Interaction of surfactants with vesicle membrane of dipalmitoylphosphatidylcholine: fluorescence depolarization study

The effect of surfactants on the "fluidity" of dipalmitoylphosphatidylcholine (DPPC) vesicle membrane was studied by means of the fluorescence depolarization technique with fatty acid fluorescent probes, in which the anthroyloxy group is introduced at different positions along the acyl chain. Three types of surfactants were examined; anionic sodium alkylsulfates, cationic alkyltrimethylammonium chlorides, and non-ionic alkanoyl-N-methylglucamides (MEGA-n). Perturbing effects of the surfactants depended on both the alkyl chain-length and the type of head group. Sodium alkylsulfates with octyl- and decyl-chain and alkyltrimethylammonium chlorides with octyl-, decyl- and dodecyl-chain did not affect the membrane fluidity when incorporated in the membrane, whereas sodium dodecylsulfate and tetradecyltrimethylammonium chloride decreased the membrane fluidity at both gel and liquid crystalline states of the membrane. All the MEGA series surfactants decreased the membrane fluidity, whose perturbing potency was in the order of MEGA-8 less than MEGA-9 approximately equal to MEGA-10. The perturbation at different depths in the membrane by sodium dodecylsulfate and MEGA-9 was also examined. No significant change in the fluidity gradient across the membrane was induced by the addition of these surfactants.     

Video fluorescence microscopy studies of phospholipid vesicle fusion with a planar phospholipid membrane. Nature of membrane-membrane interactions and detection of release of contents

Video fluorescence microscopy was used to study adsorption and fusion of unilamellar phospholipid vesicles to solvent-free planar bilayer membranes. Large unilamellar vesicles (2-10 microns diam) were loaded with 200 mM of the membrane-impermeant fluorescent dye calcein. Vesicles were ejected from a pipette brought to within 10 microns of the planar membrane, thereby minimizing background fluorescence and diffusion times through the unstirred layer. Vesicle binding to the planar membrane reached a maximum at 20 mM calcium. The vesicles fused when they were osmotically swollen by dissipating a KCl gradient across the vesicular membrane with the channel-forming antibiotic nystatin or, alternatively, by making the cis compartment hyperosmotic. Osmotically induced ruptures appeared as bright flashes of light that lasted several video fields (each 1/60 s). Flashes of light, and therefore swelling, occurred only when channels were present in the vesicular membrane. The flashes were observed when nystatin was added to the cis compartment but not when added to the trans. This demonstrates that the vesicular and planar membranes remain individual bilayers in the region of contact, rather than melding into a single bilayer. Measurements of flash duration in the presence of cobalt (a quencher of calcein fluorescence) were used to determine the side of the planar membrane to which dye was released. In the presence of 20 mM calcium, 50% of the vesicle ruptures were found to result in fusion with the planar membrane. In 100 mM calcium, nearly 70% of the vesicle ruptures resulted in fusion. The methods of this study can be used to increase significantly the efficiency of reconstitution of channels into planar membranes by fusion techniques.     

Direct determination of free fatty acid transport across the adipocyte plasma membrane using quantitative fluorescence microscopy.

Movement of free fatty acids (FFA) across the plasma membrane has been directly measured for the first time, using fluorescent FFA analogs and quantitative fluorescence microscopy. The rate of short chain FFA (less than or equal to 12 carbons) transport from the extracellular medium into intracellular lipid droplets of 3T3F442A adipocytes was more than 40-fold faster than long chain FFA (16 and 18 carbons). The membrane-impermeable amino reagent 4,4'-diisothiocyanostilbene-2,2'-disulfonate, inhibited greater than or equal to 50% of the long chain FFA transport but had no effect on short chain FFA transport. Oleic acid (2 microM) inhibited 90% of the fluorescent oleate transport but had no effect on the 11-carbon analog. These results indicate that a large fraction of long chain FFA uptake is mediated by a plasma membrane protein (s).     

A light-scattering measurement of membrane vesicle permeability.

Light-scattering/intensity autocorrelation measurements of vesicle diffusivity were used to follow the time course of the osmotic response of lobster abdominal sarcoplasmic reticulum vesicles to five lipophobic nonelectrolytes. Steady-state portions of the resulting time traces show these vesicles to be permeable to ethylene glycol and glycerol and impermeable to erythritol, glucose, and sucrose. Using measured values of the hydrodynamic radii of these nonelectrolytes, it is concluded that under passive transport conditions, these vesicles may be thought of as having pores whose radii lie between 3.1 and 3.5 A. In addition, the results presented here indicated that above a certain impermeable nonelectrolyte concentration, vesicles did not respond osmotically even though they had not collapsed. This suggests that at least under the experimental conditions reported here, vesicles behaved as if rigid when their average volume had decreased to about 50% of its original isotonic value.     

Electron cryomicroscopy of bacteriorhodopsin vesicles: mechanism of vesicle formation.

We obtained vesicles from purple membrane of Halobacterium halobium at different suspension compositions (pH, electrolytes, buffers), following the procedure of Kouyama et al. (1994) (J. Mol. Biol. 236:990-994). The vesicles contained bacteriorhodopsin (bR) and halolipid, and spontaneously formed during incubation of purple membrane suspension in the presence of detergent octylthioglucoside (OTG) if the protein:OTG ratio was 2:1 by weight. The size distribution of the vesicles was precisely determined by electron cryomicroscopy and was found to be almost independent on the incubation conditions (mean radius 17.9-19 nm). The size distribution in a given sample was close to the normal one, with a standard deviation of approximately +/- 1 nm. During dialysis for removal of the detergent, the vesicles diminished their radius by 2-2.5 nm. The results allow us to conclude that the driving force for the formation of bR vesicles is the preferential incorporation of OTG molecules in the cytoplasmic side of the membrane (with possible preferential delipidation of the extracellular side), which creates spontaneous curvature of the purple membrane. From the size distribution of the vesicles, we calculated the elasticity bending constant, K(B) approximately 9 x 10(-20) J, of the vesicle wall. The results provide some insight into the possible formation mechanisms of spherical assembles in living organisms. The conditions for vesicle formation and the mechanical properties of the vesicles could also be of interest with respect to the potential technological application of the bR vesicles as light energy converters.     

Phospholipid vesicle fusion monitored by fluorescence energy transfer.

A method is presented which allows the observation of phospholipid vesicle fusion by the occurrence of Förster resonance energy transfer between the amphiphilic probes dansyldipalmitoylphosphatidylethanolamine and 3-[4-($\text{p}$-N,N-didecylaminostyryl)-1-pyridinium]-propylsulfonate. This method is applied to the Ca⁺⁺ mediated fusion of phosphatidyl serine vesicles.     

The mechanism of Pseudomonas aeruginosa outer membrane vesicle biogenesis determines their protein composition

Gram-negative bacteria produce outer membrane vesicles (OMVs) and contain bacterial cargo including nucleic acids and proteins. The proteome of OMVs can be altered by various factors including bacterial growth stage, growth conditions, and environmental factors. However, it is currently unknown if the mechanism of OMV biogenesis can determine their proteome. In this study, we examined whether the mechanisms of OMV biogenesis influenced the production and protein composition of Pseudomonas aeruginosa OMVs. OMVs were isolated from three P. aeruginosa strains that produced OMVs either by budding alone, by explosive cell lysis, or by both budding and explosive cell lysis. We identified that the mechanism of OMV biogenesis dictated OMV quantity. Furthermore, a global proteomic analysis comparing the proteome of OMVs to their parent bacteria showed significant differences in the identification of proteins in bacteria and OMVs. Finally, we determined that the mechanism of OMV biogenesis influenced the protein composition of OMVs, as OMVs released by distinct mechanisms of biogenesis differed significantly from one another in their proteome and functional enrichment analysis. Overall, our findings reveal that the mechanism of OMV biogenesis is a main factor that determines the OMV proteome which may affect their subsequent biological functions.     

Surfactant partition between bulk water and DPPC vesicle membrane: solid-gel vs. liquid-crystalline membrane

The main phase transition temperature, Tm, of dipalmitoylphosphatidylcholine (DPPC) vesicle membrane was measured in the presence of the cationic surfactants tetradecyltrimethylammonium bromide and hexadecyltrimethylammonium bromide. Variation of the perturbing effect of these surfactants on Tm with the lipid concentration was analyzed according to the theory recently proposed by Kaminoh et al. (Y. Kaminoh, C. Tashiro, H. Kamaya and I. Ueda (1988) Biochim. Biophys. Acta 946, 215-220), and the partition coefficients of the surfactant into solid-gel and liquid-crystalline membranes were estimated.     

Visualization of synaptic vesicle movement in intact synaptic boutons using fluorescence fluctuation spectroscopy

Not much is known about the mobility of synaptic vesicles inside small synapses of the central nervous system, reflecting a lack of methods for visualizing these dynamics. We adapted confocal spot detection with fluctuation analysis to monitor the mobility of fluorescently labeled synaptic vesicles inside individual boutons of cultured hippocampal neurons. Using Monte Carlo simulations we were able to propose a simple quantitative model that can describe vesicle mobility in small hippocampal boutons under resting conditions and different pharmacological treatments. We find that vesicle mobility in a time window of 20 s can be well described by caged diffusion (D approximately 5 x 10(-5) microm(2)/s, cage sizes of approximately 50 nm). Mobility can be upregulated by phosphatase blockage and increased further by actin disruption in a dose-dependent manner. Inhibition of the myosin light chain kinase slows down vesicle mobility 10-fold, whereas other kinases like protein kinase C (PKC), A (PKA), and calmodulin kinase II (caMKII) do not affect mobility in unstimulated boutons.     

The parasitophorous vacuole membrane of Plasmodium falciparum: demonstration of vesicle formation using an immunoprobe

We have applied several immunolabeling techniques using a monoclonal antibody to a Plasmodium falciparum antigen to differentiate morphologically dissimilar membranous structures present in infected erythrocytes. Evidence is presented that cytoplasmic clefts, multimembranous structures and vesicles within the infected cell originate from the parasitophorous vacuole membrane by a process described as budding off. The parasitophorous vacuole membrane and related structures in infected, parasitized erythrocytes reacted with the cyanine dye Merocyanine 540, demonstrating that they are accessible to molecules from the extracellular environment. Immunogold labeling of freeze-fractured preparations and of thin sections of parasitized cells using pre- and post-embedding techniques revealed that each of the membranous structures carried a common parasite antigen, QF 116, which was identified by monoclonal antibody 8E7/55.     

In situ determination of intracellular membrane physical state heterogeneity in renal epithelial cells using fluorescence ratio microscopy

6-Lauroyl-2-dimethylaminonaphtalene (laurdan) shows a spectral sensitivity to the lipid phase state with a 50 nm red shift of the emission maximum when passing from the gel to the liquid crystalline phase. This spectral sensitivity allows one to determine the membrane physical state using Generalized Polarization (GP). In the present experiments, we used fluorescence ratio imaging microscopy to determine the laurdan GP in living kidney cells. Two renal epithelial cells lines, MDCK and LLC-PK1 cells, and CV-1 cells, a fibroblast-like renal cell line were investigated. In these cells, laurdan labels both the plasma membrane and intracellular membranes. Comparison of spectrofluorimetry and fluorescence ratio imaging data obtained from liposomes and cells suspensions labeled with laurdan demonstrates that the GP can be accurately determined using common fluorescence microscopy equipment. The GP mean values determined from individual cells varied from 0.2 to 0.4 for the epithelial cells as compared to 0.0 – 0.1 for CV1 cells. Using living MDCK cells grown as a monolayer, the GP maps indicated that, within a single cell, the intracellular GP values varied from 0.0 to 0.6, i. e., from the equivalent of a liquid-crystalline state to a gel or a lipid-ordered state, and that there was a marked heterogeneity in the spatial distribution of the GP values. To further characterize this intracellular heterogeneity, co-localization experiments with specific organelle markers were undertaken. The results strongly suggest that in intact cells at physiological temperature, GP values decrease in the following order: plasma membranes > endosomes > mitochondria > Golgi apparatus. Received: 3 June 1997 / Revised version: 6 March 1998 / Accepted: 7 March 1998     

Photosynthetic membrane development studied using picosecond fluorescence kinetics

Using measurements of the kinetics of chlorophyll a fluorescence emission, we have investigated the development of the photosynthetic membrane during etioplast-to-chloroplast differentiation. The chlorophyll fluorescence decay kinetics of pea chloroplasts from plants grown under intermittent (2 min light-118 min dark) and continuous light regimes were monitored with a single-photon timing system with picosecond resolution. We have associated the changes in the fluorescence yields and decay kinetics with known structural and organizational developmental phenomena in the chloroplast. This correlation provides a more detailed assignment of the origins of the fluorescence decay components than has been previously obtained by studying only mature chloroplasts. In particular, our analysis of the variable kinetics and multiexponential character of the fluorescence emission during thylakoid development focuses on the organization of photosynthetic units and the degree of communication between reaction centers in the same photosystem. Our results further demonstrate that the age of etiolated tissue is critical to plastid development.     

Analysis of cell membrane micro-heterogeneity using the fluorescence lifetime of DPH-type fluorophores.

Heterogeneity in the lipid organization in lipid bilayers and cell membranes was probed by using the fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH) and DPH attached to the sn-2 position of phosphatidylcholine (DPH-PC). In the presence of protein, it is proposed that the bulk lipids and boundary lipids can potentially provide distinct enough fluorophore environments for two different lifetime centers to be recovered from the analysis of the fluorescence decay. To test this model experiments were performed with cytochrome b5 in 1-palmitoyl-2-oleoylphosphatidylcholine bilayers. The number of boundary lipids of cytochrome b5 is known from the literature or can be calculated from known dimensions, so that for a known protein:lipid ratio the fraction of lipids in the bulk and boundary lipid regions is known. These values were found to closely correspond to the fractions associated with the lifetime centers recovered from an analysis of the fluorescence decay assuming two major fluorophore populations. This indicated that the DPH distributed in a similar manner to the lipids and that its boundary lipid residency time was greater than the excited state lifetime, showing the validity of the approach. An important requirement was that the protein should influence the fluorophore decay sufficiently enough to enable separate lifetime centers for the bulk and boundary lipid fluorophores to be recovered by the analysis. Attempts were made to analyze the fluorescence decay of DPH in liver plasma membranes and microsomes as arising from two distinct fluorophore populations, however, the basic condition was not satisfied. By contrast, using DPH-PC it was possible to extract two separate lifetime centers. The limitations and potential of this approach are critically assessed and it is concluded that in certain circumstances information pertaining to the protein-lipid interfacial region of membranes can be extracted from fluorescence decay heterogeneity properties.     

Stalk mechanism of vesicle fusion

Å mechanism for rupture of a separating bilayer, resulting from vesicle monolayer fusion is investigated theoretically. The stalk mechanism of monolayer fusion, assuming the formation and expansion of a stalk between two interacting membranes is considered. The stalk evolution leads to formation of a separating bilayer and mechanical tension appearance in the system. This tension results in rupture of the separating bilayer and hydrophilic pore formation. Competition between the mechanical tension and hydrophilic pore energy defines the criteria of contacting bilayer rupture. The tension increases with an increase of the absolute value of the negative spontaneous curvature of the outer membrane monolayer, K _s ^o . The pore edge energy decreases with an increase of the positive spontaneous curvature of the inner membrane monolayer, K _s ⁱ . The relations of spontaneous curvatures of outer and inner monolayers, leading to separating bilayer rupture, is calculated. It is demonstrated that his process is possible, provided spontaneous curvatures of membrane monolayers have opposite signs: K _s ^o <0, K _s ⁱ <0. Experimental data concerning the fusion process are analysed.     

Synaptic vesicle membrane fusion complex: action of clostridial neurotoxins on assembly.

Clostridial neurotoxins inhibit neurotransmitter release by selective and specific intracellular proteolysis of synaptobrevin/VAMP, synaptosomal-associated protein of 25 kDa (SNAP-25) or syntaxin. Here we show that in binary reactions synaptobrevin binds weakly to both SNAP-25 and syntaxin, and SNAP-25 binds to syntaxin. In the presence of all three components, a dramatic increase in the interaction strengths occurs and a stable sodium dodecyl sulfate-resistant complex forms. Mapping of the interacting sequences reveals that complex formation correlates with the presence of predicted alpha-helical structures, suggesting that membrane fusion involves intermolecular interactions via coiled-coil structures. Most toxins only attack the free, and not the complexed, proteins, and proteolysis of the proteins by different clostridial neurotoxins has distinct inhibitory effects on the formation of synaptobrevin-syntaxin-SNAP-25 complexes. Our data suggest that synaptobrevin, syntaxin and SNAP-25 associate into a unique stable complex that functions in synaptic vesicle exocytosis.     

Investigation of membrane structure using fluorescence quenching by spin-labels

Summary Fluorescence quenching is the loss of fluorescence intensity which is observed when a fluorescent molecule or group interacts with another molecule or group, called the quencher. By use of tryptophan residues of proteins, together with specific probe molecules, quenching can be applied to problems of biological and model membrane structure. Quenching interactions are short range (<50 Å) so that structure on the scale of molecular dimensions can be examined. This review summarizes the recent applications of fluorescence quenching by spin (nitroxide)-labeled molecules to problems of membrane structure, including determination of the distance of membrane-bound molecules from the membrane surface, the strength of lipid-protein interactions and the strength of protein-protein interactions within membranes. The unique advantages and the limitations of this powerful method are examined.     

Time-resolved distance determination by tryptophan fluorescence quenching: probing intermediates in membrane protein folding

The mechanism of insertion and folding of an integral membrane protein has been investigated with the beta-barrel forming outer membrane protein A (OmpA) of Escherichia coli. This work describes a new approach to this problem by combining structural information obtained from tryptophan fluorescence quenching at different depths in the lipid bilayer with the kinetics of the refolding process. Experiments carried out over a temperature range between 2 and 40 degrees C allowed us to detect, trap, and characterize previously unidentified folding intermediates on the pathway of OmpA insertion and folding into lipid bilayers. Three membrane-bound intermediates were found in which the average distances of the Trps were 14-16, 10-11, and 0-5 A, respectively, from the bilayer center. The first folding intermediate is stable at 2 degrees C for at least 1 h. A second intermediate has been isolated at temperatures between 7 and 20 degrees C. The Trps move 4-5 A closer to the center of the bilayer at this stage. Subsequently, in an intermediate that is observable at 26-28 degrees C, the Trps move another 5-10 A closer to the center of the bilayer. The final (native) structure is observed at higher temperatures of refolding. In this structure, the Trps are located on average about 9-10 A from the bilayer center. Monitoring the evolution of Trp fluorescence quenching by a set of brominated lipids during refolding at various temperatures therefore allowed us to identify and characterize intermediate states in the folding process of an integral membrane protein.