In a previous study (Spanova et al., 2010, J. Biol. Chem., 285, 6127–6133) we demonstrated that squalene, an intermediate of sterol biosynthesis, accumulates in yeast strains bearing a deletion of ...the HEM1 gene. In such strains, the vast majority of squalene is stored in lipid particles/droplets together with triacylglycerols and steryl esters. In mutants lacking the ability to form lipid particles, however, substantial amounts of squalene accumulate in organelle membranes. In the present study, we investigated the effect of squalene on biophysical properties of lipid particles and biological membranes and compared these results to artificial membranes. Our experiments showed that squalene together with triacylglycerols forms the fluid core of lipid particles surrounded by only a few steryl ester shells which transform into a fluid phase below growth temperature. In the hem1∆ deletion mutant a slight disordering effect on steryl esters was observed indicated by loss of the high temperature transition. Also in biological membranes from the hem1∆ mutant strain the effect of squalene per se is difficult to pinpoint because multiple effects such as levels of sterols and unsaturated fatty acids contribute to physical membrane properties. Fluorescence spectroscopic studies using endoplasmic reticulum, plasma membrane and artificial membranes revealed that it is not the absolute squalene level in membranes but rather the squalene to sterol ratio which mainly affects membrane fluidity/rigidity. In a fluid membrane environment squalene induces rigidity of the membrane, whereas in rigid membranes there is almost no additive effect of squalene. In summary, our results demonstrate that squalene (i) can be well accommodated in yeast lipid particles and organelle membranes without causing deleterious effects; and (ii) although not being a typical membrane lipid may be regarded as a mild modulator of biophysical membrane properties.
► Deletion of HEM1 causes squalene accumulation in yeast. ► Squalene accumulated in lipid droplets has a disordering effect. ► In biological membranes squalene modulates fluidity/rigidity.
Neurotransmitters are released through nascent fusion pores, which ordinarily dilate after bilayer fusion, preventing consistent biochemical studies. We used lipid bilayer nanodiscs as fusion ...partners; their rigid protein framework prevents dilation and reveals properties of the fusion pore induced by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor). We found that although only one SNARE per nanodisc is required for maximum rates of bilayer fusion, efficient release of content on the physiologically relevant time scale of synaptic transmission apparently requires three or more SNARE complexes (SNAREpins) and the native transmembrane domain of vesicle-associated membrane protein 2 (VAMP2). We suggest that several SNAREpins simultaneously zippering their SNARE transmembrane helices within the freshly fused bilayers provide a radial force that prevents the nascent pore from resealing during synchronous neurotransmitter release.
Purpose: The effect of extremely low frequency electromagnetic fields (ELF-EMF) on bacteria has attracted attention due to its potential for beneficial uses. This research aimed to determine the ...effect of ELF-EMF on bacterial membrane namely the membrane potential, surface potential, hydrophobicity, respiratory activity and growth.
Materials and methods: Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli were subjected to ELF-EMF, 50 Hz, 1 mT for 2 h. Membrane potential was determined by fluorescence spectroscopy with or without EDTA (Ethylenediaminetetraacetic acid) with DisC3(5) (3,3-dipropylthiacarbocyanine iodide), zeta potential measurements were performed by electrophoretic mobility, hydrophobicity of the membrane was measured with MATH (Microbial Adhesion to Hydrocarbons) test, respiratory activity was determined with CTC (5-Cyano-2,3-ditolyl tetrazolium chloride), colony forming unit (CFU) and DAPI (4′,6-diamidino-2-phenylindole, dihydrochloride) was used for growth determinations.
Results: ELF-EMF caused changes in physicochemical properties of both Gram-positive and Gram-negative bacteria. Hyperpolarization was seen in S. aureus and EDTA-treated E. coli. Surface potential showed a positive shift in S. aureus contrariwise to the negative shift seen in EDTA-untreated E. coli. Respiratory activity increased in both bacteria. A slight decrease in growth was observed.
Conclusion: These results show that ELF-EMF affects the crucial physicochemical processes in both Gram-positive and Gram-negative bacteria which need further research.
Membrane tethering is a highly regulated event occurring during the initial physical contact between membrane-bounded transport carriers and their target subcellular membrane compartments, thereby ...ensuring the spatiotemporal specificity of intracellular membrane trafficking. Although Rab-family small GTPases and specific Rab-interacting effectors, such as coiled-coil tethering proteins and multisubunit tethering complexes, are known to be involved in membrane tethering, how these protein components directly act upon the tethering event remains enigmatic. Here, using a chemically defined reconstitution system, we investigated the molecular basis of membrane tethering by comprehensively and quantitatively evaluating the intrinsic capacities of 10 representative human Rab-family proteins (Rab1a, -3a, -4a, -5a, -6a, -7a, -9a, -11a, -27a, and -33b) to physically tether two distinct membranes via homotypic and heterotypic Rab-Rab assembly. All of the Rabs tested, except Rab27a, specifically caused homotypic membrane tethering at physiologically relevant Rab densities on membrane surfaces (e.g. Rab/lipid molar ratios of 1:100–1:3,000). Notably, endosomal Rab5a retained its intrinsic potency to drive efficient homotypic tethering even at concentrations below the Rab/lipid ratio of 1:3,000. Comprehensive reconstitution experiments further uncovered that heterotypic combinations of human Rab-family isoforms, including Rab1a/6a, Rab1a/9a, and Rab1a/33b, can directly and selectively mediate membrane tethering. Rab1a and Rab9a in particular synergistically triggered very rapid and efficient membrane tethering reactions through their heterotypic trans-assembly on two opposing membranes. In conclusion, our findings establish that, in the physiological context, homotypic and heterotypic trans-assemblies of Rab-family small GTPases can provide the essential molecular machinery necessary to drive membrane tethering in eukaryotic endomembrane systems.
Photosynthetic processes, including light capture, electron transfer, and energy conversion, are not only ensured by the activities of individual photosynthetic complexes but also substantially ...determined and regulated by the composition and assembly of the overall photosynthetic apparatus at the supramolecular level. In recent years, atomic force microscopy (AFM) has matured as a unique and powerful tool for directly assessing the supramolecular assembly of integral membrane protein complexes in their native membrane environment at submolecular resolution. This review highlights the major contributions and advances of AFM studies to our understanding of the structure of the bacterial photosynthetic machinery and its regulatory arrangement during chromatic adaptation. AFM topographs of other biological membrane systems and potential future applications of AFM are also discussed.
The Ca²⁺ ATPases of the plasma membrane (PMCA pumps) export Ca²⁺ from all eukaryotic cells. In mammals they are the products of four separate genes. PMCA types 1 and 4 are distributed ubiquitously; ...PMCA types 2 and 3 are restricted to some tissues, the most important being the nervous system. Alternative splicing at two sites greatly increases the number of pump isoforms. The two ubiquitous isoforms are no longer considered as only housekeeping pumps as they also perform tissue‐specific functions. The PMCAs are classical P‐type pumps, their reaction cycle repeating that of all other pumps of the family. Their 3D structure has not been solved, but molecular modeling on SERCA pump templates shows the essential structural pattern of the latter. PMCAs are regulated by calmodulin, which interacts with high affinity with their cytosolic C‐terminal tail. A second calmodulin‐binding domain with lower affinity is present in some splicing variants of the pump. The PMCAs are essential to the regulation of cellular Ca²⁺, but the all‐important Ca²⁺ signal is ambivalent: defects in its control generate various pathologies, the most thoroughly studied being those of genetic origin. Genetic defects of PMCA function produce disease phenotypes: the best characterized is a form of deafness in mice and in humans linked to PMCA2 mutations. A cerebellar X‐linked human ataxia has recently been found to be caused by a mutation in the calmodulin‐binding domain of PMCA3.
Membrane fusion is required to establish the morphology and cellular distribution of the mitochondrial compartment. In Drosophila, mutations in the fuzzy onions (fzo) GTPase block a developmentally ...regulated mitochondrial fusion event during spermatogenesis. Here we report that the yeast orthologue of fuzzy onions, Fzo1p, plays a direct and conserved role in mitochondrial fusion. A conditional fzo1 mutation causes the mitochondrial reticulum to fragment and blocks mitochondrial fusion during yeast mating. Fzo1p is a mitochondrial integral membrane protein with its GTPase domain exposed to the cytoplasm. Point mutations that alter conserved residues in the GTPase domain do not affect Fzo1p localization but disrupt mitochondrial fusion. Suborganellar fractionation suggests that Fzo1p spans the outer and is tightly associated with the inner mitochondrial membrane. This topology may be required to coordinate the behavior of the two mitochondrial membranes during the fusion reaction. We propose that the fuzzy onions family of transmembrane GTPases act as molecular switches to regulate a key step in mitochondrial membrane docking and/or fusion.
As a thermally induced membrane separation process, membrane distillation (MD) has drawn more and more attention to the advantages of treating hypersaline wastewaters, especially the concentrate from ...the reverse osmosis (RO) process. One of the major obstacles in widespread MD application is the membrane fouling. We investigated the feasibility of direct contact membrane distillation (DCMD) for landfill leachate reverse osmosis concentrate (LFLRO) brine treatment and systematically assessed the efficiency of chemical cleaning for DCMD after processing LFLRO brine. The results showed that 80% water recovery rate was achieved when processing the LFLRO brine by DCMD, but membrane fouling occurred during the DCMD process, and manifested as the decreasing of permeate flux and the increasing of permeate conductivity. Analysis revealed that the serious flux reduction was primarily caused by the fouling layer, which consisted of organic matter and inorganic salts. Five cleaning methods were investigated for membrane cleaning, including hydrogen chloride (HCl)-sodium hydroxide (NaOH), ethylene diamine tetraacetic acid (EDTA)-NaOH, citric acid, sodium hypochlorite (NaClO) and sodium dodecyl sulphate (SDS) cleaning. Among the chemical cleaning methods investigated, the 3 wt.% SDS cleaning showed the best efficiency at recovering the performance of fouled membranes.
•Zirconia ceramic membrane shows underwater superoleophobicity.•Enhanced alkalinity of oily emulsion effectively mitigated membrane fouling.•Membrane robustness and stable performance were achieved ...under long-term operation.•Efficient separation was realized for real industrial oily wastewater.
While membrane-based oil-water separation has been widely explored, using conventional membranes to treat oily wastewaters remains practically challenging especially when such wastewaters contain more stable nano-sized oil droplets and are of high oil content, and harsh chemical conditions. Herein, we report a novel protocol of efficiently separating both synthetic and real oil nano-emulsions via specially designed robust zirconia membranes. The best-performing zirconia membrane, fabricated at low sintering temperature, has relatively uniform sub-100 nm pores and is underwater superoleophobic. Such zirconia membranes possess not only outstanding separation performance under long-term operation but robust structural stability at harsh conditions. At different cross-flow velocities, a combined model of intermediate pore blocking and cake filtration dominated membrane fouling behavior. Specifically, at high pH value (especially > pH(IEP)), membrane fouling was effectively mitigated due to a dominant role of electrostatic repulsion interaction at membrane–oil interface. Compared with conventional and commercial ceramic membranes, our zirconia membrane is the first reported in literature that can effectively reject nano-sized oil droplets (∼18 nm) with over 99% rejection. Moreover, the zirconia membrane has also been challenged with real degreasing wastewater with very high oil content (∼4284 mg L–1) and pH (∼12.4) and delivered consistently high separation performance over many operation cycles.
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Several studies have demonstrated interactions between the two leaflets in membrane bilayers and the importance of specific lipid species for such interaction and membrane function. We here discuss ...these investigations with a focus on the sphingolipid and cholesterol-rich lipid membrane domains called lipid rafts, including the small flask-shaped invaginations called caveolae, and the importance of such membrane structures in cell biology and cancer. We discuss the possible interactions between the very long-chain sphingolipids in the outer leaflet of the plasma membrane and the phosphatidylserine species PS 18:0/18:1 in the inner leaflet and the importance of cholesterol for such interactions. We challenge the view that lipid rafts contain a large fraction of lipids with two saturated fatty acyl groups and argue that it is important in future studies of membrane models to use asymmetric membrane bilayers with lipid species commonly found in cellular membranes. We also discuss the need for more quantitative lipidomic studies in order to understand membrane function and structure in general, and the importance of lipid rafts in biological systems. Finally, we discuss cancer-related changes in lipid rafts and lipid composition, with a special focus on changes in glycosphingolipids and the possibility of using lipid therapy for cancer treatment.
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