Astrocyte swelling leads to membrane unfolding, not membrane insertion

The mechanisms mediating the release of chemical transmitters from astrocytes are the subject of intense research. Recent experiments have shown that hypotonic conditions stimulate the release of glutamate and ATP from astrocytes, but a mechanistic understanding of this process is not available. To determine whether hypotonicity activates the process of regulated exocytosis, we monitored membrane capacitance by the whole-cell patch-clamp technique whilst a hypotonic medium was applied to cultured astrocytes. If exocytosis is triggered under hypotonic conditions, as it is following increases in cytosolic calcium, a net increase in membrane surface area, monitored by measuring the whole-cell membrane capacitance, is expected. Simultaneous measurements of cell size and whole-cell membrane conductance and surface area demonstrated that hypotonic medium (210 mOsm for 200 s) resulted in an increase in membrane conductance and in the swelling of cultured astrocytes by an average of 40%, as monitored by cell cross-sectional area, but without any corresponding change in membrane surface area. As we have demonstrated that capacitance measurements have the sensitivity to detect increases in cell surface area as small as 0.5%, we conclude that cell swelling occurs via an exocytosis-independent mechanism, probably involving the unfolding of the plasma membrane.     

Role of membrane organization and membrane domains in endocytic lipid trafficking

Lipid compositions vary greatly among organelles, and specific sorting mechanisms are required to establish and maintain these distinct compositions. In this review, we discuss how the biophysical properties of the membrane bilayer and the chemistry of individual lipid molecules play a role in the intracellular trafficking of the lipids themselves, as well as influencing the trafficking of transmembrane proteins. The large diversity of lipid head groups and acyl chains lead to a variety of weak interactions, such as ionic and hydrogen bonding at the lipid/water interfacial region, hydrophobic interactions, and van-der-Waals interactions based on packing density. In simple model bilayers, these weak interactions can lead to large-scale phase separations, but in more complex mixtures, which mimic cell membranes, such phase separations are not observed. Nevertheless, there is growing evidence that domains (i.e., localized regions with non-random lipid compositions) exist in biological membranes, and it is likely that the formation of these domains are based on interactions similar to those that lead to phase separations in model systems. Sorting of lipids appears to be based in part on the inclusion or exclusion of certain types of lipids in vesicles or tubules as they bud from membrane organelles.     

Interplay between the electrostatic membrane potential and conformational changes in membrane proteins

Transmembrane electrostatic membrane potential is a major energy source of the cell. Importantly, it determines the structure as well as function of charge‐carrying membrane proteins. Here, we discuss the relationship between membrane potential and membrane proteins, in particular whether the conformation of these proteins is integrally connected to the membrane potential. Together, these concepts provide a framework for rationalizing the types of conformational changes that have been observed in membrane proteins and for better understanding the electrostatic effects of the membrane potential on both reversible as well as unidirectional dynamic processes of membrane proteins.     

SNAREs--engines for membrane fusion

Since the discovery of SNARE proteins in the late 1980s, SNAREs have been recognized as key components of protein complexes that drive membrane fusion. Despite considerable sequence divergence among SNARE proteins, their mechanism seems to be conserved and is adaptable for fusion reactions as diverse as those involved in cell growth, membrane repair, cytokinesis and synaptic transmission. A fascinating picture of these robust nanomachines is emerging.     

Non-covalent binding of membrane lipids to membrane proteins

Polar lipids and membrane proteins are major components of biological membranes, both cell membranes and membranes of enveloped viruses. How these two classes of membrane components interact with each other to influence the function of biological membranes is a fundamental question that has attracted intense interest since the origins of the field of membrane studies. One of the most powerful ideas that driven the field is the likelihood that lipids bind to membrane proteins at specific sites, modulating protein structure and function. However only relatively recently has high resolution structure determination of membrane proteins progressed to the point of providing atomic level structure of lipid binding sites on membrane proteins. Analysis of X-ray diffraction, electron crystallography and NMR data over 100 specific lipid binding sites on membrane proteins. These data demonstrate tight lipid binding of both phospholipids and cholesterol to membrane proteins. Membrane lipids bind to membrane proteins by their headgroups, or by their acyl chains, or binding is mediated by the entire lipid molecule. When headgroups bind, binding is stabilized by polar interactions between lipid headgroups and the protein. When acyl chains bind, van der Waals effects dominate as the acyl chains adopt conformations that complement particular sites on the rough protein surface. No generally applicable motifs for binding have yet emerged. Previously published biochemical and biophysical data link this binding with function. This Article is Part of a Special Issue Entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.     

Bacterial membrane proteomics

About one quarter to one third of all bacterial genes encode proteins of the inner or outer bacterial membrane. These proteins perform essential physiological functions, such as the import or export of metabolites, the homeostasis of metal ions, the extrusion of toxic substances or antibiotics, and the generation or conversion of energy. The last years have witnessed completion of a plethora of whole-genome sequences of bacteria important for biotechnology or medicine, which is the foundation for proteome and other functional genome analyses. In this review, we discuss the challenges in membrane proteome analysis, starting from sample preparation and leading to MS-data analysis and quantification. The current state of available proteomics technologies as well as their advantages and disadvantages will be described with a focus on shotgun proteomics. Then, we will briefly introduce the most abundant proteins and protein families present in bacterial membranes before bacterial membrane proteomics studies of the last years will be presented. It will be shown how these works enlarged our knowledge about the physiological adaptations that take place in bacteria during fine chemical production, bioremediation, protein overexpression, and during infections. Furthermore, several examples from literature demonstrate the suitability of membrane proteomics for the identification of antigens and different pathogenic strains, as well as the elucidation of membrane protein structure and function.     

Immobilized artificial membrane chromatography: supports composed of membrane lipids

Cell membranes provide an environment for several types of molecular processes and we are attempting to mimic the cell membranes' environment on a chromatography solid support. Chromatography solid supports utilizing lecithin as the bonded phase were synthesized and the HPLC behavior of hydrophilic peptides evaluated. A diC14 lecithin containing a terminal carboxy group on the C2 fatty acid chain was amidated with the surface amines of Nucleosil-300 (7NH2) silica particles. Based on elemental analysis, lecithin was coupled to Nucleosil-300 (7NH2) at a surface density near that of lecithin found in biological membranes and this novel chromatographic support material is denoted as Nucleosil-lecithin, the prototype immobilized artificial membrane. Infrared difference spectra of Nucleosil-lecithin minus Nucleosil-300 (7NH2) clearly showed amide I (1653.1 cm-1) and amide II (1550.9 cm-1) bands, giving direct spectroscopic evidence for the amide linkage. Spectral deconvolution resolved two peaks for the amide I band, and three peaks for the amide II band. This demonstrates lecithin interchain amide hydrogen bonding and/or hydrogen bonds between the lecithin amide link and unreacted silica surface amines. Nucleosil-lecithin as a solid phase mimics membranes and can be used to study the interactions of biomolecules with membranes. Our primary objective is to develop HPLC methods for studying the interaction between cell membranes and peptide sequences found near the interfaces of cell membranes. A frequency distribution of amino acids bracketing approximately 400 transmembrane peptide sequences showed Cys to be the least frequently occurring amino acid at this putative interfacial membrane region. Hydrophilic peptide analogs bearing Cys were used as model compounds to test Nucleosil-lecithin solid supports. Small peptides, six to eight amino acids in length, containing Cys bind approximately 2X tighter to Nucleosil-lecithin compared to identical peptides without the Cys residue. Thus, Cys at the interface of cells may stabilize protein-lipid interactions.     

Surfactant-free purification of membrane proteins with intact native membrane environment

In order to study the structure and function of a protein, it is generally required that the protein in question is purified away from all others. For soluble proteins, this process is greatly aided by the lack of any restriction on the free and independent diffusion of individual protein particles in three dimensions. This is not the case for membrane proteins, as the membrane itself forms a continuum that joins the proteins within the membrane with one another. It is therefore essential that the membrane is disrupted in order to allow separation and hence purification of membrane proteins. In the present review, we examine recent advances in the methods employed to separate membrane proteins before purification. These approaches move away from solubilization methods based on the use of small surfactants, which have been shown to suffer from significant practical problems. Instead, the present review focuses on methods that stem from the field of nanotechnology and use a range of reagents that fragment the membrane into nanometre-scale particles containing the protein complete with the local membrane environment. In particular, we examine a method employing the amphipathic polymer poly(styrene-co-maleic acid), which is able to reversibly encapsulate the membrane protein in a 10?nm disc-like structure ideally suited to purification and further biochemical study.     

Origin of magnetosome membrane: proteomic analysis of magnetosome membrane and comparison with cytoplasmic membrane

Prokaryotes are known to have evolved one or more unique organelles. Although several hypotheses have been proposed concerning the biogenesis of these intracellular components, the majority of these proposals remains unclear. Magnetotactic bacteria synthesize intracellular magnetosomes that are enclosed by lipid bilayer membranes. From the identification and characterization of several surface and transmembrane magnetosome proteins, we have postulated that magnetosomes are derived from the cytoplasmic membrane (CM). To confirm this hypothesis, a comparative proteomic analysis of the magnetosome membrane (MM) and CM of the magnetotactic bacterium, Magnetospirillum magneticum AMB-1, was undertaken. Based on the whole genome sequence of M. magneticum AMB-1, 78 identified MM proteins were also found to be prevalent in the CM, several of which are related to magnetosome biosynthesis, such as Mms13, which is tightly bound on the magnetite surface. Fatty acid analysis was also conducted, and showed a striking similarity between the CM and MM profiles. These results suggest that the MM is derived from the CM.     

Localization of membrane proteins in membrane vesicles of Bacillus subtilis.

Electrons can be transferred to the respiratory chain in whole cells and in membrane vesicles of Bacillus subtilis W 23 by the membrane impermeable electron donor reduced 5-N-methyl-phenazonium-3-sulfonate as efficiently as by the membrane permeable electron donor reduced 5-N-methyl-phenazonium methyl-sulfate, indicating that the respiratory chain is accessible from the outside of the membrane.Succinate is oxidized by whole cells and membrane vesicles at a low rate and does not energize transport of l-glutamate. In the presence of 5-N-methyl-phenazonium-3-sulfonate or 5-N-methyl-phenazonium methyl-sulfate, the oxidation rate and the rate of l-glutamate transport are increased considerably. The electrons are transferred directly from succinic dehydrogenase to these acceptors. Succinic dehydrogenase must therefore be exposed to the outside surface of the membrane in both membrane vesicles and whole cells. The exposure of succinic dehydrogenase to the outside is also indicated by the observations that only a 5% increase in the oxidation rates of succinate-5-N-methyl-phenazonium methylsulfate and succinate-5-N-methyl-phenazonium-3-sulfonate is observed upon solubilization of the membrane with the nonionic detergent Brij-58. Furthermore, treatment of membrane vesicles with trypsin decreases by more than 95% these oxidation rates.NADH is oxidized at a high rate and energizes transport of l-glutamate in whole cells and membrane vesicles effectively. The NADH-oxidation is not effected by trypsin treatment of the vesicles indicating that the oxidation occurs at the inside-surface of the membrane. Trypsin treatment of the vesicles, however, significantly decreases the rate of l-glutamate transport driven by NADH. Therefore component(s) of the transport system for l-glutamate must be effected by trypsin treatment. No apparent differences could be observed in the localization of membrane-bound functions between membrane vesicles and whole cells. This strongly supports the contention that the vesicle membrane of B. subtilis has the same orientation as the cytoplasmic membrane of whole cells.     

Membrane proteins and membrane proteomics

Biological membranes form an essential barrier between living cells and their external environments, as well as serve to compartmentalize intracellular organelles within eukaryotes. The latter includes membranes that envelope the nucleus, the outer and inner membranes of the mitochondria, membrane cisternae complex of the ER, Golgi apparatus, as well as lysosomes and secretory vesicles. Depending on their localizations in the whole organism and also within the cell, these membranes have different, highly specialized functions. Although 30% of naturally occurring proteins are predicted to be embedded in biological membranes, membrane proteomics is traditionally understudied due to difficulties in solubilizing, separating, and identifying membrane proteins. Given the importance of membrane proteins in the various cellular processes listed in this review, as well as the roles they play in diseases and their potential as drug targets, it is imperative that this class of proteins be better studied. With the recent advancement in technology, it is expected that some of the difficulties in membrane proteomics will be overcome, yielding new data on membrane proteins.     

Junctional membrane permeability

Summary Substitution of extracellular Na⁺ by Li⁺ causes depression of junctional membrane permeability inChironomus salivary gland cells; within 3 hr, permeability falls to so low a level that neither fluorescein nor the smaller inorganic ions any longer traverse the junctional membrane in detectable amounts (uncoupling). The effect is Li-specific: if choline⁺ is the Na⁺ substitute, coupling is unchanged. The Li-produced uncoupling is not reversed by restitution of Na⁺. Long-term exposure (>1 hr) of the cells to Ca, Mg-free medium leads also to uncoupling. This uncoupling is fully reversible by early restitution of Ca⁺⁺ or Mg⁺⁺. Coupling is maintained in the presence of either Ca⁺⁺ or Mg⁺⁺, so long as the total divalent concentration is about 12mm. The uncoupling in Ca, Mg-free medium ensues regardless of whether the main monovalent cation is Na, Li or choline.The uncouplings are accompanied by cell depolarization. Repolarization of the cells by inward current causes restoration of coupling; the junctional conductance rises again to its normal level. The effect was shown for Li-produced uncoupling, for uncoupling by prolonged absence of external Ca⁺⁺ and Mg⁺⁺, and for uncoupling produced by dinitrophenol. In all cases, the recoupling has the same features: (1) it develops rapidly upon application of the polarizing current; (2) it is cumulative; (3) it is transient, but outlasts the current; and (4) it appears not to depend on the particular ions carrying the current from the electrodes to the cell. The recoupling is due to repolarization of nonjunctional cell membrane; recoupling can be produced at zero net currernt through the junctional membrane. Recoupling takes place also as a result of chemically produced repolarization; restoration of theK gradients in uncoupled cells causes partial recoupling during the repolarization phase.An explanation of the results on coupling is proposed in terms of known mechanisms of regulation of Ca⁺⁺ flux in cells. The uncouplings are explained by actions raising the Ca⁺⁺ level in the cytoplasmic environment of the junctional membranes; the recoupling is explained by actions lowering this Ca⁺⁺ level.     

The human platelet membrane proteome reveals several new potential membrane proteins

We present the first focused proteome study on human platelet membranes. Due to the removal of highly abundant cytoskeletal proteins a wide spectrum of known platelet membrane proteins and several new and hypothetical proteins were accessible. In contrast to other proteome studies we focused on prefractionation and purification of membranes from human platelets according to published protocols to reduce sample complexity and enrich interesting membrane proteins. Subsequently protein separation by common one-dimensional SDS-PAGE as well as the combined benzyldimethyl-n-hexadecylammonium chloride/SDS separation technique was performed prior to mass spectrometry analysis by nano-LC-ESI-MS/MS. We demonstrate that the application of both separation systems in parallel is required for maximization of protein tagging out of a complex sample. Furthermore the identification of several potential membrane proteins in human platelets yields new potential targets in functional platelet research.     

Relations between plasma membrane and lysosomal membrane. 1. Fate of covalently labelled plasma membrane protein

To quantify the kinetics of the plasma membrane flow into lysosomes, we covalently labelled at 4 degrees C the pericellular membrane of rat fibroblasts and followed label redistribution to the lysosomal membrane using purified lysosomal preparations. The polypeptides were, either labelled with 125I by the lactoperoxidase procedure, or conjugated to [3H]peroxidase using bisdiazobenzidine as a bifunctional reagent. Both labels were initially bound to plasma membrane, as indicated by their equilibrium density in sucrose or Percoll gradients and their displacement by digitonin, as well as by electron microscopy. Upon cell incubation at 37 degrees C, both covalent labels were lost from cells with diphasic kinetics: a minor component (35% of cell-associated labels) was rapidly released (half-life less than 1 h), and most label (65%) was released slowly (half-life was 20 h for incorporated 125I and 27 h for 3H). Immediately after labelling up to 30 h after incubation at 37 degrees C, the patterns of 125I-polypeptides quantified by autoradiography after SDS-PAGE were indistinguishable, indicating no preferential turnover for the major plasma membrane polypeptides. The redistribution of both labels to lysosomes was next quantified by cell fractionation. At equilibrium (between 6 and 25 h of cell incubation) 2-4% of cell-associated 125I label was recovered with the purified lysosomal membranes. By contrast, when 3H-labelled cells were incubated for 16 h, most of the label codistributed with lysosomes. However, only 6% of cell-associated 3H was bound to lysosomal membrane. These results indicate that in cultured rat fibroblasts, a minor fraction of plasma membrane polypeptides becomes associated with the lysosomal membrane and is constantly equilibrated by membrane traffic.     

Small scale membrane mechanics

Large scale changes to lipid bilayer shapes are well represented by the Helfrich model. However, there are membrane processes that take place at smaller length scales that this model cannot address. In this work, we present a one-dimensional continuum model that captures the mechanics of the lipid bilayer membrane at the length scale of the lipids themselves. The model is developed using the Cosserat theory of surfaces with lipid orientation, or ‘tilt’, as the fundamental degree of freedom. The Helfrich model can be recovered as a special case when the curvatures are small and the lipid tilt is everywhere zero. We use the tilt model to study local membrane deformations in response to a protein inclusion. Parameter estimates and boundary conditions are obtained from a coarse-grained molecular model using dissipative particle dynamics (DPD) to capture the same phenomenon. The continuum model is able to reproduce the membrane bending, stretch and lipid tilt as seen in the DPD model. The lipid tilt angle relaxes to the bulk tilt angle within 5–6 nm from the protein inclusion. Importantly, for large tilt gradients induced by the proteins, the tilt energy contribution is larger than the bending energy contribution. Thus, the continuum model of tilt accurately captures behaviors at length scales shorter than the membrane thickness.     

金鱼精子质膜和核膜的区域特异性

Combined SEM and TEM technique including thin sectioning, freeze-fracture and etching as well as cytochemical staining have been used for ultrastructural study on goldfish (Carassius auratus) sperm. It has been shown that primitive sperm plasma membrane and nuclear membrane are differentiated with regional specificity. The results from this study can be summarized as follows: 1. Intercalated protein particles are highly organized in the plasma membrane in the certain region of the head to form crystalline-like structure, in contrast rest of the area is rich in randomly or clustered particles. 2. Many vesicles in different size are often tightly packed in the head, neck and tail regions. Only these plasma membrane covering the vesicles contain almost no protein particles. 3. The vesicles can be densely stained cytochemically, suggesting the existence of glycoprotein. 4. Most of the nuclear membrane have no nuclear pores on it except the area near the neck part where many nuclear pores concentrate.     

Excimer-forming lipids in membrane research

Pyrenedecanoic acid and pyrene lecithin are optical probes well suited to investigate lipid bilayer membranes. The method is based on the determination of the formation of excited dimers or excimers. The rate of excimer formation yields information on the dynamic molecular properties of artificial as well as of natural membranes. This article will review applications of the excimer-forming probes.Pyrene lipid probes are used to determine the coefficient of the lateral diffusion in fluid lipid membranes. Results in artificial membranes are comparable to the values obtained in erythrocyte membranes.Moreover, the excimer formation rate is a very sensitive measure of changes in membrane fluidity. Membrane fluidity is an important regulator of membrane functional proteins. For example, there is a correlation between membrane fluidity and enzyme activities of the adenylate cyclase system.The excimer formation technique is not restricted to the measurement of lateral mobility in membranes. It can also be used to determine the transversal mobility, that is, the lipid exchange between the lipid layers of one bilayer or between bilayers of different vesicles. Again, artificial as well as natural membranes can be investigated by this technique.Another important area of investigation in membrane research is the interaction between lipids and proteins. Lipids, in the presence of a protein, show a different dynamic behavior from free lipids. Because of changes in fluidity and a modified solubility of the pyrene probes within different membrane regions, our methods could also be applied to the examination of phase separation phenomena and to lipid-protein interactions.     

Tethering the Z ring to the membrane through a conserved membrane targeting sequence in FtsA

The cytokinetic Z ring is required for bacterial cell division. It consists of polymers of FtsZ, the bacterial ancestor of eukaryotic tubulin, linked to the cytoplasmic membrane. Formation of a Z ring in Escherichia coli occurs as long as one of two proteins, ZipA or FtsA, is present. Both of these proteins bind FtsZ suggesting that they might function to tether FtsZ filaments to the membrane. Although ZipA has a transmembrane domain and therefore can function as a membrane anchor, interaction of FtsA with the membrane has not been explored. In this study we demonstrate that FtsA, which is structurally related to eukaryotic actin, has a conserved C-terminal amphipathic helix that is essential for FtsA function. It is required to target FtsA to the membrane and subsequently to the Z ring. As FtsA is much more widely conserved in bacteria than ZipA, it is likely that FtsA serves as the principal membrane anchor for the Z ring.     

The secretory carrier membrane protein family: structure and membrane topology

Secretory carrier membrane proteins (SCAMPs) are integral membrane proteins found in secretory and endocytic carriers implicated to function in membrane trafficking. Using expressed sequence tag database and library screens and DNA sequencing, we have characterized several new SCAMPs spanning the plant and animal kingdoms and have defined a broadly conserved protein family. No obvious fungal homologue has been identified, however. We have found that SCAMPs share several structural motifs. These include NPF repeats, a leucine heptad repeat enriched in charged residues, and a proline-rich SH3-like and/or WW domain-binding site in the N-terminal domain, which is followed by a membrane core containing four putative transmembrane spans and three amphiphilic segments that are the most highly conserved structural elements. All SCAMPs are 32-38 kDa except mammalian SCAMP4, which is approximately 25 kDa and lacks most of the N-terminal hydrophilic domain of other SCAMPs. SCAMP4 is authentic as determined by Northern and Western blotting, suggesting that this portion of the larger SCAMPs encodes the functional domain. Focusing on SCAMP1, we have characterized its structure further by limited proteolysis and Western blotting with the use of isolated secretory granules as a uniformly oriented source of antigen and by topology mapping through expression of alkaline phosphatase gene fusions in Escherichia coli. Results show that SCAMP1 is degraded sequentially from the N terminus and then the C terminus, yielding an approximately 20-kDa membrane core that contains four transmembrane spans. Using synthetic peptides corresponding to the three conserved amphiphilic segments of the membrane core, we have demonstrated their binding to phospholipid membranes and shown by circular dichroism spectroscopy that the central amphiphilic segment linking transmembrane spans 2 and 3 is alpha-helical. In the intact protein, these segments are likely to reside in the cytoplasm-facing membrane interface. The current model of SCAMP1 suggests that the N and C termini form the cytoplasmic surface of the protein overlying a membrane core, which contains a functional domain located at the cytoplasmic interface with little exposure of the protein on the ectodomain.     

Unconventional roles for membrane traffic proteins in response to muscle membrane stress

In skeletal muscle fibers, ubiquitous membrane trafficking pathways responsible for transporting newly synthesized proteins, recycling cell surface receptors, and organizing membrane compartmentation have adapted to the high needs of an extremely specialized cell under constant mechanical stress. Membrane remodeling proteins involved in ubiquitous mechanisms such as clathrin-mediated endocytosis, caveolae formation, and membrane fusion have evolved to produce new pathways with sometimes completely different functions such as adhesion and mechanoprotection. In this review, I discuss recent advances in understanding the specialized features of skeletal muscle clathrin-coated plaques, caveolae, and dysferlin-mediated membrane repair. A special emphasis is given on recent findings suggesting that membrane trafficking pathways have evolved to participate into the mechanisms responsible for sarcolemma resistance to mechanical stress and discuss how defects in these pathways result in muscle disease.