scholarly journals Hydrostatic pressures developed by osmotically swelling vesicles bound to planar membranes.

1989 ◽  
Vol 93 (2) ◽  
pp. 211-244 ◽  
Author(s):  
W D Niles ◽  
F S Cohen ◽  
A Finkelstein
Keyword(s):  
Contact Region ◽  
Irreversible Thermodynamics ◽  
Open Channels ◽  
Vesicle Membrane ◽  
Osmotic Swelling ◽  
Solute Permeability ◽  
Membrane Permeabilities ◽  
Membrane Contact ◽  
Planar Membrane ◽  
Planar Membranes

When phospholipid vesicles bound to a planar membrane are osmotically swollen, they develop a hydrostatic pressure (delta P) and fuse with the membrane. We have calculated the steady-state delta P, from the equations of irreversible thermodynamics governing water and solute flows, for two general methods of osmotic swelling. In the first method, vesicles are swollen by adding a solute to the vesicle-containing compartment to make it hyperosmotic. delta P is determined by the vesicle membrane's permeabilities to solute and water. If the vesicle membrane is devoid of open channels, then delta P is zero. When the vesicle membrane contains open channels, then delta P peaks at a channel density unique to the solute permeability properties of both the channel and the membrane. The solute enters the vesicle through the channels but leaks out through the region of vesicle-planar membrane contact. delta P is largest for channels having high permeabilities to the solute and for solutes with low membrane permeabilities in the contact region. The model predicts the following order of solutes producing pressures of decreasing magnitude: KCl greater than urea greater than formamide greater than or equal to ethylene glycol. Differences between osmoticants quantitatively depend on the solute permeability of the channel and the density of channels in the vesicle membrane. The order of effectiveness is the same as that experimentally observed for solutes promoting fusion. Therefore, delta P drives fusion. When channels with small permeabilities are used, coupling between solute and water flows within the channel has a significant effect on delta P. In the second method, an impermeant solute bathing the vesicles is isosmotically replaced by a solute which permeates the channels in the vesicle membrane. delta P resulting from this method is much less sensitive to the permeabilities of the channel and membrane to the solute. delta P approaches the theoretical limit set by the concentration of the impermeant solute.

scholarly journals Fusion of phospholipid vesicles with planar phospholipid bilayer membranes. II. Incorporation of a vesicular membrane marker into the planar membrane.

1980 ◽  
Vol 75 (3) ◽  
pp. 251-270 ◽  
Author(s):  
F S Cohen ◽  
J Zimmerberg ◽  
A Finkelstein
Keyword(s):  
Divalent Cations ◽  
Phospholipid Bilayer ◽  
Phospholipid Vesicles ◽  
Osmotic Gradient ◽  
Bilayer Membranes ◽  
Fusion Procedure ◽  
Vesicular Membrane ◽  
Membrane Contact ◽  
Planar Membrane ◽  
Planar Membranes

Fusion of multilamellar phospholipid vesicles with planar phospholipid bilayer membranes was monitored by the rate of appearance in the planar membrane of an intrinsic membrane protein present in the vesicle membranes. An essential requirement for fusion is an osmotic gradient across the planar membrane, with the cis side (the side containing the vesicles) hyperosmotic to the opposite (trans) side; for substantial fusion rates, divalent cation must also be present on the cis side. Thus, the low fusion rates obtained with 100 mM excess glucose in the cis compartment are enhanced orders of magnitude by the addition of 5-10 mM CaCl2 to the cis compartment. Conversely, the rapid fusion rates induced by 40 mM CaCl2 in the cis compartment are completely suppressed when the osmotic gradient (created by the 40 mM CaCl2) is abolished by addition of an equivalent amount of either CaCl2, NaCl, urea, or glucose to the trans compartment. We propose that fusion occurs by the osmotic swelling of vesicles in contact with the planar membrane, with subsequent rupture of the vesicular and planar membranes in the region of contact. Divalent cations catalyze this process by increasing the frequency and duration of vesicle-planar membrane contact. We argue that essentially this same osmotic mechanism drives biological fusion processes, such as exocytosis. Our fusion procedure provides a general method for incorporating and reconstituting transport proteins into planar phospholipid bilayer membranes.

scholarly journals Fusion of phospholipid vesicles with a planar membrane depends on the membrane permeability of the solute used to create the osmotic pressure.

1989 ◽  
Vol 93 (2) ◽  
pp. 201-210 ◽  
Author(s):  
F S Cohen ◽  
W D Niles ◽  
M H Akabas
Keyword(s):  
Osmotic Pressure ◽  
Lipid Bilayer ◽  
Lipid Membrane ◽  
Contact Region ◽  
Companion Paper ◽  
Phospholipid Vesicles ◽  
Osmotic Gradient ◽  
Vesicle Membrane ◽  
Permeable Channel ◽  
Planar Membrane

Phospholipid vesicles fuse with a planar membrane when they are osmotically swollen. Channels in the vesicle membrane are required for swelling to occur when the vesicle-containing compartment is made hyperosmotic by adding a solute (termed an osmoticant). We have studied fusion using two different channels, porin, a highly permeable channel, and nystatin, a much less permeable channel. We report that an osmoticant's ability to support fusion (defined as the magnitude of osmotic gradient necessary to obtain sustained fusion) depends on both its permeability through lipid bilayer as well as its permeability through the channel by which it enters the vesicle interior. With porin as the channel, formamide requires an osmotic gradient about ten times that required with urea, which is approximately 1/40th as permeant as formamide through bare lipid membrane. When nystatin is the channel, however, fusion rates sustained by osmotic gradients of formamide are within a factor of two of those obtained with urea. Vesicles containing a porin-impermeant solute can be induced to swell and fuse with a planar membrane when the impermeant bathing the vesicles is replaced by an isosmotic quantity of a porin-permeant solute. With this method of swelling, formamide is as effective as urea in obtaining fusion. In addition, we report that binding of vesicles to the planar membrane does not make the contact region more permeable to the osmoticant than is bare lipid bilayer. In the companion paper, we quantitatively account for the observation that the ability of a solute to promote fusion depends on its permeability properties and the method of swelling. We show that the intravesicular pressure developed drives fusion.

scholarly journals Separation of the osmotically driven fusion event from vesicle-planar membrane attachment in a model system for exocytosis.

1984 ◽  
Vol 98 (3) ◽  
pp. 1063-1071 ◽  
Author(s):  
M H Akabas ◽  
F S Cohen ◽  
A Finkelstein
Keyword(s):  
Divalent Cations ◽  
Model System ◽  
Second Step ◽  
Planar Bilayer ◽  
Fusion Event ◽  
Osmotic Swelling ◽  
Bilayer Membranes ◽  
Membrane Attachment ◽  
Planar Membrane ◽  
Planar Membranes

We demonstrate that there are two experimentally distinguishable steps in the fusion of phospholipid vesicles with planar bilayer membranes. In the first step, the vesicles form a stable, tightly bound pre-fusion state with the planar membrane; divalent cations (Ca++) are required for the formation of this state if the vesicular and/or planar membrane contain negatively charged lipids. In the second step, the actual fusion of vesicular and planar membranes occurs. The driving force for this step is the osmotic swelling of vesicles attached (in the pre-fusion state) to the planar membrane. We suggest that osmotic swelling of vesicles may also be crucial for biological fusion and exocytosis.

scholarly journals The role of N-acetylneuraminic (sialic) acid in the pH dependence of influenza virion fusion with planar phospholipid membranes.

1991 ◽  
Vol 97 (6) ◽  
pp. 1121-1140 ◽  
Author(s):  
W D Niles ◽  
F S Cohen
Keyword(s):  
Influenza Virus ◽  
Sialic Acid ◽  
Ph Dependence ◽  
Phospholipid Composition ◽  
Fusion Rate ◽  
Influenza Virion ◽  
Order Of Magnitude ◽  
Ph Dependent ◽  
Planar Membrane ◽  
Planar Membranes

It is known that fusion of influenza virus to host cell membranes is strongly promoted by acidic pH. We have determined conditions required to obtain pH-dependent fusion of influenza virus to planar bilayer membranes. The rate of viral fusion was determined from the flash rate of R18-labeled virions delivered to the surface of the planar membrane by pressure-ejection from a pipette. For a bilayer formed only of phospholipids and cholesterol, the fusion rate was independent of pH and unaffected by the phospholipid composition. When the gangliosides GD1a + GT1b were included in the planar membrane, however, the fusion rate varied steeply with pH. The rate at pH 7.4 in the presence of the gangliosides was about an order of magnitude less than in their absence. At pH less than approximately 5.5, the rate was about an order of magnitude greater in the presence of gangliosides than in their absence. The fusion rate with planar membranes containing globoside, a ceramide-backboned glycolipid, was also independent of pH, indicating that the pH dependence required sialic acid on the carbohydrate moiety of the glycolipid. The gangliosides GM1a and GM3, both of which possess sialic acid, produced the same pH-dependent fusion rate as seen with GD1a + GT1b, indicating that the presence, but not the location, of terminal sialic acids is critical. Incubating virus with soluble sialyllactose blocked fusion to both ganglioside-free and ganglioside-containing planar membranes. These results show that the pH dependence of influenza virion fusion arises from the interaction of the sialic acid receptor with the influenza hemagglutinin. A model for sialic acid-hemagglutinin interactions accounting for pH-dependent fusion is presented.

scholarly journals A hemifused complex is the hub in a network of pathways to membrane fusion

2021 ◽  
Author(s):  
Jason Warner ◽  
Dong An ◽  
Benjamin Stratton ◽  
Ben O'Shaughnessy
Keyword(s):  
Membrane Fusion ◽  
Experimental Studies ◽  
Cell Penetrating Peptides ◽  
Vesicle Membrane ◽  
Membrane Rupture ◽  
Dead End ◽  
High Tension ◽  
Delivery Strategies ◽  
Planar Membranes ◽  
Over 40 Years

Membrane fusion is required for essential processes from neurotransmission to fertilization. For over 40 years protein-free fusion driven by calcium or other cationic species has provided a simplified model of biological fusion, but the mechanisms remain poorly understood. Cation-mediated membrane fusion and permeation are essential in their own right to drug delivery strategies based on cell-penetrating peptides or cation-bearing lipid nanoparticles. Experimental studies suggest cations drive anionic membranes to a hemifused intermediate which serves as a hub for a network of pathways, but the pathway selection mechanism is unknown. Here we develop a mathematical model that identifies the network hub as a highly dynamical hemifusion complex. We find multivalent cations drive expansion of a high tension hemifusion interface between interacting vesicles during a brief transient. During this window, rupture of the interface competes with vesicle membrane rupture to determine the outcome, either fusion, dead-end hemifusion or vesicle lysis. The model reproduces the unexplained finding that fusion of vesicles with planar membranes typically stalls at hemifusion, and we show that the equilibrated hemifused state is a novel lens-shaped complex. Thus, membrane fusion kinetics follow a stochastic trajectory within a network of pathways, with outcome weightings set by fusogen concentration, vesicle size, lipid composition and geometry.

scholarly journals Resonance energy transfer imaging of phospholipid vesicle interaction with a planar phospholipid membrane: undulations and attachment sites in the region of calcium-mediated membrane--membrane adhesion.

1996 ◽  
Vol 107 (3) ◽  
pp. 329-351 ◽  
Author(s):  
W D Niles ◽  
J R Silvius ◽  
F S Cohen
Keyword(s):  
Energy Transfer ◽  
Membrane Fusion ◽  
Contact Region ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Phospholipid Vesicle ◽  
Osmotic Gradient ◽  
Attachment Sites ◽  
Donor And Acceptor ◽  
Planar Membrane

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 microns 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 RET. The total area of the contact region depended biphasically on the Ca2+ concentration, but the distance between the bilayers in this zone decreased with increasing [Ca2+]. 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 < or = 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.

scholarly journals Parameters affecting the fusion of unilamellar phospholipid vesicles with planar bilayer membranes.

1984 ◽  
Vol 98 (3) ◽  
pp. 1054-1062 ◽  
Author(s):  
F S Cohen ◽  
M H Akabas ◽  
J Zimmerberg ◽  
A Finkelstein
Keyword(s):  
Divalent Cation ◽  
Phospholipid Bilayer ◽  
Fusion Process ◽  
Phospholipid Vesicles ◽  
Osmotic Gradient ◽  
Planar Bilayer ◽  
Unilamellar Vesicles ◽  
Bilayer Membranes ◽  
Planar Membrane ◽  
Planar Membranes

It was previously shown (Cohen, F. S., J. Zimmerberg, and A. Finkelstein, 1980, J. Gen. Physiol., 75:251-270) that multilamellar phospholipid vesicles can fuse with decane-containing phospholipid bilayer membranes. An essential requirement for fusion was an osmotic gradient across the planar membrane, with the vesicle-containing (cis) side hyperosmotic with respect to the opposite (trans) side. We now report that unilamellar vesicles will fuse with "hydrocarbon-free" membranes subject to these same osmotic conditions. Thus the same conditions that apply to fusion of multilamellar vesicles with planar bilayer membranes also apply to fusion of unilamellar vesicles with these membranes, and hydrocarbon is not required for the fusion process. If the vesicles and/or planar membrane contain negatively charged lipids, divalent cation (approximately 15 mM Ca++) is required in the cis compartment (in addition to the osmotic gradient across the membrane) to obtain substantial fusion rates. On the other hand, vesicles made from uncharged lipids readily fuse with planar phosphatidylethanolamine planar membranes in the near absence of divalent cation with just an osmotic gradient. Vesicles fuse much more readily with phosphatidylethanolamine-containing than with phosphatidylcholine-containing planar membranes. Although hydrocarbon (decane) is not required in the planar membrane for fusion, it does affect the rate of fusion and causes the fusion process to be dependent on stirring in the cis compartment.

scholarly journals Membrane fusion during secretion. A hypothesis based on electron microscope observation of Phytophthora Palmivora zoospores during encystment.

1977 ◽  
Vol 73 (1) ◽  
pp. 161-181 ◽  
Author(s):  
P Pinto da Silva ◽  
M L Nogueira
Keyword(s):  
Membrane Fusion ◽  
Freeze Fracture ◽  
Bilayer Membrane ◽  
Phytophthora Palmivora ◽  
Electron Microscope Observation ◽  
Fusion Event ◽  
Vesicle Membrane ◽  
Membrane Bound ◽  
Membrane Contact ◽  
Outer Half

Interpretation of freeze-fracture and thin-section results shows that fusion of the peripheral vesicle with the plasmalemma of a Phytophthora palmivora zoospore occurs at several discrete sites and results in the formation and expansion of a particle-free bilayer membrane diaphragm and in the appearance of a polymorphic network of membrane-bounded tunnels, the lumina of which are continuous with the cytoplasm. The outer half of the bilayer membrane diaphragm appears continuous with the outer half of the plasma membrane; the inner half of the bilayer membrane diaphragm with the inner half of the peripheral vesicle membrane; and the inner half of the plasmalemma with the outer half of the peripheral vesicle membrane. Interpretation of our results leads us to formulate a hypothesis for a sequence of several intermediate stages involved in membrane fusion. The initial fusion event is viewed as a local catastrophe (Thom, R. 1972. Stabilité Structurelle et Morphogenèse. W. A. Benjamin Inc., Reading, Mass.) involving the sudden reorganization of apposed elements of the inner half of the plasmalemma and the outer half of the peripheral vesicle membrane. Fusion of apposed components at the rim of the perimeter of fusion results in the formation of a toroid hemi-micelle which provides continuity between the inner half of the plasmalemma and the outer half of the peripheral vesicle membrane. Simultaneously, apposed components at the site of fusion may reorganize into an inverted membrane micelle. A bilayer membrane diaphragm is then formed by apposition and flowing of components form the outer half of the plasmalemma and the inner (exoplasmic) half of the peripheral vesicle membrane. The existence of large areas of membrane contact before fusion may lead to several fusion events and the formation of a polymorphic network of membrane-bound tunnels.

scholarly journals Fusion of phospholipid vesicles with planar phospholipid bilayer membranes. I. Discharge of vesicular contents across the planar membrane.

1980 ◽  
Vol 75 (3) ◽  
pp. 241-250 ◽  
Author(s):  
J Zimmerberg ◽  
F S Cohen ◽  
A Finkelstein
Keyword(s):  
Bilayer Membrane ◽  
Convincing Evidence ◽  
Water Soluble ◽  
Phospholipid Bilayer ◽  
Phospholipid Vesicles ◽  
Biological Phenomenon ◽  
Vesicle Membrane ◽  
Bilayer Membranes ◽  
Phospholipid Bilayer Membrane ◽  
Planar Membrane

Multilamellar phospholipid vesicles are introduced into the cis compartment on one side of a planar phospholipid bilayer membrane. The vesicles contain a water-soluble fluorescent dye trapped in the aqueous phases between the lamellae. If a vesicle containing n lamellae fuses with a planar membrane, an n-1 lamellar vesicle should be discharged into the opposite trans compartment, where it would appear as a discernible fluorescent particle. Thus, fusion events can be assayed by counting the number of fluorescent particles appearing in the trans compartment. In the absence of divalent cation, fusion does not occur, even after vesicles have been in the cis compartment for 40 min. When CaCl2 is introduced into the cis compartment to a concentration of greater than or equal to 20 mM, fusion occurs within the next 20 min; it generally ceases thereafter because of vesicle aggregation in the cis compartment. With approximately 3 x 10(8) vesicles/cm3 in the cis compartment, about 25-50 fusion events occur following CaCl2 addition. The discharge of vesicular contents across the planar membrane is the most convincing evidence of vesicle-membrane fusion and serves as a model for that ubiquitous biological phenomenon--exocytosis.

scholarly journals A voltage-dependent K+ channel in the lysosome is required for refilling lysosomal Ca2+ stores

2017 ◽  
Vol 216 (6) ◽  
pp. 1715-1730 ◽  
Author(s):  
Wuyang Wang ◽  
Xiaoli Zhang ◽  
Qiong Gao ◽  
Maria Lawas ◽  
Lu Yu ◽  
...  
Keyword(s):  
Bk Channels ◽  
K Channel ◽  
Resting Membrane Potential ◽  
Genetic Ablation ◽  
Membrane Contact Sites ◽  
Selective Permeability ◽  
Voltage Dependent ◽  
Cytosolic Side ◽  
Membrane Permeabilities ◽  
Membrane Contact

The resting membrane potential (Δψ) of the cell is negative on the cytosolic side and determined primarily by the plasma membrane’s selective permeability to K+. We show that lysosomal Δψ is set by lysosomal membrane permeabilities to Na+ and H+, but not K+, and is positive on the cytosolic side. An increase in juxta-lysosomal Ca2+ rapidly reversed lysosomal Δψ by activating a large voltage-dependent and K+-selective conductance (LysoKVCa). LysoKVCa is encoded molecularly by SLO1 proteins known for forming plasma membrane BK channels. Opening of single LysoKVCa channels is sufficient to cause the rapid, striking changes in lysosomal Δψ. Lysosomal Ca2+ stores may be refilled from endoplasmic reticulum (ER) Ca2+ via ER–lysosome membrane contact sites. We propose that LysoKVCa serves as the perilysosomal Ca2+ effector to prime lysosomes for the refilling process. Consistently, genetic ablation or pharmacological inhibition of LysoKVCa, or abolition of its Ca2+ sensitivity, blocks refilling and maintenance of lysosomal Ca2+ stores, resulting in lysosomal cholesterol accumulation and a lysosome storage phenotype.

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