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Reconstituted lipid vesicles

Karlo Komorowski, Annalena Salditt, Yihui Xu, Halenur Yavuz, Martha Brennich, Reinhard Jahn, Tim Salditt
We have studied the adhesion state (also denoted by docking state) of lipid vesicles as induced by the divalent ions Ca2+ or Mg2+ at well-controlled ion concentration, lipid composition, and charge density. The bilayer structure and the interbilayer distance in the docking state were analyzed by small-angle x-ray scattering. A strong adhesion state was observed for DOPC:DOPS vesicles, indicating like-charge attraction resulting from ion correlations. The observed interbilayer separations of ∼1.6 nm agree quantitatively with the predictions of electrostatics in the strong coupling regime...
April 24, 2018: Biophysical Journal
Sathish Kumar Ramakrishnan, Andrea Gohlke, Feng Li, Jeff Coleman, Weiming Xu, James E Rothman, Frederic Pincet
In vivo membrane fusion primarily occurs between highly curved vesicles and planar membranes. A better understanding of fusion entails an accurate in vitro reproduction of the process. To date, supported bilayers have been commonly used to mimic the planar membranes. SNARE proteins, that induce membrane fusion, usually have limited fluidity when embedded in supported bilayers. This alters the kinetics and prevents correct reconstitution of the overall fusion process. Also, observing content release across the membrane is hindered by the lack of second aqueous compartment...
April 25, 2018: Langmuir: the ACS Journal of Surfaces and Colloids
Katharina Dürre, Felix C Keber, Philip Bleicher, Fridtjof Brauns, Christian J Cyron, Jan Faix, Andreas R Bausch
Arp2/3 complex-mediated actin assembly at cell membranes drives the formation of protrusions or endocytic vesicles. To identify the mechanism by which different membrane deformations can be achieved, we reconstitute the basic membrane deformation modes of inward and outward bending in a confined geometry by encapsulating a minimal set of cytoskeletal proteins into giant unilamellar vesicles. Formation of membrane protrusions is favoured at low capping protein (CP) concentrations, whereas the formation of negatively bent domains is promoted at high CP concentrations...
April 24, 2018: Nature Communications
Mikhail A Galkin, Aidan N Russell, Steven B Vik, Richard M Berry, Robert R Ishmukhametov
Detergents are indispensable for delivery of membrane proteins into 30-100 nm small unilamellar vesicles, while more complex, larger model lipid bilayers are less compatible with detergents. Here we describe a strategy for bypassing this fundamental limitation using fusogenic oppositely charged liposomes bearing a membrane protein of interest. Fusion between such vesicles occurs within 5 min in a low ionic strength buffer. Positively charged fusogenic liposomes can be used as simple shuttle vectors for detergent-free delivery of membrane proteins into biomimetic target lipid bilayers, which are negatively charged...
April 5, 2018: Journal of Visualized Experiments: JoVE
Daichi Yamaura, Daisuke Tadaki, Shun Araki, Miyu Yoshida, Kohei Arata, Takeshi Ohori, Ken-Ichi Ishibashi, Miki Kato, Teng Ma, Ryusuke Miyata, Hideaki Yamamoto, Ryugo Tero, Masao Sakuraba, Toshio Ogino, Michio Niwano, Ayumi Hirano-Iwata
Artificial bilayer lipid membranes (BLMs) provide well-defined systems for investigating the fundamental properties of membrane proteins, including ion channels, and in screening the effect of drugs that act on them. However, the application of this technique is limited due to the low stability and low reconstitution efficiency of the process. We previously reported on improving the stability of BLM based on the fabrication of microapertures having a tapered edge in SiO2/Si3N4 septa and efficient ion channel incorporation based on vesicle fusion accelerated by a centrifugal force...
April 17, 2018: Langmuir: the ACS Journal of Surfaces and Colloids
Rianne Bartelds, Mohammad Hadi Nematollahi, Tjeerd Pols, Marc C A Stuart, Abbas Pardakhty, Gholamreza Asadikaram, Bert Poolman
Niosomes are used in studies for drug delivery or gene transfer. However, their physical properties and features relative to liposomes are not well documented. To characterize and more rationally optimize niosome formulations, the properties of these vesicle systems are compared to those of liposomes composed of phosphatidylcholine and phosphatidylethanolamine lipids plus cholesterol. Niosomes are highly stable and only slightly more leaky than liposomes as assayed by calcein leakage; the permeability for ions (KCl) is higher than that of liposomes...
2018: PloS One
Danilo Donnarumma, Claudio Maestri, Pietro Ivan Giammarinaro, Luigi Capriotti, Erika Bartolini, Daniele Veggi, Roberto Petracca, Maria Scarselli, Nathalie Norais
Hydrogen Deuterium exchange (HDx) associated with Mass Spectrometry (MS) is emerging as a powerful tool to provide conformational information on membrane proteins. Unfortunately, as for X-ray diffraction and NMR, HDx performed on reconstituted in vitro systems might not always reflect the in vivo environment. Outer Membrane Vesicles naturally released by E. coli were used to carry out analysis of native OmpF through HDx-MS. A new protocol compatible with HDx analysis and avoiding hindrance from the lipid contents was set-up...
April 5, 2018: Journal of Proteome Research
Yuval Mulla, Anders Aufderhorst-Roberts, Gijsje Koenderink
How do the cells in our body reconfigure their shape to achieve complex tasks like migration and mitosis, yet maintain their shape in response to the forces exerted by for instance blood flow and muscle action? Cell shape control is defined by a delicate mechanical balance between active force generation and passive material properties of the plasma membrane and the cytoskeleton. The cytoskeleton forms a space-spanning fibrous network comprising three subsystems; actin, microtubules and intermediate filaments...
March 23, 2018: Physical Biology
Julia P Steringer, Walter Nickel
As illustrated by a diverse set of examples in this special issue, multiple mechanisms of protein secretion have been identified in eukaryotes that do not involve the endoplasmic reticulum (ER) and the Golgi apparatus. Here we focus on the type I pathway with Fibroblast Growth Factor 2 (FGF2) being the most prominent example. Unconventional secretion of FGF2 from cells is mediated by direct protein translocation across the plasma membrane. A unique feature of this process is the ability of FGF2 to form its own membrane translocation intermediate through oligomerization and membrane insertion...
March 5, 2018: Seminars in Cell & Developmental Biology
Dimitri Fayolle, Michele Fiore, Pasquale Stano, Peter Strazewski
Giant lipid vesicles (GVs) are emerging models for investigating the properties and reactivity of cell-like microcompartments, providing useful information about plausible protocellular structures in primitive times, as well as for the modern synthetic biology goal of constructing the first artificial cell from its reconstituted and partly modified components. Here we explore a novel methodology of GV purification by microfiltration under reduced pressure, operated by a simple apparatus. The method has been characterized in terms of flow rate, amount of lipid loss, quality of recovered GVs, and size distribution...
2018: PloS One
Huan Bao, Debasis Das, Nicholas A Courtney, Yihao Jiang, Joseph S Briguglio, Xiaochu Lou, Daniel Roston, Qiang Cui, Baron Chanda, Edwin R Chapman
The fusion pore is the first crucial intermediate formed during exocytosis, yet little is known about the mechanisms that determine the size and kinetic properties of these transient structures. Here, we reduced the number of available SNAREs (proteins that mediate vesicle fusion) in neurons and observed changes in transmitter release that are suggestive of alterations in fusion pores. To investigate these changes, we employed reconstituted fusion assays using nanodiscs to trap pores in their initial open state...
February 8, 2018: Nature
Aymeric Chorlay, Abdou Rachid Thiam
Cells store excess energy in the form of neutral lipids that are synthesized and encapsulated within the endoplasmic reticulum intermonolayer space. The lipids next demix to form lipid droplets (LDs), which, surprisingly, bud off mostly toward the cytosol. This directional LD formation is critical to energy metabolism, but its mechanism remains poorly understood. Here, we reconstituted the LD formation topology by embedding artificial LDs into the intermonolayer space of bilayer vesicles. We provide experimental evidence that the droplet behavior in the membrane is recapitulated by the physics of three-phase wetting systems, dictated by the equilibrium of surface tensions...
February 6, 2018: Biophysical Journal
Rashmi Seneviratne, Sanobar Khan, Ellen Moscrop, Michael Rappolt, Stephen P Muench, Lars J C Jeuken, Paul A Beales
Hybrid vesicles composed of lipids and block copolymers hold promise for increasing liposome stability and providing a stable environment for membrane proteins. Recently we reported the successful functional reconstitution of the integral membrane protein cytochrome bo3 (ubiquinol oxidase) into hybrid vesicles composed of a blend of phospholipids and a block copolymer (PBd-PEO). We demonstrated that these novel membrane environments stabilise the enzymes' activity, prolonging their functional lifetime [Chem...
February 2, 2018: Methods: a Companion to Methods in Enzymology
Nicholas A Ferraro, Michael Cascio
The glycine receptor (GlyR) belongs to a superfamily of pentameric ligand-gated ion channels (pLGICs) that mediate fast neurotransmission. GlyR typically modulates inhibitory transmission by antagonizing membrane depolarization through anion influx. Allosteric interactions between the receptor and its lipid surroundings affect receptor function, and cholesterol is essential for pLGIC activity. Cholesterol at compositions below ∼33 mol percent has been shown to have negligible chemical activity, suggesting that specific interactions between membrane proteins and cholesterol become significant only at concentrations above this stoichiometric threshold...
February 20, 2018: Analytical Chemistry
Rahul Chadda, Lucy Cliff, Marley Brimberry, Janice L Robertson
The thermodynamic reasons why membrane proteins form stable complexes inside the hydrophobic lipid bilayer remain poorly understood. This is largely because of a lack of membrane-protein systems amenable for equilibrium studies and a limited number of methods for measuring these reactions. Recently, we reported the equilibrium dimerization of the CLC-ec1 Cl- /H+ transporter in lipid bilayers (Chadda et al. 2016. eLife, which provided a new type of model system for studying protein association in membranes...
February 5, 2018: Journal of General Physiology
Sunyia Hussain, Harris D Bernstein
Most proteins that reside in the bacterial outer membrane (OM) have a distinctive "β-barrel" architecture, but the assembly of these proteins is poorly understood. The spontaneous assembly of OM proteins (OMPs) into pure lipid vesicles has been studied extensively but often requires non-physiological conditions and time scales and is strongly influenced by properties of the lipid bilayer, including surface charge, thickness, and fluidity. Furthermore, the membrane insertion of OMPs in vivo is catalyzed by a heterooligomer called the β-<u>b</u>arrel <u>a</u>ssembly <u>m</u>achinery (Bam) complex...
February 23, 2018: Journal of Biological Chemistry
Giulia Morra, Asghar M Razavi, Kalpana Pandey, Harel Weinstein, Anant K Menon, George Khelashvili
Several class-A G protein-coupled receptor (GPCR) proteins act as constitutive phospholipid scramblases catalyzing the transbilayer translocation of >10,000 phospholipids per second when reconstituted into synthetic vesicles. To address the molecular mechanism by which these proteins facilitate rapid lipid scrambling, we carried out large-scale ensemble atomistic molecular dynamics simulations of the opsin GPCR. We report that, in the process of scrambling, lipid head groups traverse a dynamically revealed hydrophilic pathway in the region between transmembrane helices 6 and 7 of the protein while their hydrophobic tails remain in the bilayer environment...
February 6, 2018: Structure
Coline Prévost, Feng-Ching Tsai, Patricia Bassereau, Mijo Simunovic
The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many different proteins associate with curved membranes because of their ability to sense or induce membrane curvature. Typically, these processes involve a multitude of proteins making them too complex to study quantitatively in the cell. We describe a protocol to reconstitute a curved membrane in vitro, mimicking a curved cellular structure, such as the endocytic neck...
December 7, 2017: Journal of Visualized Experiments: JoVE
Pasquale Stano, Emiliano Altamura, Fabio Mavelli
Important progresses have been achieved in the past years in the field of bottom-up synthetic biology, especially aiming at constructing cell-like systems based on lipid vesicles (liposomes) entrapping both biomolecules or synthetic compounds. These "synthetic cells" mimic the behaviour of biological cells but are constituted by a minimal number of components. One key aspect related to this research is the energetic needs of synthetic cells. Up to now, high-energy compounds have been given in order to drive biochemical reactions inside the vesicle lumen...
2017: Communicative & Integrative Biology
Anders Lundgren, Björn Johansson Fast, Stephan Block, Björn Agnarsson, Erik Reimhult, Anders Gunnarsson, Fredrik Höök
The function of integral membrane proteins is critically dependent on their naturally surrounding lipid membrane. Detergent-solubilized and purified membrane proteins are therefore often reconstituted into cell-membrane mimics and analyzed for their function with single-molecule microscopy. Expansion of this approach toward a broad range of pharmaceutically interesting drug targets and biomarkers however remains hampered by the fact that these proteins have low expression levels, and that detergent solubilization and reconstitution often cause protein conformational changes and loss of membrane-specific cofactors, which may impair protein function...
January 10, 2018: Nano Letters
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