Variations In Membrane Composition Research Articles

Membrane permeabilization of colistin toward pan-drug resistant Gram-negative isolates.

Pan-drug resistant Gram-negative bacteria, being resistant to most available antibiotics, represent a huge threat to the medical community. Colistin is considered the last therapeutic option for patients in hospital settings. Thus, we were concerned in this study to demonstrate the membrane permeabilizing activity of colistin focusing on investigating its efficiency toward those pan-drug resistant isolates which represent a critical situation. We determined the killing dynamics of colistin against pan-drug resistant isolates. The permeability alteration was confirmed by different techniques as: leakage, electron microscopy and construction of an artificial membrane model; liposomes. Moreover, selectivity of colistin against microbial cells was also elucidated. Colistin was proved to be rapid bactericidal against pan-drug resistant isolates. It interacts with the outer bacterial membrane leading to deformation of its outline, pore formation, leakage of internal contents, cell lysis and finally death. Furthermore, variations in membrane composition of eukaryotic and microbial cells provide a key for colistin selectivity toward bacterial cells. Colistin selectively alters membrane permeability of pan-drug resistant isolates which leads to cell lysis. Colistin was proved to be an efficient last line treatment for pan-drug resistant infections which are hard to treat.

Platelet membrane variations and their effects on δ-granule secretion kinetics and aggregation spreading among different species

Platelet exocytosis is regulated partially by the granular/cellular membrane lipids and proteins. Some platelets contain a membrane-bound tube, called an open canalicular system (OCS), which assists in granular release events and increases the membrane surface area for greater spreading. The OCS is not found in all species, and variations in membrane composition can cause changes in platelet secretion. Since platelet studies use various animal models, it is important to understand how platelets differ in both their composition and granular release to draw conclusions among various models. The relative phospholipid composition of the platelets with (mouse, rabbit) and without (cow) an OCS was quantified using UPLC-MS/MS. Cholesterol and protein composition was measured using an Amplex Red Assay and BCA Assay. TEM and dark field platelet images were gathered and analyzed with Image J. Granular release was monitored with single cell carbon fiber microelectrode amperometry. Cow platelets contained greater amounts of cholesterol and sphingomyelin. In addition, they yield greater serotonin release and longer δ granule secretion times. Finally, they showed greater spreading area with a greater range of spread. Platelets containing an OCS had more similarities in their membrane composition and secretion kinetics compared to cow platelets. However, cow platelets showed greater fusion pore stability which could be due to extra sphingomyelin and cholesterol, the primary components of lipid rafts. In addition, their greater stability may lead to many granules assisting in spreading. This study highlights fundamental membrane differences and their effects on platelet secretion.

The Many Faces of Lipid Rafts

The Many Faces of Lipid Rafts

Minimal mesoscale model for protein-mediated vesiculation in clathrin-dependent endocytosis.

In eukaryotic cells, the internalization of extracellular cargo via the endocytic machinery is an important regulatory process required for many essential cellular functions. The role of cooperative protein-protein and protein-membrane interactions in the ubiquitous endocytic pathway in mammalian cells, namely the clathrin-dependent endocytosis, remains unresolved. We employ the Helfrich membrane Hamiltonian together with surface evolution methodology to address how the shapes and energetics of vesicular-bud formation in a planar membrane are stabilized by presence of the clathrin-coat assembly. Our results identify a unique dual role for the tubulating protein epsin: multiple epsins localized spatially and orientationally collectively play the role of a curvature inducing capsid; in addition, epsin serves the role of an adapter in binding the clathrin coat to the membrane. Our results also suggest an important role for the clathrin lattice, namely in the spatial- and orientational-templating of epsins. We suggest that there exists a critical size of the coat above which a vesicular bud with a constricted neck resembling a mature vesicle is stabilized. Based on the observed strong dependence of the vesicle diameter on the bending rigidity, we suggest that the variability in bending stiffness due to variations in membrane composition with cell type can explain the experimentally observed variability on the size of clathrin-coated vesicles, which typically range 50–100 nm. Our model also provides estimates for the number of epsins involved in stabilizing a coated vesicle, and without any direct fitting reproduces the experimentally observed shapes of vesicular intermediates as well as their probability distributions quantitatively, in wildtype as well as CLAP IgG injected neuronal cell experiments. We have presented a minimal mesoscale model which quantitatively explains several experimental observations on the process of vesicle nucleation induced by the clathrin-coated assembly prior to vesicle scission in clathrin dependent endocytosis.

Retinoid Chromophores as Probes of Membrane Lipid Order

There is a great need for development of independent methods to study the structure and function of membrane-associated proteins and peptides. Polarized light spectroscopy (linear dichroism, LD) using shear-aligned lipid vesicles as model membranes has emerged as a promising tool for the characterization of the binding geometry of membrane-bound biomolecules. Here we explore the potential of retinoic acid, retinol, and retinal to function as probes of the macroscopic alignment of shear-deformed 100 nm liposomes. The retinoids display negative LD, proving their preferred alignment perpendicular to the membrane surface. The magnitude of the LD indicates the order retinoic acid > retinol > retinal regarding the degree of orientation in all tested lipid vesicle types. It is concluded that mainly nonspecific electrostatic interactions govern the apparent orientation of the retinoids within the bilayer. We propose a simple model for how the effective orientation may be related to the polarity of the end groups of the retinoid probes, their insertion depths, and their angular distribution of configurations around the membrane normal. Further, we provide evidence that the retinoids can sense subtle structural differences due to variations in membrane composition and we explore the pH sensitivity of retinoic acid, which manifests in variations in absorption maximum wavelength in membranes of varying surface charge. Based on LD measurements on cholesterol-containing liposomes, the influence of membrane constituents on bending rigidity and vesicle deformation is considered in relation to the macroscopic alignment, as well as to lipid chain order on the microscopic scale.

Phase Separation of Lipid Membranes Analyzed with High-Resolution Secondary Ion Mass Spectrometry

Lateral variations in membrane composition are postulated to play a central role in many cellular events, but it has been difficult to probe membrane composition and organization on length scales of tens to hundreds of nanometers. We present a high-resolution imaging secondary ion mass spectrometry technique to reveal the lipid distribution within a phase-separated membrane with a lateral resolution of approximately 100 nanometers. Quantitative information about the chemical composition within small lipid domains was obtained with the use of isotopic labels to identify each molecular species. Composition variations were detected within some domains.

Lateral Pressures in Cell Membranes: A Mechanism for Modulation of Protein Function

Variations in the composition of cell membranes can strongly influence the function of proteins embedded therein. However, in most cases it is not known whether lipids and other membrane components act by binding directly to proteins or indirectly through changes in a structural or thermodynamic property of the fluid bilayer. In the present work, we develop a simple thermodynamic analysis based on the hypothesis that variations in membrane composition induce changes in the transverse pressure profile in lipid bilayers. If protein function involves a conformational transition accompanied by a depth-dependent change in its cross-sectional area, we predict that small changes in the lateral pressure can induce a large shift in the conformational distribution. The sensitivity of the conformational equilibrium to the lateral pressure profile arises in part from the localization of the large interfacial free energy within a domain of molecular thickness and also from the difference between the logarithmic depend...

28] Depalmitoylation of rhodopsin with hydroxylamine

Intrinsic membrane proteins that to date have been investigated with respect to the function of palmitoylation are the beta-adrenergic receptor, rhodopsin, the alpha 2A-adrenergic receptor, and the influenza virus spike glycoprotein. As described above, the studies have led to differing conclusions with respect to the influence of palmitoylation on physiological activity. The basis of the differences remains unclear, but it may relate at least in part to the membrane environment of the protein during these studies, that is, the presence of a native membrane, the membrane composition of the expression cell line (in the case of mutant proteins), or the absence of membrane (in the case of detergent-purified proteins). For example, in the case of rhodopsin, the composition of the ROS disk membrane differs from that of the rod plasma membrane, and presumably also from the plasma membranes of cell lines in which mutant rhodopsins are expressed. Variation in membrane composition is known to have marked effects on the ability of rhodopsin to mediate the photic activation of PDE. Thus, although Karnik et al. clearly demonstrated the absence of an absolute requirement for palmitate in activating transducin, the influence of detergent on tertiary protein structure may have masked the full effect of the elimination of palmitate on the transducin-activating property of rhodopsin. Alternatively, the differing results obtained in the studies of rhodopsin could be a consequence of differences in amino acid sequences of the proteins studied. The precise functional role of the palmitate groups of rhodopsin remains an important question for further research. It was suggested by Ovchinnikov et al. that the hydrolysis of covalently bound palmitate might occur during the process of rhodopsin bleaching, but more recent data argue against this hypothesis. Experiments using synthetic peptides (representing cytoplasmic loop regions of rhodopsin) to identify the sites of interaction of R* and transducin provide support for an alternative possibility, namely, that palmitoylation and the resulting cytoplasmic loop play a role in the coupling of rhodopsin with transducin. The finding that the binding of transducin to R* occurs independently of the presence of palmitate argues against an essential requirement of palmitoylation on the binding step itself. However, available data indicate an enhancement, by depalmitoylation, of light-dependent GTPase activity in ROS preparations, although not in assays of unpalmitoylated, purified mutant rhodopsins (see above).(ABSTRACT TRUNCATED AT 400 WORDS)

Differences in composition and fluidity of intestinal microvillus membrane vesicles prepared by different methods

Multiple methods have been developed to isolate the intestinal microvillus membrane and facilitate the study of its composition and fuction. Variations in membrane composition and fluidity may result from different preparative techniques. This study shows that the use of MgCl 2 and/or KSCN in vesicle preparation alters phospholipid and protein composition of the membrane compared to CaCl 2 precipitation. The use of MgCl 2 in membrane preparation increased phosphatidylethanolamine and decreased phosphatidylinositol content. The use of KSCN in membrane preparation decreased the protein content. The structural changes seen with the use MgCl 2 alone are accompanied by an increase in both static and dynamic membrane fluidity. These results suggest that different methods of membrane vesicle preparation affect membrane phospholipid and protein content as well as membrane fluidity.

Cellular effects of endotoxin in vitro: mobility of endotoxin in the plasma membrane of hepatocytes and neuroblastoma cells

Lipopolysaccharide labeled with fluorescein isothiocyanate (FITC-LPS) was used to examine interactions between endotoxin and plasma membrane in isolated rat hepatocytes and mouse neuroblastoma NB41A3 cells. At the same endotoxin to cell ratio, hepatocytes bound more toxin than did neuroblastoma cells. At a dose of 12 μg/mg dry wt, a bound mobile fraction of between 60 and 75% of FITC-LPS was found on hepatocytes at 25°C with a lateral diffusion coefficient ( D) of 4.0·10 −9 cm 2/s. In neuroblastoma cells, the mobile fraction was larger (85–90%), with D 1.0·10 −8 cm 2/s. D was temperature-dependent between 10 and 37°C and increased from 1.8·10 −9 to 1.0·10 −8 cm 2/s in hepatocytes and from 9.4·10 −9 to 1.9·1 cm 2/s in neuroblastoma cells. In both types of cell, nonviable (cells which did not exclude Trypan blue) as compared to viable cells showed different recovery patterns and 100% of the probe molecules were mobile. These results suggest that: (1) endotoxin binding to mammalian cells consists of two subpopulations with different mobilities; (2) binding of the immobile fraction is dependent on cellular integrity; and (3) the differences in binding, lateral mobility, and size of the immobile fraction in hepatocytes and neuroblastoma cells may be due to variations in membrane composition and/or number of binding sites.

15] Artificial cells containing multienzyme systems

An artificial cell is a spherical ultrathin membrane of cellular dimensions enveloping biologically active material. The enveloping membrane of each artificial cell separates the contents of the artificial cells from the external environment. Artificial cells can also be made with other variations in membrane composition, permeability, and other characteristics. It is possible to enclose almost any combination of enzymes, multienzyme systems, cofactor regenerating enzyme systems, cell extracts, whole cells, proteins, adsorbents, magnetic materials, and multicompartmental systems. This chapter discusses the basic methods used for the preparation of artificial cells and presents the physical examples for demonstrating the principle of artificial cells. Artificial cells are being used in detoxifiers, artificial kidneys, artificial livers, immunosorbents, blood substitutes, drug carriers, cell cultures, and hybrid organs. Artificial cells containing enzymes and proteins have been used in a number of experimental and therapeutic conditions. Artificial cells with red blood cell hemolysate have been tested for use as red blood cell substitutes.

Adenine nucleotide transport in hepatoma mitochondria. Characterization of factors influencing the kinetics of ADP and ATP uptake

Initial velocity measurements of [ 3H]ADP and [ 3H]ATP uptake have been made with mitochondria isolated from Morris hepatomas of differing growth rates, and factors known to influence the rates of nucleotide exchange have been examined in an effort to determine whether the elevated rates of aerobic glycolysis in these tumors can be attributed to altered carrier activity. These studies included the determination of the apparent kinetic constants for nucleotide uptake as a function of the mitochondrial energy state and the dependence of transport rates on temperature. Also included in these studies were measurements of the mitochondrial levels of endogenous inhibitors, divalent cations and internal adenine nucleotides. Results obtained showed that with mitochondria isolated from the various tumor lines, the apparent kinetic constants for nucleotide uptake are different from those of control rat or regenerating liver mitochondria; the apparent V max values for both ADP and ATP uptake are significantly lower. Furthermore, under conditions of a high-energy state, the K m and V max values for ATP uptake are greater than the K m and V max value for ADP uptake but that under uncoupled conditions, the opposite is observed. Comparison of the levels of mitochondrial Ca 2+, Mg 2+, long-chain acyl-CoA ester and adenine nucleotide from the various mitochondria showed that important differences exist between liver and hepatoma mitochondria in the levels of Ca 2+, long-chain acyl-CoA ester and AMP. Mitochondrial Ca 2+ levels are elevated 3–5-fold in all tumor lines, and for Morris 7777 hepatoma (a rapidly growing tumor) by a remarkable 70-fold; whereas the levels of acyl-CoA ester and AMP are significantly lower in the more rapidly growing tumors. Arrhenius plots for nucleotide uptake in mitochondria from liver and hepatoma are characterized as being biphasic, having similar activation energies above and below the break point temperature (28–38 and 6–16 kcal/mol, respectively). However, the transition temperature for mitochondria from the various hepatomas is uniformly 4–5°C lower than mitochondria from control liver. The latter difference may reflect a variation in membrane composition, most probably lipid components. It is concluded that the presence of elevated levels of Ca 2+ and lower levels of AMP in hepatoma mitochondria and difference of membrane compositions may play an important role in limiting adenine nucleotide transport activity in vivo and that the impaired carrier activity may contribute to higher rates of aerobic glycolysis observed in these tumors.

Influence of different fixing agents on lectin binding by the Tetrahymena

Binding studies on membranes of unicellulars are most conveniently performed in fixed preparations, in which phagocytotic activity does not interfere with the membrane-ligand inter-actions. The fixing procedure may, however, considerably alter the interrelationships between membrane components, and the quality and quantity of the groups capable of binding. We examined the influence of 8 different fixatives on binding of 7 lectins of dissimilar sugar specificity to the membrane of Tetrahymena pyriformis GL strain. The quantitative relations of binding were found to vary markedly with the type of fixing agent, and disparities of the binding pattern between body regions have suggested regional variations in membrane composition. It follows that the results of binding experiments are not comparable unless the same fixing agents has been used.

A Mycoplasma Mutant Resistant to Lysis by C: Variations in Membrane Composition and Altered Response to the Terminal C Complex

Abstract Mutant strains of Acholeplasma laidlawii resistant to immune lysis by the classical C pathway have been obtained after the exposure of wild-type cells to nitrosoguanidine. The lysis of one mutant strain, comr-328 is less than 20% of that of the wild-type strain when incubated with antiserum prepared against wild-type cells and guinea pig serum as a source of C. The osmotic stability of comr-328 is similar to that of the wild-type strain. Absorption of anti-wild-type antiserum with mutant or wild-type cells results in the complete loss of C fixing antibody as shown by the inability of the absorbed antiserum to sensitize wild-type cells for immune lysis. Both strains convert 125I-labeled C3 to C3b to the same extent and bind equivalent amounts of 125I-C3b. Whereas the wild-type strain is sensitive to lysis by reactive lysis, comr-328 is resistant. Although both cell types are lysed equally well by amphotericin B, comr-328 is less sensitive than the wild-type strain to lysis by melittin. The membrane lipids of both strains are qualitatively identical, as judged by thin-layer chromatography of chloroform-methanol extracts, but the amount of each lipid varies according to the strain. The most significant variation is found in the glycolipids; the monoglucosyldiglyceride to diglucosyldiglyceride ratio of comr-328 is 2-fold higher than that of the wild-type strain. The difference in the phospholipids is less marked. While more than 35 Coomassie Blue-stained polypeptides are detected by SDS-polyacrylamide gel electrophoresis of solubilized membranes, major quantitative differences occur in only three of these bands. Band 11 is more prominent in comr-328 than in the wild-type strain whereas bands 14 and 26 appear to be less abundant in the mutant.