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Giant plasma membrane vesicles derived from mammalian cells are shown to exhibit phase separation at sub-physiological temperatures. The phase partitioning behavior of fluorescent lipids and labeled proteins are also described.
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Networks of GUV's connected by lipid tubules are used to mix enzyme and substrate incorporated into the vesicle contents. This approaches the level of organization found in biological systems, and allows controlled sequences of reactions to be observed.
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Immunological synapses are formed between T-cells exposed to a patterned supported lipid bilayer displaying complementary protein ligands simulating an antigen presenting cell. The patterned surface constrains T-cell receptor reorganization, and a dependence of T-cell receptor signaling on position is observed.
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Mossman K.D., Campi G., Groves J.T., and Dustin M.L. Altered TCR signaling from geometrically repatterned immunological synapses. Science 310 (2005) 1191-1193. Immunological synapses are formed between T-cells exposed to a patterned supported lipid bilayer displaying complementary protein ligands simulating an antigen presenting cell. The patterned surface constrains T-cell receptor reorganization, and a dependence of T-cell receptor signaling on position is observed.
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SecYEG complexes in lipid nanodiscs are found to bind only the monomeric form of their partner SecA. The binding is also found to be dependent on a key amino acid in the SecY protein as well as the lipid composition of the nanodisc.
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This paper is an excellent synthesis of experiments elucidating the mechanism of the vesicle fusion reaction to form supported lipid bilayers and its dependence on factors including lipid composition, electrostatics, divalent cations, and surface properties.
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Phase separation of lipid membranes analyzed with high-resolution secondary ion mass spectrometry
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NanoSIMS imaging was performed on two-component, phase-separated lipid bilayers. Isotopic labeling of the lipids allowed composition to be quantified based on the collection of molecular ions, leading to images with high spatial resolution and information content.
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Kraft M.L., Weber P.K., Longo M.L., Hutcheon I.D., and Boxer S.G. Phase separation of lipid membranes analyzed with high-resolution secondary ion mass spectrometry. Science 313 (2006) 1948-1951. NanoSIMS imaging was performed on two-component, phase-separated lipid bilayers. Isotopic labeling of the lipids allowed composition to be quantified based on the collection of molecular ions, leading to images with high spatial resolution and information content.
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Kraft, M.L.1
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Lateral heterogeneity of dipalmitoylphosphatidylethanolamine-cholesterol Langmuir-Blodgett films investigated with imaging time-of-flight secondary ion mass spectrometry and atomic force microscopy
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Mass spectrometric imaging of highly curved membranes during Tetrahymena mating
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TOF-SIMS composition imaging is performed on a complex biological sample, mating Tetrahymena cells. The junction between cells shows depleted levels of phosphocholine lipids and elevation of the cone-shaped 2-aminoethylphosphonolipid.
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Ostrowski S.G., Van Bell C.T., Winograd N., and Ewing A.G. Mass spectrometric imaging of highly curved membranes during Tetrahymena mating. Science 305 (2004) 71-73. TOF-SIMS composition imaging is performed on a complex biological sample, mating Tetrahymena cells. The junction between cells shows depleted levels of phosphocholine lipids and elevation of the cone-shaped 2-aminoethylphosphonolipid.
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Ostrowski, S.G.1
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Danelon C., Perez J.-B., Santschi C., Brugger J., and Vogel H. Cell membranes suspended across nanoaperture arrays. Langmuir 22 (2006) 22-25
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Supported cell-membrane sheets for functional fluorescence imaging of membrane proteins
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Poly-l-lysine coated slides are placed on top of cultured cells then removed, creating a supported membrane taken directly from biological sources for further study. This is a general method for capturing complex membrane composition in a planar format.
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Perez J.-B., Martinez K.L., Segura J.-M., and Vogel H. Supported cell-membrane sheets for functional fluorescence imaging of membrane proteins. Adv Funct Mater 16 (2006) 306-312. Poly-l-lysine coated slides are placed on top of cultured cells then removed, creating a supported membrane taken directly from biological sources for further study. This is a general method for capturing complex membrane composition in a planar format.
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Adv Funct Mater
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Perez, J.-B.1
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Asymmetric bilayers are formed on a polymer cushion where the proximal leaflet is expected to phase separate but the distal leaflet is not. Induced domain formation in the distal leaflet was observed for certain compositions indicating coupling between the bilayer leaflets.
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Kiessling V., Crane J.M., and Tamm L.K. Transbilayer effects of raft-like lipid domains in asymmetric planar bilayers measured by single molecule tracking. Biophys J 91 (2006) 3313-3326. Asymmetric bilayers are formed on a polymer cushion where the proximal leaflet is expected to phase separate but the distal leaflet is not. Induced domain formation in the distal leaflet was observed for certain compositions indicating coupling between the bilayer leaflets.
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A defect free tethered bilayer incorporating the pore forming domain of an ion channel is assembled on a gold electrode. Ions of different size can be distinguished, and the tether identity also affects the performance of the device.
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Vockenroth I.K., Atanasova P.P., Long J.R., Jenkins A.T.A., Knoll W., and Köper I. Functional incorporation of the pore forming segment of AChR M2 into tethered bilayer lipid membranes. Biochim Biphys Acta 1768 (2007) 1114-1120. A defect free tethered bilayer incorporating the pore forming domain of an ion channel is assembled on a gold electrode. Ions of different size can be distinguished, and the tether identity also affects the performance of the device.
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The αHL protein membrane pore protein is assembled in a bilayer spanning a small Teflon hole on an electrode. This device is able to detect the presence of chemical analytes based on electrical current, and can be developed into robust sensors.
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Kang X.-F., Cheley S., Rice-Ficht A.C., and Bayley H. A storable encapsulated bilayer chip containing a single protein nanopore. J Am Chem Soc 129 (2007) 4701-4705. The αHL protein membrane pore protein is assembled in a bilayer spanning a small Teflon hole on an electrode. This device is able to detect the presence of chemical analytes based on electrical current, and can be developed into robust sensors.
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J Am Chem Soc
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Vesicles are tethered to a SLB using complementary DNA which has been covalently coupled a lipid-like tail group. The sequence specificity of DNA allows arrays of vesicles to be made using patterned bilayers and their lateral mobility can be exploited for studies of vesicle-vesicle interactions.
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Yoshina-Ishii C., Miller G.P., Kraft M.L., Kool E.T., and Boxer S.G. General method for modification of liposomes for encoded assembly on supported bilayers. J Am Chem Soc 127 (2005) 1356-1357. Vesicles are tethered to a SLB using complementary DNA which has been covalently coupled a lipid-like tail group. The sequence specificity of DNA allows arrays of vesicles to be made using patterned bilayers and their lateral mobility can be exploited for studies of vesicle-vesicle interactions.
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J Am Chem Soc
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Yoshina-Ishii, C.1
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Indriati P., and Höök F. Bivalent cholesterol-based coupling of oligonucleotides to lipid membrane assemblies. J Am Chem Soc 126 (2004) 10224-10225
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Vesicles presenting yeast SNARE proteins are tethered to an SLB and reacted with vesicles in solution presenting the cognate proteins. Several mechanisms for outer then inner leaflet mixing (many including hemifusion intermediates) are classified based on FRET analysis of lipid labels.
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Yoon T.-Y., Okumus B., Zhang F., Shin Y.-K., and Ha T. Multiple intermediates in SNARE-induced membrane fusion. Proc Natl Acad Sci U S A 103 (2006) 19731-19736. Vesicles presenting yeast SNARE proteins are tethered to an SLB and reacted with vesicles in solution presenting the cognate proteins. Several mechanisms for outer then inner leaflet mixing (many including hemifusion intermediates) are classified based on FRET analysis of lipid labels.
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Proc Natl Acad Sci U S A
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Bolinger P.-Y., Stamou D., and Vogel H. Integrated nanoreactor systems: Triggering the release and mixing of compounds inside single vesicles. J Am Chem Soc 126 (2004) 8594-8595
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Yoshina-Ishii C., Chan Y-HM, Johnson J.M., Kung L.A., Lenz P., and Boxer S.G. Diffusive dynamics of vesicles tethered to a fluid supported bilayer by single-particle tracking. Langmuir 22 (2006) 5682-5689
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The dynamics of vesicles tethered on a patterned SLB and subjected to an external electric field were observed. A subtle balance between electrophoretic and electroosmotic forces, in conjunction with electrophoretic reorganization of the lipids in the SLB, can lead to focusing effects where the steady-state position of the vesicles is dependent on their charge.
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Yoshina-Ishii C., and Boxer S.G. Controlling two-dimensional tethered vesicle motion using an electric field: Interplay of electrophoresis and electro-osmosis. Langmuir 22 (2006) 2384-2391. The dynamics of vesicles tethered on a patterned SLB and subjected to an external electric field were observed. A subtle balance between electrophoretic and electroosmotic forces, in conjunction with electrophoretic reorganization of the lipids in the SLB, can lead to focusing effects where the steady-state position of the vesicles is dependent on their charge.
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DNA-induced programmable fusion of phospholipid vesicles
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Cholesterol-anchored DNA introduced into lipid vesicles can mediate inner and outer leaflet mixing as shown by FRET dequenching. This can lead to insights into the mechanisms for fusion.
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Stengel G., Zahn R., and Höök F. DNA-induced programmable fusion of phospholipid vesicles. J Am Chem Soc 129 (2007) 9584-9585. Cholesterol-anchored DNA introduced into lipid vesicles can mediate inner and outer leaflet mixing as shown by FRET dequenching. This can lead to insights into the mechanisms for fusion.
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Multiscale simulation of transmembrane proteins
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This simulation study uses a combined coarse-grained and continuum model to describe membrane proteins and the lipid bilayer, respectively. The protein shows a subtle conformational change when the mechanical properties of the bilayer are adjusted.
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Ayton G.S., and Voth G.A. Multiscale simulation of transmembrane proteins. J Struct Biol 157 (2007) 570-578. This simulation study uses a combined coarse-grained and continuum model to describe membrane proteins and the lipid bilayer, respectively. The protein shows a subtle conformational change when the mechanical properties of the bilayer are adjusted.
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Shi Q., Izvekov S., and Voth G.A. Mixed atomistic and coarse-grained molecular dynamics: Simulation of a membrane-bound ion channel. J Phys Chem B 110 (2006) 15045-15048
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Molecular dynamics simulations of lipid vesicle fusion in atomic detail
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Atomistic simulations of vesicle fusion show progression from stalk formation to a stable hemifusion to the appearance fusion pore on the order of 10 ns. The density of lipids in the bilayer junction is reduced during hemifusion, but voids are not observed.
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Knecht V., and Marrink S.-J. Molecular dynamics simulations of lipid vesicle fusion in atomic detail. Biophys J 92 (2007) 4254-4261. Atomistic simulations of vesicle fusion show progression from stalk formation to a stable hemifusion to the appearance fusion pore on the order of 10 ns. The density of lipids in the bilayer junction is reduced during hemifusion, but voids are not observed.
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Ensemble molecular dynamics yields submillisecond kinetics and intermediates of membrane fusion
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A coarse-grained model is used to simulated the fusion of lipid vesicles. Many mechanistic pathways are observed, and the timescale of fusion depends on the separation distance between the vesicles' surfaces.
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Kasson P.M., Kelley N.W., Singhal N., Vrljic M., Brunger A.T., and Pande V.S. Ensemble molecular dynamics yields submillisecond kinetics and intermediates of membrane fusion. Proc Natl Acad Sci U S A 103 (2006) 11916-11921. A coarse-grained model is used to simulated the fusion of lipid vesicles. Many mechanistic pathways are observed, and the timescale of fusion depends on the separation distance between the vesicles' surfaces.
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Kasson, P.M.1
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Brunger, A.T.5
Pande, V.S.6
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