Formation of supported planar bilayers by fusion of vesicles to supported phospholipid monolayers

Kalb, Edwin; Frey, Sammy; Tamm, Lukas K.

doi:10.1016/0005-2736(92)90101-q

Cited by 612 publications

(575 citation statements)

References 27 publications

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“…LangmuirBlodgett methods can then be used to deposit a lipid monolayer onto the PEG layer 55 . Liposomes introduced above this hydrophobic surface fuse with it, forming a bilayer 56 . It is hoped that this approach will be combined with microfluidics in the future.…”

Section: Discussionmentioning

confidence: 99%

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

Nikolaus

Karatekin

2016

JoVE

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In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. During neurotransmitter or hormone release via exocytosis, the fusion pore can transiently open and close repeatedly, regulating cargo release kinetics. Pore dynamics also determine the mode of vesicle recycling; irreversible resealing results in transient, "kiss-and-run" fusion, whereas dilation leads to full fusion. To better understand what factors govern pore dynamics, we developed an assay to monitor membrane fusion using polarized total internal reflection fluorescence (TIRF) microscopy with single molecule sensitivity and ~15 msec time resolution in a biochemically well-defined in vitro system. Fusion of fluorescently labeled small unilamellar vesicles containing v-SNARE proteins (v-SUVs) with a planar bilayer bearing t-SNAREs, supported on a soft polymer cushion (t-SBL, t-supported bilayer), is monitored. The assay uses microfluidic flow channels that ensure minimal sample consumption while supplying a constant density of SUVs. Exploiting the rapid signal enhancement upon transfer of lipid labels from the SUV to the SBL during fusion, kinetics of lipid dye transfer is monitored. The sensitivity of TIRF microscopy allows tracking single fluorescent lipid labels, from which lipid diffusivity and SUV size can be deduced for every fusion event. Lipid dye release times can be much longer than expected for unimpeded passage through permanently open pores. Using a model that assumes retardation of lipid release is due to pore flickering, a pore "openness", the fraction of time the pore remains open during fusion, can be estimated. A soluble marker can be encapsulated in the SUVs for simultaneous monitoring of lipid and soluble cargo release. Such measurements indicate some pores may reseal after losing a fraction of the soluble cargo. Video LinkThe video component of this article can be found at

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Section: Discussionmentioning

confidence: 99%

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

Nikolaus

Karatekin

2016

JoVE

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“…A possible application is to use the film as supporting layer for solid supported membranes. On top of a hydrophobic SAM, a second lipid monolayer can be deposited by either vesicle fusion, 19 solvent exchange, 20 or horizontal Langmuir film transfer. 21 Depending on the nature of the surface and the external parameters such as temperature, pH, and ionic strength, vesicles interact with the surface in different ways.…”

Section: Introductionmentioning

confidence: 99%

Formation of tethered bilayer lipid membranes probed by various surface sensitive techniques

et al. 2009

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Tethered bilayer lipid membranes are promising biomimetic architectures. Their formation has been investigated using four different surface sensitive techniques, including optical, acoustic, and electrical methods. The lipid bilayers are built in a two-step procedure; the proximal layer is formed by self-assembly and is then completed to a bilayer by fusion with small vesicles. The different technical approaches revealed specific aspects of the layer formation processes, namely, first a fast adsorption process followed by a longer rearrangement period. Similar phenomena have been observed for the vesicle fusion process. The results allow for a more controlled assembly protocol for the preparation of highly insulating lipid membranes.

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“…SLBs are commonly prepared by vesicle fusion, a method developed in the McConnell laboratory, 15,16 where sonicated vesicles in contact with an appropriately cleaned glass surface rupture and spread out to form a continuous bilayer. 17 The difference in surface free energy between a bare and lipid bilayer-coated glass surfaces under water is the driving force that drags the bilayer from an area of excess lipid to uncovered areas. 18,19 Other methods to prepare supported membranes include the Langmuir-Blodgett deposition, 15 spin coating, 20 evaporation induced self-assembly, 21 and more recently, dip-pen technology.…”

mentioning

confidence: 99%

Controlled solvent-exchange deposition of phospholipid membranes onto solid surfaces

Hohner¹,

David²,

Rädler³

2010

Biointerphases

115

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Formation of supported planar bilayers by fusion of vesicles to supported phospholipid monolayers

Cited by 612 publications

References 27 publications

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

Formation of tethered bilayer lipid membranes probed by various surface sensitive techniques

Controlled solvent-exchange deposition of phospholipid membranes onto solid surfaces

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