The Role of Lateral Tension in Calcium-Induced DPPS Vesicle Rupture (original) (raw)
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The Journal of General Physiology, 1996
Membrane fusion of a phospholipid vesicle with a planar lipid bilayer is preceded by an initial prefusion stage in which a region of the vesicle membrane adheres to the planar membrane. A resonance energy transfer (RET) imaging microscope, with measured spectral transfer functions and a pair of radiometrically calibrated video cameras, was used to determine both the area of the contact region and the distances between the membranes within this zone. Large vesicles (5-20 ~m diam) were labeled with the donor fluorophore coumarin-phosphatidylethanolamine (PE), while the planar membrane was labeled with the acceptor rhodamine-PE. The donor was excited with 390 nm light, and separate images of donor and acceptor emission were formed by the microscope. Distances between the membranes at each location in the image were determined from the RET rate constant (kt) computed from the acceptor:donor emission intensity ratio. In the absence of an osmotic gradient, the vesicles stably adhered to the planar membrane, and the dyes did not migrate between membranes. The region of contact was detected as an area of planar membrane, coincident with the vesicle image, over which rhodamine fluorescence was sensitized by PET. The total area of the contact region depended biphasically on the Ca 2+ concentration, but the distance between the bilayers in this zone decreased with increasing [Ca'2+]. The changes in area and separation were probably related to divalent cation effects on electrostatic screening and binding to charged membranes. At each [Ca2+], the intermembrane separation varied between 1 and 6 nm within each contact region, indicating membrane undulation prior to adhesion. Intermembrane separation distances <~2 nm were localized to discrete sites that formed in an ordered arrangement throughout the contact region. The area of the contact region occupied by these punctate attachment sites was increased at high [Ca2+]. Membrane fusion may be initiated at these sites of closest membrane apposition. pholipid membranes, each demarcating separate aqueous compartments, meld into a contiguous lamella, and compartments initially delineated by the original membranes are joined . Although these different biological processes are regulated by distinct sets of proteins , membrane fusion, the common feature controlled by these proteins, is essentially the coalescence of two highly anisotropic, viscoelastic fluids.
Calcium-induced interaction of phospholipid vesicles and bilayer lipid membranes
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1977
The interaction of unilamellar phospholipid vesicles with bilayer lipid membranes has been studied by observing the electrical conductance of the planar membrane. The presence of phosphatidylcholine vesicles as well as phosphatidylcholine/phosphatidylserine (1 : 1) vesicles on one side compartment of the bilayer membrane, but not phosphatidylserine vesicles, causes discrete fluctuations in the phosphatidylserine membrane conductance, which is also increased by at least an order of magnitude. These events are dependent on vesicle concentration as well as the presence of Ca 2÷. The results are interpreted in terms of the incorporation of domains of phosphatidylcholine into the membrane, which confer a higher conductance state.
Biophysical Journal, 2012
Synaptic vesicles (SVs) are small, membrane-bound organelles that are found in the synaptic terminal of neurons. Although tremendous progress has been made in understanding the protein machinery that drives fusion of SVs with the presynaptic membrane, little progress has been made in understanding changes in the membrane structure that accompany this process. We used lipid monolayers of defined composition to mimic biological membranes, which were probed by x-ray reflectivity and grazing incidence x-ray diffraction. These techniques allowed us to successfully monitor structural changes in the membranes at molecular level, both in response to injection of SVs in the subphase below the monolayer, as well as to physiological cues involved in neurotransmitter release, such as increases in the concentration of the membrane lipid PIP 2 , or addition of physiological levels of Ca 2þ. Such structural changes may well modulate vesicle fusion in vivo.
Small-angle neutron scattering (SANS) curves of unilamellar dipalmitoylphosphatidylcholine (DPPC) vesicles in 1-60 mM CaCl 2 were analyzed using a strip-function model of the phospholipid bilayer. The fraction of Ca 2+ ions bound in the DPPC polar head group region was determined using Langmuir adsorption isotherm. In the gel phase, at 20 • C, the lipid bilayer thickness, d L , goes through a maximum as a function of CaCl 2 concentration (d L = 54.4 Å at ∼2.5 mM of CaCl 2 ). Simultaneously, both the area per DPPC molecule A L , and the number of water molecules n W located in the polar head group region decrease ( A L = A L(DPPC) − A L(DPPC+Ca) = 2.3 Å 2 and n = n W(DPPC) − n W(DPPC+Ca) = 0.8 mol/mol at ∼2.5 mM of CaCl 2 ). In the fluid phase, at 60 • C, the structural parameters d L , A L , and n W show evident changes with increasing Ca 2+ up to a concentration c Ca 2+ ≤ 10 mM. DPPC bilayers affected by the calcium binding are compared to unilamellar vesicles prepared by extrusion. The structural parameters of DPPC vesicles prepared in 60 mM CaCl 2 (at 20 and 60 • C) are nearly the same as those for unilamellar vesicles without Ca 2+ .
pH-dependent lipid packing, membrane permeability and fusion in phosphatidylcholine vesicles
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1991
We have studied the rate of membrane fusion, the lipid dynamics and order and the membrane permeability of phosphatidylcholine vesicles as a function of pH. Acidification induced very different effects depending on the state of the bilayer. In liquid-crystalline hilayers, acidification decreased the rate of membrane fusion, the acyl chain motion and disorder and the rate of K + release, whereas in solid bilayers acidification increased the rate of membrane fusion, the lipid aryl chain disorder and the rate of K + release. These pH-dependent modifications are interpreted in terms of conformational and/or packing changes of the phosphatidylcholine head group in the membrane. In solid Uilayers, these changes are not easily accommodated by the rigid structure, and the resulting stress leads to an unstable bilayer.
Cationic, O-alkylphosphatidylcholines, recently developed as DNA transfection agents, form bilayers indistinguishable from those of natural phospholipids and undergo fusion with anionic bilayers. Membrane merging (lipid mixing), contents release, and contents mixing between populations of positive vesicles containing O-ethylphosphatidylcholine (EDOPC) and negative vesicles containing dioleolylphosphatidylglycerol (DOPG) have been determined with standard fluorometric vesiclepopulation assays. Surface-charge densities were varied from zero to full charge. All interactions depended critically on surface-charge density, as expected from the adhesion-condensation mechanism. Membrane mixing ranged from zero to 100%, with significant mixing (>10 <70%) occurring between cationic vesicles that were fully charged and anionic vesicles that had fractional surface charges as low as 0.1. Such mixing with membranes as weakly charged as cell membranes should be relevant to transfection with cationic lipids. Unexpectedly, lipid mixing was higher at high than at low ionic strength when one lipid dispersion was prepared from EDOPC plus DOPG (in different proportions), especially when the other vesicles were of EDOPC; this may somehow be a consequence of the ability of the former mixture to assume non-lamellar phases. Leakage of aqueous contents was also a strong function of charge, with fully charged vesicles releasing essentially all of their contents less than 1 min after mixing. EDOPC was more active in this regard than was DOPG, which probably reflects stronger intermolecular interactions of DOPG. Fusion, as measured by contents mixing, exhibited maximal values of 10% at intermediate surface charge. Reduced fusion at higher charge is attributed to multiple vesicle interactions leading to rupture. The existence of previously published data on individual interactions of vesicles of the same composition made it possible for the first time to compare pairwise with population interactions, confirming the likelihood of population studies to overestimate rupture and hemifusion and underestimate true vesicle fusion.
2000
A B S T R A C T 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 ~m diam) were loaded with 200 mM of the membrane-impermeant fluorescent dye calcein. Vesicles were ejected from a pipette brought to within 10 #m 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 KCI gradient across the vesicular membrane with the channel-forming antibiotic nystatin or, alternatively, by making the c/s 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 c/s 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 caicein 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.
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1981
Small unilamellar phosphatidylseriae/phosphatidylcholine liposomes incubated on one side of planar phosphatidylserine bilayer membranes induced fluctuations and a sharp increase in the membrane conductance when the Ca ~÷ concentration was increased to a threshold of 3--5 mM in 100 mM NaC1, pH 7.4. Under the same ionic conditions, these liposomes fused with large (0.2 tim diameter) single-bilayer phosphatidylserine vesicles, as shown by a fluorescence assay for the mixing of internal aqueous contents of the two vesicle populations. The conductance behavior of the planar membranes was interpreted to be a consequence of the structural rearrangement of phospholipids during individual fusion events and the incorporation of domains of phosphatidylcholine into the Ca2÷-complexed phosphatidylserine membrane. The small vesicles did not aggregate or fuse with one another at these Ca :÷ concentrations, but fused preferentially with the phosphatidylserine membrane, analogous to simple exocytosis in biological membranes. Phosphatidylserine vesicles containing gramicidin A as a probe interacted with the planar membranes upon raising the Ca 2÷ concentration from 0.9 to 1.2 mM, as detected by an abrupt increase in the membrane conductance. In parallel experiments, these vesicles were shown to fuse with the large phosphatidylserine liposomes at the same Ca 2÷ concentration. Abbreviation: Hepes, N-2-hydroxyethylpiperazine-N~-2-ethanesulfonic acid.
The formation and annealing of structural defects in lipid bilayer vesicles
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1976
It is shown that sonication of phospholipid-water dispersions b.-Iow the crystalline ~ liquid crystalline phase transition temperature (Te) produces bila.,~cr vesicles with structuraldefects within the bilayer memberane, which permit rapid permeation of ions and. catalyze vesicle-vesicle fusion. These structural defects age annih/l&ted simply by annealing the vesicle suspension above T.. The rate of aaaeal~ag wes found tob( slow, ofthe order of an hour for T = 3 °C above 7"., but mmea~ng is complete withitb 10 rain for T=, 10 °C above ~. It is proposed that these defectsare far~tt-Cfislocations in the bilayer structure, which arise from a popelatiou defect .in the distribution of the lipid mokceks between the outer and inner moaolayers, :whea small bilayer :fms~'nts rcs~'mb~ to form the small bila3~n" during,the sonicttion proccdure. Such a popelation defect can only he remedied by lipid transport via the i!udde ~-outside flip-flop mechanism, which would account for the slow kinetics of annealing observed even at 3 °C above the phase transition.