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Reconstituted lipid vesicles

Andrew Scott, Marek J Noga, Paul de Graaf, Ilja Westerlaken, Esengul Yildirim, Christophe Danelon
The goal of bottom-up synthetic biology culminates in the assembly of an entire cell from separate biological building blocks. One major challenge resides in the in vitro production and implementation of complex genetic and metabolic pathways that can support essential cellular functions. Here, we show that phospholipid biosynthesis, a multiple-step process involved in cell membrane homeostasis, can be reconstituted starting from the genes encoding for all necessary proteins. A total of eight E. coli enzymes for acyl transfer and headgroup modifications were produced in a cell-free gene expression system and were co-translationally reconstituted in liposomes...
2016: PloS One
J Lemière, F Valentino, C Campillo, C Sykes
Lipid membranes define the boundaries of living cells and intracellular compartments. The dynamic remodelling of these membranes by the cytoskeleton, a very dynamic structure made of active biopolymers, is crucial in many biological processes such as motility or division. In this review, we present some aspects of cellular membranes and how they are affected by the presence of the actin cytoskeleton. We show that, in parallel with the direct study of membranes and cytoskeleton in vivo, biomimetic in vitro systems allow reconstitution of biological processes in a controlled environment...
September 28, 2016: Biochimie
Koki Kamiya, Ryuji Kawano, Toshihisa Osaki, Kazunari Akiyoshi, Shoji Takeuchi
Asymmetric lipid giant vesicles have been used to model the biochemical reactions in cell membranes. However, methods for producing asymmetric giant vesicles lead to the inclusion of an organic solvent layer that affects the mechanical and physical characteristics of the membrane. Here we describe the formation of asymmetric giant vesicles that include little organic solvent, and use them to investigate the dynamic responses of lipid molecules in the vesicle membrane. We formed the giant vesicles via the inhomogeneous break-up of a lipid microtube generated by applying a jet flow to an asymmetric planar lipid bilayer...
September 2016: Nature Chemistry
Frans Bianchi, Joury S van 't Klooster, Stephanie J Ruiz, Katja Luck, Tjeerd Pols, Ina L Urbatsch, Bert Poolman
The import of basic amino acids in Saccharomyces cerevisiae has been reported to be unidirectional, which is not typical of how secondary transporters work. Since studies of energy coupling and transport kinetics are complicated in vivo, we purified the major lysine transporter (Lyp1) of yeast and reconstituted the protein into lipid vesicles. We show that the Michaelis constant (KM) of transport from out-to-in is well in the millimolar range and at least 3 to 4-orders of magnitude higher than that of transport in the opposite direction, disfavoring the efflux of solute via Lyp1...
2016: Scientific Reports
Rodrigo Cuevas Arenas, Johannes Klingler, Carolyn Vargas, Sandro Keller
Copolymers of styrene and maleic acid (SMA) have gained great attention as alternatives to conventional detergents, as they offer decisive advantages for studying membrane proteins and lipids in vitro. These polymers self-insert into artificial and biological membranes and, at sufficiently high concentrations, solubilise them into disc-shaped nanostructures containing a lipid bilayer core surrounded by a polymer belt. We have used (31)P nuclear magnetic resonance spectroscopy and dynamic light scattering to systematically study the solubilisation of vesicles composed of saturated or unsaturated phospholipids by an SMA copolymer with a 3 : 1 styrene/maleic acid molar ratio at different temperatures...
August 11, 2016: Nanoscale
Younes F Barooji, Andreas Rørvig-Lund, Szabolcs Semsey, S Nader S Reihani, Poul M Bendix
Membrane deformation is a necessary step in a number of cellular processes such as filopodia and invadopodia formation and has been shown to involve membrane shaping proteins containing membrane binding domains from the IRSp53-MIM protein family. In reconstituted membranes the membrane shaping domains can efficiently deform negatively charged membranes into tubules without any other proteins present. Here, we show that the IM domain (also called I-BAR domain) from the protein ABBA, forms semi-flexible nanotubes protruding into Giant Unilamellar lipid Vesicles (GUVs)...
2016: Scientific Reports
Ida Louise Jørgensen, Gerdi Christine Kemmer, Thomas Günther Pomorski
Studying membrane proteins at the molecular level represents a major challenge in biochemistry due to the complexity of the membrane in which they are embedded. As an important step towards a detailed understanding of their action and molecular functioning, current studies focus on membrane proteins reconstituted into artificial lipid environments. Such reconstituted systems allow for a more flexible choice of biochemical, biophysical, and microscopy techniques for characterizing the proteins. This review gives an overview of the methods currently available for reconstituting membrane proteins in a functional state into giant unilamellar vesicles, and discusses some key methods to verify successful reconstitution...
July 20, 2016: European Biophysics Journal: EBJ
Robert J Rawle, Steven G Boxer, Peter M Kasson
Enveloped viruses must bind to a receptor on the host membrane to initiate infection. Membrane fusion is subsequently initiated by a conformational change in the viral fusion protein, triggered by receptor binding, an environmental change, or both. Here, we present a strategy to disentangle the two processes of receptor binding and fusion using synthetic DNA-lipid conjugates to bind enveloped viruses to target membranes in the absence of receptor. This permits direct testing of whether receptor engagement affects the fusion mechanism as well as a comparison of fusion behavior across viruses with different receptor binding specificities...
July 12, 2016: Biophysical Journal
Marco Emanuele, Alessandro Esposito, Serena Camerini, Flavia Antonucci, Silvia Ferrara, Silvia Seghezza, Tiziano Catelani, Marco Crescenzi, Roberto Marotta, Claudio Canale, Michela Matteoli, Elisabetta Menna, Evelina Chieregatti
Alpha-synuclein (αSyn) interferes with multiple steps of synaptic activity at pre-and post-synaptic terminals, however the mechanism/s by which αSyn alters neurotransmitter release and synaptic potentiation is unclear. By atomic force microscopy we show that human αSyn, when incubated with reconstituted membrane bilayer, induces lipid rafts' fragmentation. As a consequence, ion channels and receptors are displaced from lipid rafts with consequent changes in their activity. The enhanced calcium entry leads to acute mobilization of synaptic vesicles, and exhaustion of neurotransmission at later stages...
May 2016: EBioMedicine
Ayumi Hirano-Iwata, Yutaka Ishinari, Miyu Yoshida, Shun Araki, Daisuke Tadaki, Ryusuke Miyata, Kenichi Ishibashi, Hideaki Yamamoto, Yasuo Kimura, Michio Niwano
Artificially formed bilayer lipid membranes (BLMs) provide well-defined systems for functional analyses of various membrane proteins, including ion channels. However, difficulties associated with the integration of membrane proteins into BLMs limit the experimental efficiency and usefulness of such BLM reconstitution systems. Here, we report on the use of centrifugation to more efficiently reconstitute human ion channels in solvent-free BLMs. The method improves the probability of membrane fusion. Membrane vesicles containing the human ether-a-go-go-related gene (hERG) channel, the human cardiac sodium channel (Nav1...
May 24, 2016: Biophysical Journal
Alex J B Kreutzberger, Binyong Liang, Volker Kiessling, Lukas K Tamm
Neuronal exocytotic membrane fusion occurs on a fast timescale and is dependent on interactions between the vesicle SNARE synaptobrevin-2 and the plasma membrane SNAREs syntaxin-1a and SNAP-25 with a 1:1:1 stoichiometry. Reproducing fast fusion rates as observed in cells by reconstitution in vitro has been hindered by the spontaneous assembly of a 2:1 syntaxin-1a:SNAP-25 complex on target membranes that kinetically alters the binding of synaptobrevin-2. Previously, an artificial SNARE acceptor complex consisting of 1:1:1 syntaxin-1a(residues 183-288):SNAP-25:syb(residues 49-96) was found to greatly accelerate the rates of lipid mixing of reconstituted target and vesicle SNARE proteoliposomes...
May 24, 2016: Biophysical Journal
Konrad Sandhoff
Glycosphingolipids and sphingolipids of cellular plasma membranes (PMs) reach luminal intra-lysosomal vesicles (LVs) for degradation mainly by pathways of endocytosis. After a sorting and maturation process (e.g. degradation of sphingomyelin (SM) and secretion of cholesterol), sphingolipids of the LVs are digested by soluble enzymes with the help of activator (lipid binding and transfer) proteins. Inherited defects of lipid-cleaving enzymes and lipid binding and transfer proteins cause manifold and fatal, often neurodegenerative diseases...
May 5, 2016: Biochimie
Keita Takagi, Takashi Ohgita, Takenori Yamamoto, Yasuo Shinohara, Kentaro Kogure
Liposomes are closed-membrane vesicles comprised of lipid bilayers, in which the inside of the vesicles is isolated from the external environment. Liposomes are therefore often used as models for biomembranes and as drug delivery carriers. However, materials encapsulated within liposomes often cannot respond to changes in the external environment. The ability of enclosed materials to maintain their responsiveness to changes in the external environment following encapsulation into liposomes would greatly expand the applicability of such systems...
2016: Chemical & Pharmaceutical Bulletin
Ulrike Rost, Yihui Xu, Tim Salditt, Ulf Diederichsen
Transmembrane β-peptides are promising candidates for the design of well-controlled membrane anchors in lipid membranes. Here, we present the synthesis of transmembrane β-peptides with and without tryptophan anchors, as well as a novel iodine-labeled d-β(3) -amino acid. By using one or more of the heavy-atom labeled amino acids as markers, the orientation of the helical peptide was inferred based on the electron-density profile determined by X-ray reflectivity. The β-peptides were synthesized through manual Fmoc-based solid-phase peptide synthesis (SPPS) and reconstituted in unilamellar vesicles forming a right-handed 314 -helix secondary structure, as shown by circular dichroism spectroscopy...
August 18, 2016: Chemphyschem: a European Journal of Chemical Physics and Physical Chemistry
Benjamin S Stratton, Jason M Warner, Zhenyong Wu, Joerg Nikolaus, George Wei, Emma Wagnon, David Baddeley, Erdem Karatekin, Ben O'Shaughnessy
Flickering of fusion pores during exocytotic release of hormones and neurotransmitters is well documented, but without assays that use biochemically defined components and measure single-pore dynamics, the mechanisms remain poorly understood. We used total internal reflection fluorescence microscopy to quantify fusion-pore dynamics in vitro and to separate the roles of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and lipid bilayer properties. When small unilamellar vesicles bearing neuronal v-SNAREs fused with planar bilayers reconstituted with cognate t-SNARES, lipid and soluble cargo transfer rates were severely reduced, suggesting that pores flickered...
April 12, 2016: Biophysical Journal
Qian Peter Su, Wanqing Du, Qinghua Ji, Boxin Xue, Dong Jiang, Yueyao Zhu, Jizhong Lou, Li Yu, Yujie Sun
Intracellular membrane nanotube formation and its dynamics play important roles for cargo transportation and organelle biogenesis. Regarding the regulation mechanisms, while much attention has been paid on the lipid composition and its associated protein molecules, effects of the vesicle size has not been studied in the cell. Giant unilamellar vesicles (GUVs) are often used for in vitro membrane deformation studies, but they are much larger than most intracellular vesicles and the in vitro studies also lack physiological relevance...
2016: Scientific Reports
Tripta Bhatia, Flemming Cornelius, Jonathan Brewer, Luis A Bagatolli, Adam C Simonsen, John H Ipsen, Ole G Mouritsen
We have reconstituted functional Na(+)/K(+)-ATPase (NKA) into giant unilamellar vesicles (GUVs) of well-defined binary and ternary lipid composition including cholesterol. The activity of the membrane system can be turned on and off by ATP. The hydrolytic activity of NKA is found to depend on membrane phase, and the water relaxation in the membrane on the presence of NKA. By collapsing and fixating the GUVs onto a solid support and using high-resolution atomic-force microscopy (AFM) imaging we determine the protein orientation and spatial distribution at the single-molecule level and find that NKA is preferentially located at lo/ld interfaces in two-phase GUVs and homogeneously distributed in single-phase GUVs...
June 2016: Biochimica et Biophysica Acta
Katrine Hommelhoff Jensen, Sami Sebastian Brandt, Hideki Shigematsu, Fred J Sigworth
This paper describes steps in the single-particle cryo-EM 3D structure determination of membrane proteins in their membrane environment. Using images of the Kv1.2 potassium-channel complex reconstituted into lipid vesicles, we describe procedures for the merging of focal-pairs of exposures and the removal of the vesicle-membrane signal from the micrographs. These steps allow 3D reconstruction to be performed from the protein particle images. We construct a 2D statistical model of the vesicle structure based on higher-order singular value decomposition (HOSVD), by taking into account the structural symmetries of the vesicles in polar coordinates...
April 2016: Journal of Structural Biology
Mark J Richards, Chih-Yun Hsia, Rohit R Singh, Huma Haider, Julia Kumpf, Toshimitsu Kawate, Susan Daniel
Membrane protein interactions with lipids are crucial for their native biological behavior, yet traditional characterization methods are often carried out on purified protein in the absence of lipids. We present a simple method to transfer membrane proteins expressed in mammalian cells to an assay-friendly, cushioned, supported lipid bilayer platform using cell blebs as an intermediate. Cell blebs, expressing either GPI-linked yellow fluorescent proteins or neon-green fused transmembrane P2X2 receptors, were induced to rupture on glass surfaces using PEGylated lipid vesicles, which resulted in planar supported membranes with over 50% mobility for multipass transmembrane proteins and over 90% for GPI-linked proteins...
March 29, 2016: Langmuir: the ACS Journal of Surfaces and Colloids
Ane Landajuela, Javier H Hervás, Zuriñe Antón, L Ruth Montes, David Gil, Mikel Valle, J Francisco Rodriguez, Felix M Goñi, Alicia Alonso
Autophagy, an important catabolic pathway involved in a broad spectrum of human diseases, implies the formation of double-membrane-bound structures called autophagosomes (AP), which engulf material to be degraded in lytic compartments. How APs form, especially how the membrane expands and eventually closes upon itself, is an area of intense research. Ubiquitin-like ATG8 has been related to both membrane expansion and membrane fusion, but the underlying molecular mechanisms are poorly understood. Here, we used two minimal reconstituted systems (enzymatic and chemical conjugation) to compare the ability of human ATG8 homologs (LC3, GABARAP, and GATE-16) to mediate membrane fusion...
January 19, 2016: Biophysical Journal
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