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

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.

Download Full-text

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

The Journal of Cell Biology ◽

10.1083/jcb.98.3.1054 ◽

1984 ◽

Vol 98 (3) ◽

pp. 1054-1062 ◽

Cited By ~ 84

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.

Download Full-text

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

The Journal of Cell Biology ◽

10.1083/jcb.98.3.1063 ◽

1984 ◽

Vol 98 (3) ◽

pp. 1063-1071 ◽

Cited By ~ 59

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.

Download Full-text

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

The Journal of General Physiology ◽

10.1085/jgp.75.3.241 ◽

1980 ◽

Vol 75 (3) ◽

pp. 241-250 ◽

Cited By ~ 91

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.

Download Full-text

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

The Journal of General Physiology ◽

10.1085/jgp.90.5.703 ◽

1987 ◽

Vol 90 (5) ◽

pp. 703-735 ◽

Cited By ~ 38

Author(s):

W D Niles ◽

F S Cohen

Keyword(s):

Fluorescence Microscopy ◽

Phospholipid Vesicle ◽

Flash Duration ◽

Bilayer Membranes ◽

Large Unilamellar Vesicles ◽

Video Fluorescence Microscopy ◽

Vesicular Membrane ◽

Planar Membrane ◽

Planar Membranes ◽

And Diffusion

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.

Download Full-text

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

The Journal of General Physiology ◽

10.1085/jgp.93.2.201 ◽

1989 ◽

Vol 93 (2) ◽

pp. 201-210 ◽

Cited By ~ 25

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.

Download Full-text

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

The Journal of General Physiology ◽

10.1085/jgp.93.2.211 ◽

1989 ◽

Vol 93 (2) ◽

pp. 211-244 ◽

Cited By ~ 21

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.

Download Full-text

Diffusion in hydrogel-supported phospholipid bilayer membranes

Journal of Fluid Mechanics ◽

10.1017/jfm.2013.128 ◽

2013 ◽

Vol 723 ◽

pp. 352-373 ◽

Cited By ~ 2

Author(s):

Chih-Ying Wang ◽

Reghan J. Hill

Keyword(s):

Tracer Diffusion ◽

Membrane Dynamics ◽

The Self ◽

Phospholipid Bilayer ◽

Total Force ◽

Membrane Thickness ◽

Bilayer Membranes ◽

Self Diffusion ◽

Planar Membrane ◽

Self Diffusion Coefficient

AbstractWe model a cylindrical inclusion (lipid or membrane protein) translating with velocity$U$in a thin planar membrane (phospholipid bilayer) that is supported above and below by Brinkman media (hydrogels). The total force$F$, membrane velocity, and solvent velocity are calculated as functions of three independent dimensionless parameters:$\Lambda = \eta a/ ({\eta }_{m} h)$,${\ell }_{1} / a$and${\ell }_{2} / a$. Here,$\eta $and${\eta }_{m} $are the solvent and membrane shear viscosities,$a$is the particle radius,$h$is the membrane thickness, and${ \ell }_{1}^{2} $and${ \ell }_{2}^{2} $are the upper and lower hydrogel permeabilities. As expected, the dimensionless mobility$4\mathrm{\pi} \eta aU/ F= 4\mathrm{\pi} \eta aD/ ({k}_{B} T)$(proportional to the self-diffusion coefficient,$D$) decreases with decreasing gel permeabilities (increasing gel concentrations), furnishing a quantitative interpretation of how porous, gel-like supports hinder membrane dynamics. The model also provides a means of inferring hydrogel permeability and, perhaps, surface morphology from tracer diffusion measurements.

Download Full-text