Formation of inside-out vesicles of Bacillus licheniformis. Dependence on buffer composition and lysis procedure.

1. The extent to which the cytoplasmic membrane of the Gram-positive bacterium Bacillus licheniformis formed inside-out vesicles was studied with the freeze-fracture technique. The membrane orientation appeared to be dependent on the buffer compositon as well as on the lysis procedure used. 2. By manipulating these conditions, membrane preparations were obtained with the percentage of inside-out vesicles varying from 15 to 80%. 3. More vesicles had the opposite orientation when the cells were lysed in potassium phosphate buffer than when they were lysed in sodium phosphate buffer. Tris-HCl buffer favoured the formation of inside-out vesicles more than phosphate buffer. 4. Lysis of protoplasts in hypotonic buffers resulted in more inside-out vesicles than did direct lysis of cells in hypotonic media. 5. In an attempt to explain the observed differences, experiments were performed in which the morphology of thin-sectioned lysing cells in sodium phosphate buffer was compared with that in potassium phosphate buffer. The results from these experiments indicate that the formation of inside-out vesicles is brought about by an effect on the membrane itself rather than on the cell wall, on the cell wall membrane association, or on the cytoplasm.     

Evidence for modulation by ATP of amino acid transport in human placenta

The effect of ATP on placental amino acid transport was studied by measuring the uptake of alpha-(methylamino)-isobutyrate in brush border microvillous plasma membrane vesicles prepared from human full-term placental syncytiotrophoblasts which were incubated with or without ATP. The presence of a Na+ gradient from the outside to the inside of the vesicles prepared after incubation with ATP resulted in a higher initial rate and an increased transport of alpha-(methylamino)-isobutyrate, while Na+ gradient-independent alpha-(methylamino)-isobutyrate uptake was not different in either type of membrane vesicle. The increase in transport activity was not inhibited by cycloheximide. Kinetic analysis showed that ATP enhanced transport activity by increasing the maximal velocity (Vmax) of transport, without significant changes in the affinity (Km) of the carrier for the substrate, suggesting an increase in carrier number in placental syncytiotrophoblasts incubated with ATP.     

Calcium-regulated exocytosis is required for cell membrane resealing

Using confocal microscopy, we visualized exocytosis during membrane resealing in sea urchin eggs and embryos. Upon wounding by a laser beam, both eggs and embryos showed a rapid burst of localized Ca(2+)- regulated exocytosis. The rate of exocytosis was correlated quantitatively with successfully resealing. In embryos, whose activated surfaces must first dock vesicles before fusion, exocytosis and membrane resealing were inhibited by neurotoxins that selectively cleave the SNARE complex proteins, synaptobrevin, SNAP-25, and syntaxin. In eggs, whose cortical vesicles are already docked, vesicles could be reversibly undocked with externally applied stachyose. If cortical vesicles were undocked both exocytosis and plasma membrane resealing were completely inhibited. When cortical vesicles were transiently undocked, exposure to tetanus toxin and botulinum neurotoxin type C1 rendered them no longer competent for resealing, although botulinum neurotoxin type A was still ineffective. Cortical vesicles transiently undocked in the presence of tetanus toxin were subsequently fusion incompetent although to a large extent they retained their ability to redock when stachyose was diluted. We conclude that addition of internal membranes by exocytosis is required and that a SNARE-like complex plays differential roles in vesicle docking and fusion for the repair of disrupted plasma membrane.     

General and transport properties of hypotonic and isotonic preparations of resealed erythrocyte ghosts

Summary Resealed human erythrocyte ghosts are regarded as valuable tools for the study of membrane properties. In order to investigate to what extent preparation procedures affect the yield of ghosts, their general properties, and their permeability, ghosts prepared by lysis at low (hypotonic media) and high (isotonic media) ionic strength were compared with each other and with native erythrocytes. For isotonic lysis, cells were either subjected to dielectric breakdown or suspended in isotonic NH₄Cl solutions. In spite of very different characteristics of the lysis and the resealing process in the three types of preparations, the resulting ghosts do not differ in a number of features except for somewhat varying yields and for properties resulting from the mode of lysis.Specific transport properties, as characterized by the mediated fluxes ofm-erythritol,l-arabinose,l-lactate, and sulfate, proved to be unaltered with a few unsystematic exceptions. The simple nonmediated fluxes of all these permeants, as measured in the presence of inhibitors, however, were enhanced between 1.5- and 4-fold, indicating a somewhat increased ground permeability (of the lipid domain) in all ghost membranes.     

Sugar transport in reversibly hemolyzed avian erythrocytes.

The technique of reversible hemolysis represents one approach which may be used to study transport regulation in nucleated red cells. After 1 h of incubation at 37 degrees C, 88% of the ghosts regained their permeability barrier to L-glucose. In these ghosts, the carrier-mediated rate of entry of 3-O-methylglucose was more than 10-fold greater than the rate in intact cells. Glyceraldehyde-3-phosphate dehydrogenase prevented ghosts from resealing when it was present at the time of hemolysis. Albumin, lactic dehydrogenase and peroxidase did not have this effect. Sugar transport rate could not be tested in the unsealed ghosts. Two possible mechanisms for the effect of hypotonic hemolysis on sugar transport rate were discussed: (1) altered membrane organization and (2) loss of intracellular compounds which bind to the membrane and inhibit transport in intact cells.     

Kinetic studies of human erythrocyte membrane resealing

Following lysis in hypotonic media, human erythrocyte membranes will spontaneously reseal and regain their original low permeability for polar solutes. It is generally accepted that resealing will only occur when the membranes are heated above a critical temperature, and that the membrane lesions are stable under cold conditions. Contrary to these prevailing notions, a detailed investigation of the temperature dependence of resealing kinetics over the temperature range 0–22°C revealed that resealing occurs at measurable rates at temperatures as low as 0°C, even in buffers of low ionic strength. At all temperatures studied, initial resealing rates were approximately first-order, and Arrhenius plots of these rates revealed a sharp, singular discontinuity at approx. 7°C.     

Encapsulation of proteins into human erythrocytes: a kinetic investigation

Moderate osmotic shocks of human erythrocytes by hypotonic dialysis (0.06 mosmol/kg) induce cell swelling and formation of pores, without causing apparent lysis. Using 125I-labeled macromolecules of different molecular weight and net charge, we followed the kinetics and efficiency of their encapsulation into erythrocytes. After a 20-30 min period of cell dialysis, macromolecules of up to 50 kDa begin diffusing into the swollen cells by a process which can be described by a first-order two-compartment kinetics. Adsorption to the external cell surface was insignificant, while adsorption to the inner membrane surface was substantial (15-20%) only for positively charged proteins, at physiological pH. After resealing, pores of a 12-14 kDa cut-off might remain open allowing some release of entrapped material (20-30%), depending on the final cytocrit, while the remaining might be associated with inner membrane or cytosolic components. Although the method of hypotonic dialysis is known to affect minimally the biophysical and immunological properties of red blood cell membranes, the interaction of encapsulated material with cell constituents would need to be further assessed when considering red cells as macromolecular carriers.     

Effects of arterial pressure on endothelial transport of macromolecules

The effects of variations in transmural pressure over a range of 0 to 200 mmHg on transendothelial transport of macromolecules were studied in the canine common carotid artery. The uptake of 125I-albumin per unit artery weight increased with rising pressure. There was no significant difference in albumin permeability per unit luminal surface area between 0 and 100 mmHg, but permeability nearly doubled when pressure was raised to 200 mmHg. The contribution of an increased rate of transendothelial vesicle diffusion, as evaluated from the experimental determination of the ratio of attached-to-free vesicles and theoretical modeling, was found to be negligible. The reduction in transendothelial vesicle diffusion distance due to pressure-induced thinning of the peripheral zone contributes to a 25% increase in permeability. With the use of colloidal Ag and Au of various sizes, vesicle loading of particles with diameters greater than or equal to 15 nm was found to be severely restricted at transmural pressure less than or equal to 100 mmHg, but it was significantly enhanced at 200 mmHg, when particles as large as 25 nm became detectable in endothelial vesicles and subendothelial space. This hypertension-induced increase in macromolecular transport across the endothelium may cause an overloading of the arterial wall with low-density lipoproteins and play a significant role in atherogenesis.     

Amino acid transport by membrane vesicles of virally transformed and nontransformed cells: effects of sodium gradient and cell density.]

Mixed membrane vesicle populations composed of plasma membrane and endoplasmic reticulum were prepared from Balb/c 3T3 and simian virus 40-transformed Balb/c 3T3 mouse fibroblasts. The initial rates of uptake of L-leucine and alpha-aminoisobutyric acid by these vesicles were stimulated by a NaCl gradient (external greater than internal). Cation specificity for stimulation of L-leucine uptake was Na+ greater than Li+ greater than K+. NaSCN was as effective as NaCl. Stimulation of uptake of both amino acids by a NaCl gradient was twice as great in vesicles from transformed as compared to non-transformed cells. The NaCl gradient produced transient accumulation of both L-leucine and alpha-aminoisobutyric acid to twice the equilibrium level in vesicles from transformed cells. No such "overshoot" was observed in vesicles from nontransformed cells. In vesicles from the contact-inhibitable Balb/c 3T3 cells, transport of alpha-aminoisobutyric acid, but non L-leucine, exhibited a density-dependent decrease in Na+ gradient induced stimulation, from 248% for sub-confluent to 109% with confluent cells. No density-related changes in uptake were noted with vesicles from the transformed cells. These studies suggest that variation in amino acid uptake associated with viral transformation may be related, at least in part, to alterations in Na+ permeability of the surface membrane.     

Delta pH, H+ diffusion potentials, and Mg2+ ATPase in neurosecretory vesicles isolated from bovine neurohypophyses

The proton gradient (delta pH) and electrical potential (delta psi) across the neurosecretory vesicles were measured using the optical probes 9-aminoacridine and Oxanol VI, respectively. The addition of neurosecretory vesicles to 9-aminoacridine resulted in a rapid quenching of the dye fluorescence which was reversed when the delta pH was collapsed with ammonium chloride or K+ in the presence of nigericin. From fluorescence quenching data and the intravesicular volume, delta pH across the membrane was calculated. Mg2+ ATP caused a marked carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive change in the membrane potential measured using Oxanol VI (plus 100 mV inside positive), presumably due to H+ translocation across the neurosecretory vesicle membrane. Imposition of this membrane potential was responsible for the lysis of vesicles in the presence of permeant anions. The effectiveness of these anions to support lysis reflected the relative permeability of the anion which followed the order acetate greater than I- greater than Cl greater than F- greater than SO4- = isethionate = methyl sulfate. These data showed that the neurosecretory vesicles possess a membrane H+-translocating system and prompted the study of Mg2+-dependent ATPase activities in the vesicle fractions. In intact vesicles a Mg2+ ATPase appeared to be coupled to electrogenic proton translocation, since the enzyme activity was enhanced by uncoupling the electrical potential, using proton ionophores. Inhibition of this enzyme with dicyclohexylcarbodiimide also inhibited the carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive delta psi across the vesicle membrane caused by H+ translocation. A second Mg2+ ATPase was also found on the vesicle membranes which is sensitive to vanadate. Complete inhibition of this enzyme with vanadate had little effect on the proton ionophore-uncoupled ATPase activity or on the Mg2+ ATP-induced membrane potential change.     

Membrane permeability to macromolecules mediated by the membrane attack complex

A simple and well-defined system of purified phospholipids and human complement proteins was used to study membrane permeability to macromolecules mediated by the membrane attack complex (MAC) of complement. Large unilamellar vesicles (LUVs) of phosphatidylcholine (PC) or phosphatidylserine (PS) containing trapped macromolecules [bovine pancreatic trypsin inhibitor (BPTI), thrombin, glucose-6-phosphate dehydrogenase (G6PD), and larger molecules] were used to monitor permeability. Membrane permeability to macromolecules was measured by thrombin inhibition by an external inhibitor or by separation of released molecules by gel filtration. Membrane-bound intermediates (C5b-8 or C5b-93) were stable for hours, and macromolecular permeability occurred without fragmentation, fusion, or aggregation of the vesicles. Quantitative membrane binding by C5b-7 as well as essentially quantitative release of thrombin was obtained for PS vesicles. MAC binding to PS-LUVs approximated the theoretical Poisson distribution curve for full release of vesicle contents by one complex per vesicle. Reactions with PC-LUVs occurred with some fluid-phase MAC assembly. Therefore, results from experiments with these vesicles were interpreted in a relative manner. However, the values obtained closely corroborated those obtained with PS-LUVs. At low C9/C5b-8 ratios, the size of the lesion was proportional to the C9 content of the MAC. Half-maximum release of BPTI, thrombin, and G6PD, by a single MAC per vesicle, required approximately 3,5, and 7 C9/C5b-8 (mol/mol), respectively. Larger molecules (greater than or equal to 118-A diameter) were not released from the vesicles. Release of G6PD (95.4-A diameter) required 45% of saturating C9. Therefore, it appeared that the last half of the bound C9 molecules did not increase pore size and the pore which released G6PD approached the diameter of the closed circular lesion measured (by others) in electron micrographs (approximately 100 A). The results were consistent with the formation of a stable membrane pore by a single complex per vesicle in which C9 molecules line only one side of the pore at low C9/C5b-8 ratios and maximum pore size is attained by incomplete, noncircular polymers of C9.     

Sugar transport in reversibly hemolyzed avian erythrocytes

The technique of reversible hemolysis represents one approach which may be used to study transport regulation in nucleated red cells. After 1 h of incubation at 37°C, 88% of the ghosts regained their permeability barrier to l-glucose. In these ghosts, the carrier-mediated rate of entry of $\text{3-O-}\text{methylglucose}$ was more than 10-fold greater than the rate in intact cells. Glyceraldehyde-3-phosphate dehydrogenase prevented ghosts from resealing when it was present at the time of hemolysis. Albumin, lactic dehydrogenase and peroxidase did not have this effect. Sugar transport rate could not be tested in the unsealed ghosts. Two possible mechanisms for the effect of hypotonic hemolysis on sugar transport rate were discussed: (1) altered membrane organization and (2) loss of intracellular compounds which bind to the membrane and inhibit transport in intact cells.     

Molecular regulation of membrane resealing in 3T3 fibroblasts

Membrane resealing in mammalian cells after injury depends on Ca(2+)-dependent fusion of intracellular vesicles with the plasma membrane. When cells are wounded twice, the subsequent resealing is generally faster. Physiological and biochemical studies have shown the initiation of two different repair signaling pathways, which are termed facilitated and potentiated responses. The facilitated response is dependent on the generation and recruitment of new vesicles, whereas the potentiated response is not. Here, we report that the two responses can be differentially defined molecularly. Using recombinant fragments of synaptobrevin-2 and synaptotagmin C2 domains we were able to dissociate the molecular requirements of vesicle exocytosis for initial membrane resealing and the facilitated and potentiated responses. The initial resealing response was blocked by fragments of synaptobrevin-2 and the C2B domain of synaptotagmin VII. Both the facilitated and potentiated responses were also blocked by the C2B domain of synaptotagmin VII. Although the initial resealing response was not blocked by the C2AB domain of synaptotagmin I or the C2A domain of synaptotagmin VII, recruitment of new vesicles for the facilitated response was inhibited. We also used Ca2+ binding mutant studies to show that the effects of synaptotagmins on membrane resealing are Ca(2+)-dependent. The pattern of inhibition by synaptotagmin C2 fragments that we observed cannot be used to specify a vesicle compartment, such as lysosomes, in membrane repair.     

Alignment of Synaptic Vesicle Macromolecules with the Macromolecules in Active Zone Material that Direct Vesicle Docking

Synaptic vesicles dock at active zones on the presynaptic plasma membrane of a neuron’s axon terminals as a precondition for fusing with the membrane and releasing their neurotransmitter to mediate synaptic impulse transmission. Typically, docked vesicles are next to aggregates of plasma membrane-bound macromolecules called active zone material (AZM). Electron tomography on tissue sections from fixed and stained axon terminals of active and resting frog neuromuscular junctions has led to the conclusion that undocked vesicles are directed to and held at the docking sites by the successive formation of stable connections between vesicle membrane proteins and proteins in different classes of AZM macromolecules. Using the same nanometer scale 3D imaging technology on appropriately stained frog neuromuscular junctions, we found that ∼10% of a vesicle’s luminal volume is occupied by a radial assembly of elongate macromolecules attached by narrow projections, nubs, to the vesicle membrane at ∼25 sites. The assembly’s chiral, bilateral shape is nearly the same vesicle to vesicle, and nubs, at their sites of connection to the vesicle membrane, are linked to macromolecules that span the membrane. For docked vesicles, the orientation of the assembly’s shape relative to the AZM and the presynaptic membrane is the same vesicle to vesicle, whereas for undocked vesicles it is not. The connection sites of most nubs on the membrane of docked vesicles are paired with the connection sites of the different classes of AZM macromolecules that regulate docking, and the membrane spanning macromolecules linked to these nubs are also attached to the AZM macromolecules. We conclude that the luminal assembly of macromolecules anchors in a particular arrangement vesicle membrane macromolecules, which contain the proteins that connect the vesicles to AZM macromolecules during docking. Undocked vesicles must move in a way that aligns this arrangement with the AZM macromolecules for docking to proceed.     

Carrier-mediated transport system for cephalexin in human placental brush-border membrane vesicles

The uptake of cephalosporin antibiotics, cephalexin, was studied with brush-border microvillous plasma membrane vesicles prepared and purified from human full-term placental syncytiotrophoblasts. The uptake of cephalexin by the membrane vesicles was not stimulated in the presence of an Na+ gradient from the outside to the inside of the vesicles, whereas alpha-(methylamino)isobutyrate uptake into the vesicles of the same preparation was stimulated by an Na+ gradient. The equilibrium level of cephalexin uptake decreased with increasing osmolarity of the medium, which indicates that cephalexin is transported into the membrane vesicles. When cephalexin concentrations were varied, the initial rate of uptake obeyed Michaelis-Menten kinetics with Km and Vmax values of 2.29 mM and 2.98 nmol/mg of protein per 60 s, respectively. The uptake of cephalexin was inhibited by structural analogues and sulfhydryl modifying reagents. These results indicate the existence of a carrier-mediated transport system for cephalexin in the human placental brush-border membranes.     

Purification of microvillus membrane vesicles from pig small intestine by adsorption chromatography on sepharose

A simple, rapid method for the preparation of pure microvillus membrane vesicles from pig small intestine is described. The method is based on the ability of agarose beads to adsorb selectively the impurities, mainly basolateral membrane fragments, from a microvillus vesicle preparation isolated by hypotonic lysis, Mg²⁺ aggregation of contaminants and differential centrifugation.     

A flow EPR study of deformation and orientation characteristics of erythrocyte ghosts: Effects of lysing and resealing conditions

Summary The effects of various conditions in lysing and resealing the red cell membrane on the degree of ghost deformation and orientation in flow are investigated using the flow EPR and spin-label method. The relatively low deformability of the standard ghost, which is lysed and resealed, respectively, in hypotonic and isotonic NaCl-Tris buffer, is markedly enhanced by the presence of Mg-ATP, chlorpromazine, or Ca²⁺ ion during resealing. The effect is concentration dependent, and there is an optimal level for each treatment. Chlorpromazine and Ca²⁺ are also effective when added to the resealed ghosts. Mg²⁺ ion shows an opposite effect reducing the ghost deformability in flow at all concentrations. An isotonic lysis in NH₄HCO₃ solution with less osmotic stress substantially raises ghost deformability above that of the standard ghosts. These results are interpreted on the basis of a misalignment between the bilayer leaflets that is probably brought about during hypotonic lysis and its recovery to the nearly normal bilayer state by the agents used during or after resealing. The novel finding of deformability enhancing effect of calcium is assumed to be caused by the electrostatic expansion of the inner layer relative to the outer leaflet. The explanations are supported by the resealed ghost shapes observed before and after the treatments; shape recovery from the monoconcave spheroid toward biconcave discoid is observed in most cases concomitantly with improvements of flow characteristics.     

Functional symmetry of the beta-galactoside carrier in Escherichia coli.

Cytoplasmic membrane vesicles with either normal or inverted orientation were prepared from Escherichia coli. The lactose transport activity of these vesicle preparations was compared. The parameters measured were net efflux, counterflux, and K+/valinomycin-induced active uptake of lactose. With membrane vesicles derived from both wild-type and cytochrome-deficient strains the right-side-out and inverted membrane preparations showed similar rates of lactose flux in all assays. According to these criteria, the activity of the beta-galactoside transport protein is inherently symmetrical. One major difference was observed between the native and inverted vesicle preparations: the inverted vesicles had approximately twice the specific activity of native vesicles in the counterflux and K+/valinomycin-induced uptake assays. This difference can be largely ascribed to the presence in the normal vesicle preparation of vesicles with a high passive permeability to lactose. Such vesicles are apparently absent from the inverted vesicle preparations.     

Preparation of membrane vesicles from isolated myelin: studies on functional and structural properties.

Myelin membranes purified from bovine brain are shown to form membrane vesicles when incubated in hypotonic buffer. Following restoration of isotonicity a resealing of the membrane occurs as judged by a significant decrease in 22Na+ permeability. Electron spin resonance measurements using stearic acid spin label I indicate a small decrease in membrane fluidity with increasing ionic strength between 50 and 80 mM NaCl. Iodination of myelin membrane vesicles by lactoperoxidase shows a four-fold increase in the amount of iodine incorporation into the myeline basic protein from 0--150 mM NaCl, while the iodination of the proteolipid protein remains essentially unaffected by the change in ionic strength. This dependence of the iodination of the myelin basic protein on the ionic strength can be explained by the electrostatic interactions of this protein with membrane lipids. In view of striking analogies with studies on model membranes correlating protein binding with membrane permeability changes, we suggest a similar structure-function relationship for the myelin basic protein.