory. We illustrate the method by applying it to the adsorption of the anionic polypeptide poly-Lysine on negatively charged lipid membranes composed of binary mixtures of neutral and monovalent lipids, or onto ternary mixtures of neutral, monovalent, and multivalent lipids. Consistent with previous calculations and experiments, our results show that at steady-state multivalent lipids (such as PIP₂), but not monovalent lipid (such as phosphatidylserine), will segregate near the adsorbing macromolecules. To address the corresponding diffusion of the adsorbing protein in the membrane plane, we couple lipid mobility with the propagation of the adsorbing protein through a dynamic Monte Carlo scheme. We find that due to their higher mobility dictated by the electrochemical potential, multivalent lipids such as PIP₂ more quickly segregate near oppositely charged proteins than do monovalent lipids, even though their diffusion constants may be similar. The segregation, in turn, slows protein diffusion, as lipids introduce an effective drag on the motion of the adsorbate. In contrast, monovalent lipids such as phosphatidylserine only weakly segregate, and the diffusions of protein and lipid remain largely uncorrelated.     

Ion-selective Microelectrodes: Their Use and Importance in Modern Plant Cell Biology

The exact ion gradients across cellular membranes and their changes due to metabolic or transport processes can be best studied with the use of ion-selective microelectrodes. The last decade of research using ion-selective microelectrodes in intact cells has proven this technique to be indispensable for the investigation of a variety of physiological questions of regulatory processes, membrane transport, cellular signalling, developmental biology and plant nutrition. Their application to selected problems has led to numerous exciting observations, many of which have changed our view concerning cellular responses to environmental stimuli and in many instances have led to a new understanding of plant cell physiology. Since, with these electrodes, intracellular as well as extracellular free ion concentrations can be simultaneously detected with electrical transport parameters such as membrane potential and membrane conductance, they can be powerful tools in the hands of many plant cell biologists.     

Chemiosmotic mechanism of transport of biological macromolecules through bacterial membranes]

A general mechanism of the nucleic acids transport through bacterial membranes during genetic transformation, transfection, viral infection and bacterial conjugation, has been developed. The uptake of nucleic acid occurs due to the symport with H+ ions down to an electrochemical potential gradient ("minus" inside) generated by respiration or ATP hydrolysis within recipient cells. The nucleic acid anions of non--lethal viruses are extruded from the negatively charged host cell cytoplasm by electrostatic repulsion. The difference of electrochemical potentials between the conjugating cells cytoplasms is considered as a driving force for the transport of DNA from the donor to the recipient cell.     

Contribution of electrogenic ion transport to impedance of the algae Valonia utricularis and artificial membranes.

The cell membrane of Valonia utricularis contains an electrogenic carrier system for chloride (Wang et al., Biophys J. 59:235-248 (1991)). The electrical impedance of V. utricularis was measured in the frequency range between 1 Hz and 50 kHz. The analysis of the impedance spectra from V. utricularis and its comparison with equivalent circuit models showed that the transport system created a characteristic contribution to the impedance in the frequency range between 10 Hz and 5 kHz. The fit of the impedance spectra with the formalism derived from the theory of carrier-mediated transport allowed the determination of the kinetic parameters of chloride transport through the cell membrane of V. utricularis, and its passive electrical properties. Simultaneous measurements of the kinetic parameters with the charge pulse method demonstrated the equivalence of both experimental approaches with respect to the evaluation of the translocation rate constants of the free and the charged carriers and the total density of carriers within the membrane. Moreover, the impedance spectra of the protonophor-mediated proton transport by FCCP (carbonylcyanide p-trifluoromethoxyphenyl-hydrazone) were measured in model membranes. The carrier system made a substantial contribution to the impedance of the artificial membranes. The analysis of the spectra in terms of a simple carrier system (Benz and McLaughlin, 1983, Biophys. J. 41:381-398) allowed the evaluation of the kinetic and equilibrium parameters of the FCCP-mediated proton transport. The possible application of the measurement of impedance spectra for the study of biological transport systems is discussed.     

Functionalization of PVC membrane with ss oligonucleotides for a potentiometric biosensor

A novel application of a single stranded (ss) oligonucleotide as an active component of polymeric membrane in an ion-selective electrode (ISE) is described. The original oligonucleotides, oligo(dA)(15), modified by cholesterol, triphenylmethyl and hexadecyl derivatives, were immobilized into poly(vinyl chloride) (PVC) membrane using extraction protocol. In parallel, the adsorption protocol was used to immobilize unmodified oligo(dA)(15) on the PVC membrane based on tridodecylmethyammonium chloride (TDDMA(+)Cl(-)). Immobilization of ss oligonucleotide probe through spacer was more effective for the potentiometric detection of the hybridization between complementary oligonucleotides. It was found that cholesterol-oligo(dA)(15) modified membranes were sensitive toward complementary oligo(dT)(15) in the concentration range 2-80 nM at pH 7. An explanation for the detection mechanism is proposed.     

Cerium Oxide Decorated Polymer Nanofibers as Effective Membrane Reinforcement for Durable,High‐Performance Fuel Cells

High‐power, durable composite fuel cell membranes are fabricated here by direct membrane deposition (DMD). Poly(vinylidene fluoride‐co ‐hexafluoropropylene) (PVDF‐HFP) nanofibers, decorated with CeO₂ nanoparticles are directly electrospun onto gas diffusion electrodes. The nanofiber mesh is impregnated by inkjet‐printed Nafion ionomer dispersion. This results in 12 µm thin multicomponent composite membranes. The nanofibers provide membrane reinforcement, whereas the attached CeO₂ nanoparticles promote improved chemical membrane durability due to their radical scavenging properties. In a 100 h accelerated stress test under hot and dry conditions, the reinforced DMD fuel cell shows a more than three times lower voltage decay rate (0.39 mV h^?1) compared to a comparably thin Gore membrane (1.36 mV h^?1). The maximum power density of the DMD fuel cell drops by 9%, compared to 54% measured for the reference. Impedance spectroscopy reveals that ionic and mass transport resistance of the DMD fuel cell are unaffected by the accelerated stress test. This is in contrast to the reference, where a 90% increase of the mass transport resistance is measured. Energy dispersive X‐ray spectroscopy reveals that no significant migration of cerium into the catalyst layers occurs during degradation. This proves that the PVDF‐HFP backbone provides strong anchoring of CeO₂ in the membrane.     

Attracted to membranes: lipid-binding domains in plants

Membranes are essential for cells and organelles to function. As membranes are impermeable to most polar and charged molecules, they provide electrochemical energy to transport molecules across and create compartmentalized microenvironments for specific enzymatic and cellular processes. Membranes are also responsible for guided transport of cargoes between organelles and during endo- and exocytosis. In addition, membranes play key roles in cell signaling by hosting receptors and signal transducers and as substrates and products of lipid second messengers. Anionic lipids and their specific interaction with target proteins play an essential role in these processes, which are facilitated by specific lipid-binding domains. Protein crystallography, lipid-binding studies, subcellular localization analyses, and computer modeling have greatly advanced our knowledge over the years of how these domains achieve precision binding and what their function is in signaling and membrane trafficking, as well as in plant development and stress acclimation.

Lipid-binding domains represent essential motifs within proteins that allow them to bind specific lipids in membranes in a spatial and temporal manner for signaling and trafficking purposes.     

Biocompatible ion selective electrode for monitoring metabolic activity during the growth and cultivation of human cells

Ammonia is the main nitrogenous waste product of cellular metabolism and if accumulated in culture media may limit cell growth and affect the quality of cultured cell lines. Therefore, it is crucial to control levels of this metabolite during the in vitro expansion of human cells. This paper describes the successful application of ion selective electrodes (ISE) to continuously monitor ammonium concentrations in a perfused cell bioreactor. The polymeric membranes of the ISE were cast from carboxylated poly(vinyl chloride) (PVC-COOH) and doped with highly hydrophilic poly(ethylene glycol) (PEG). The PEG was incorporated into the surface of the sensors in order to reduce the effect of biofouling without impairing their analytical characteristics. The electrodes developed enabled fast and selective measurements of ammonia in the range 0.5-5mM, corresponding well with the concentration determined off-line. Additionally, the UV sterilised sensors were small and flexible enough to be readily inserted into the limited space of the bioreactor. Long-term analytical performance of PEG-modified ISE during continuous measurements in mammalian cell cultures was investigated. The sensors remained stable for the duration of the bioprocess, 7 days.     

Thermodynamics of the interaction of RbCl with some monosaccharides (D-glucose, D-galactose, D-xylose, and D-arabinose) in aqueous solutions at 298.15K

The Gibbs energy interaction parameters of RbCl with some monosaccharides (D-glucose, D-galactose, D-xylose, and D-arabinose) in water, g(ES), were obtained from electromotive force (emf) measurements of the electrochemical cell without liquid junction and containing two ion-selective electrodes (ISE): K-ISEmid R:RbCl(m(E))mid R:ISE-Cl and K-ISEmid R:RbCl(m(E)),saccharide (m(S))mid R:ISE-Cl, at 298.15K. The enthalpy interaction parameters of RbCl with these monosaccharides in water, h(ES), are determined according to the McMillan-Mayer theory from the measurements of the enthalpies of mixing of aqueous RbCl solutions with aqueous monosaccharide solutions, as well as the enthalpies of dilution of RbCl and monosaccharide solutions in pure water at 298.15K by a calorimetric method. Furthermore, the entropy interaction parameters, s(ES), can be evaluated through g(ES) and h(ES). The results suggest that the electrostatic interactions of these monosaccharides with RbCl in water are predominant compared with structural interactions, and these parameters are controlled primarily by the stereochemical structure of the monosaccharides in water.     

Time resolved secretion of chloride from a monolayer of mucin-secreting epithelial cells

Short-circuit current (Isc) measurement is used to quantify transepithelial ion flux. This technique provides a direct measure of net charge transport across a cell monolayer. Isc however, lacks chemical selectivity. Chemically resolved ion fluxes may be much greater than Isc, and differ in different biological processes. This work describes a novel experimental approach and deconvolution method to obtain temporally resolved ion fluxes at epithelial cell monolayers. HT29-Cl.16E cells, a sub clone of the human colonic cancer cell line HT29 was used as a model cell line to validate this approach in the context of epithelial transport studies. This cell line is known to secrete chloride in response to purinergic stimulation. Changes in chloride concentration after stimulation with 1 mM ATP plus 50 nM phorbol-myristate acetate (PMA) are recorded with a chloride ion-selective electrode (ISE) at a short distance (∼50 μm) from the monolayer. The recorded concentrations are transformed to corresponding chloride flux across the monolayer using a deconvolution algorithm for extracellular mass transport based on minimization of the shape error function (Nair and Gratzl in Anal Chem 77:2875–2888, 2005). Simultaneous voltage clamp yields the associated net electrical charge flux (Isc). The dynamics of Cl⁻ flux did correlate with that of the electrical flux, but was found to be greater in amplitude. This suggests that Cl⁻ may not be the only ion secreted. The method of simultaneously assessing ionic and electrical fluxes with a temporal resolution of seconds provides unique information about the dynamics of solute fluxes across the apical membrane. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

High-Sensitivity Peptide Mapping by Micro-LC with On-Line Membrane Blotting and Subsequent Detection by Scanning-IR-MALDI Mass Spectrometry

A novel approach to the on-line mass determination of peptides from digested proteins by scanning infrared matrix-assisted laser desorption/ionization (scanning-IR-MALDI) is described. The peptides were continuously collected directly onto a PVDF (polyvinylidene fluoride) strip during a HPLC run. Individual peptides were detected by lining up the PVDF strip with the UV trace from the HPLC run, using visible dye markers as reference points. The local resolution of the peptides on the PVDF membrane is preserved during matrix incubation for MALDI-MS as shown by comparing the UV chromatogram and the total ion current (TIC) from an on-line coupled electrospray ionization (ESI) mass spectrometer with the scanning-IR-MALDI data from the corresponding areas on the PVDF strip. The intensities of the mass profiles obtained by scanning-IR-MALDI reflect the amount of peptides present on the PVDF strip. The higher sensitivity of IR-MALDI-MS yielded mass information not detectable by ESI-MS. After the scanning-IR-MALDI experiment, the same membrane strip can be used directly for automated Edman degradation. Comparable initial and repetitive yields were obtained for blotted peptides with and without matrix incubation.     

How membrane proteins sense voltage

The ionic gradients across cell membranes generate a transmembrane voltage that regulates the function of numerous membrane proteins such as ion channels, transporters, pumps and enzymes. The mechanisms by which proteins sense voltage is diverse: ion channels have a conserved, positively charged transmembrane region that moves in response to changes in membrane potential, some G-protein coupled receptors possess a specific voltage-sensing motif and some membrane pumps and transporters use the ions that they transport across membranes to sense membrane voltage. Characterizing the general features of voltage sensors might lead to the discovery of further membrane proteins that are voltage regulated.     

Switching of membrane organelles between cytoskeletal transport systems is determined by regulation of the microtubule-based transport

Intracellular transport of membrane organelles occurs along microtubules (MTs) and actin filaments (AFs). Although transport along each type of the cytoskeletal tracks is well characterized, the switching between the two types of transport is poorly understood because it cannot be observed directly in living cells. To gain insight into the regulation of the switching of membrane organelles between the two major transport systems, we developed a novel approach that combines live cell imaging with computational modeling. Using this approach, we measured the parameters that determine how fast membrane organelles switch back and forth between MTs and AFs (the switching rate constants) and compared these parameters during different signaling states. We show that regulation involves a major change in a single parameter: the transferring rate from AFs onto MTs. This result suggests that MT transport is the defining factor whose regulation determines the choice of the cytoskeletal tracks during the transport of membrane organelles.     

Positively charged ceramide is a potent inducer of mitochondrial permeabilization

Ceramide-induced cell death is thought to be mediated by change in mitochondrial function, although the precise mechanism is unclear. Proposed models suggest that ceramide induces cell death through interaction with latent binding sites on the outer or inner mitochondrial membranes, followed by an increase in membrane permeability, as an intermediate step in ceramide signal propagation. To investigate these models, we developed a new generation of positively charged ceramides that readily accumulate in isolated and in situ mitochondria. Accumulated, positively charged ceramides increased inner membrane permeability and triggered release of mitochondrial cytochrome c. Furthermore, the positively charged ceramide-induced permeability increase was suppressed by cyclosporin A (60%) and 1,3-dicyclohexylcarbodiimide (90%). These observations suggest that the inner membrane permeability increase is due to activation of specific ion transporters, not the generalized loss of lipid bilayer barrier functions. The difference in sensitivity of ceramide-induced ion fluxes to inhibitors of mitochondrial transporters suggests activation of at least two transport systems: the permeability transition pore and the electrogenic H(+) channel. Our results indicate the presence of specific ceramide targets in the mitochondrial matrix, the occupation of which triggers permeability alterations of the inner and outer mitochondrial membranes. These findings also suggest a novel therapeutic role for positively charged ceramides.     

Membrane biosensor platforms using nano- and microporous supports

Lipid membranes are versatile and convenient models for the study of properties of natural cell membranes. In particular, surface-supported membranes have attracted considerable attention because the whole range of surface-sensitive techniques, including interface-sensitive electrochemical measurements, can be used with them. Here we describe recent advances and current directions in the development of nano- and microporous substrates for electrochemical characterization of membrane protein-containing lipid bilayers. Improved techniques for lipid membrane self-assembly and membrane protein incorporation on these substrates have led to great improvements in measurement sensitivity, membrane stability and packaging. These advances suggest that nanopore-spanning membranes are leading contenders for a breakthrough in membrane protein screening and biosensing applications.     

Measurements of interbilayer forces and protein adsorption on uncharged lipid bilayers displaying poly(ethylene glycol) chains

Poly(ethylene glycol) (PEG)-stabilized liposomes were recently shown to exhibit differences in cell uptake that were linked to the liposome charge. To determine the differences and similarities between charged and uncharged PEG-decorated liposomes, we directly measured the forces between two supported, neutral bilayers with terminally grafted PEG chains. The measurements were performed with the surface force apparatus. The force profiles were similar to those measured with negatively charged PEG conjugates of 1, 2-distearoyl-sn-glycero-3-phosphatidyl ethanolamine (DSPE), except that they lacked the longer ranged electrostatic repulsion observed with the charged compound. Theories for simple polymers describe the forces between end-grafted polymer chains on neutral bilayers. The force measurements were complemented by surface plasmon resonance studies of protein adsorption onto these layers. The lack of electrostatic forces reduced the adsorption of positively charged proteins and enhanced the adsorption of negatively charged ones. The absence of charge also allowed us to determine how membrane charge and the polymer grafting density independently affect protein adsorption on the coated membranes. Such studies suggest the physical basis of the different interactions of charged and uncharged liposomes with proteins and cells.     

Highly robust lipid membranes on crystalline S-layer supports investigated by electrochemical impedance spectroscopy

In the present work, S-layer supported lipid membranes formed by a modified Langmuir-Blodgett technique were investigated by electrochemical impedance spectroscopy (EIS). Basically two intermediate hydrophilic supports for phospholipid- (DPhyPC) and bipolar tetraetherlipid- (MPL from Thermoplasma acidophilum) membranes have been applied: first, the S-layer protein SbpA isolated from Bacillus sphaericus CCM 2177 recrystallized onto a gold electrode; and second, as a reference support, an S-layer ultrafiltration membrane (SUM), which consists of a microfiltration membrane (MFM) with deposited S-layer carrying cell wall fragments. The electrochemical properties and the stability of DPhyPC and MPL membranes were found to depend on the used support. The specific capacitances were 0.53 and 0.69 microF/cm(2) for DPhyPC bilayers and 0.75 and 0.77 microF/cm(2) for MPL monolayers resting on SbpA and SUM, respectively. Membrane resistances of up to 80 mega Ohm cm(2) were observed for DPhyPC bilayers on SbpA. In addition, membranes supported by SbpA exhibited a remarkable long-term robustness of up to 2 days. The membrane functionality could be demonstrated by reconstitution of membrane-active peptides such as valinomycin and alamethicin. The present results recommend S-layer-supported lipid membranes as promising structures for membrane protein-based biosensor technology.     

Development of a novel spatiotemporal depletion system for cellular cholesterol

Cholesterol is an essential component of mammalian cell membranes whose subcellular concentration and function are tightly regulated by de novo biosynthesis, transport, and storage. Although recent reports have suggested diverse functions of cellular cholesterol in different subcellular membranes, systematic investigation of its site-specific roles has been hampered by the lack of a methodology for spatiotemporal manipulation of cellular cholesterol levels. Here, we report the development of a new cholesterol depletion system that allows for spatiotemporal manipulation of intracellular cholesterol levels. This system utilizes a genetically encoded cholesterol oxidase whose intrinsic membrane binding activity is engineered in such a way that its membrane targeting can be controlled in a spatiotemporally specific manner via chemically induced dimerization. In combination with in situ quantitative imaging of cholesterol and signaling activity measurements, this system allows for unambiguous determination of site-specific functions of cholesterol in different membranes, including the plasma membrane and the lysosomal membrane.