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Reconstituted lipid vesicles

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https://www.readbyqxmd.com/read/28536532/phase-partitioning-of-gm1-and-its-bodipy-labeled-analog-determine-their-different-binding-to-cholera-toxin
#1
Sami Rissanen, Michal Grzybek, Adam Orłowski, Tomasz Róg, Oana Cramariuc, Ilya Levental, Christian Eggeling, Erdinc Sezgin, Ilpo Vattulainen
Driven by interactions between lipids and proteins, biological membranes display lateral heterogeneity that manifests itself in a mosaic of liquid-ordered (Lo) or raft, and liquid-disordered (Ld) or non-raft domains with a wide range of different properties and compositions. In giant plasma membrane vesicles and giant unilamellar vesicles, specific binding of Cholera Toxin (CTxB) to GM1 glycolipids is a commonly used strategy to label raft domains or Lo membrane environments. However, these studies often use acyl-chain labeled bodipy-GM1 (bdGM1), whose headgroup accessibility and membrane order or phase partitioning may differ from those of GM1, rendering the interpretation of CTxB binding data quite problematic...
2017: Frontiers in Physiology
https://www.readbyqxmd.com/read/28476622/green-fluorescence-protein-based-content-mixing-assay-of-snare-driven-membrane-fusion
#2
Paul Heo, Byoungjae Kong, Young-Hun Jung, Joon-Bum Park, Jonghyeok Shin, Myungseo Park, Dae-Hyuk Kweon
Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediate intracellular membrane fusion by forming a ternary SNARE complex. A minimalist approach utilizing proteoliposomes with reconstituted SNARE proteins yielded a wealth of information pinpointing the molecular mechanism of SNARE-mediated fusion and its regulation by accessory proteins. Two important attributes of a membrane fusion are lipid-mixing and the formation of an aqueous passage between apposing membranes. These two attributes are typically observed by using various fluorescent dyes...
June 17, 2017: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/28472750/effect-of-the-presence-of-cholesterol-in-the-interfacial-microenvironment-on-the-modulation-of-the-alkaline-phosphatase-activity-during-in-vitro-mineralization
#3
B F Favarin, M A R Andrade, M Bolean, A M S Simão, A P Ramos, M F Hoylaerts, J L Millán, P Ciancaglini
Mineralization of the skeleton starts within cell-derived matrix vesicles (MVs); then, minerals propagate to the extracellular collagenous matrix. Tissue-nonspecific alkaline phosphatase (TNAP) degrades inorganic pyrophosphate (PPi), a potent inhibitor of mineralization, and contributes Pi (Phosphate) from ATP to initiate mineralization. Compared to the plasma membrane, MVs are rich in Cholesterol (Chol) (∼32%) and TNAP, but how Chol influences TNAP activity remains unclear. We have reconstituted TNAP in liposomes of dipalmitoylphosphatidylcholine (DPPC) or dioleoylphosphatidylcholine (DOPC) combined with Chol or its derivatives Cholestenone (Achol) and Ergosterol (Ergo)...
April 26, 2017: Colloids and Surfaces. B, Biointerfaces
https://www.readbyqxmd.com/read/28415454/development-of-a-dna-liposome-complex-for-gene-delivery-applications
#4
M Rasoulianboroujeni, G Kupgan, F Moghadam, M Tahriri, A Boughdachi, P Khoshkenar, J J Ambrose, N Kiaie, D Vashaee, J D Ramsey, L Tayebi
The association structures formed by cationic liposomes and DNA (Deoxyribonucleic acid)-liposome have been effectively utilized as gene carriers in transfection assays. In this research study, cationic liposomes were prepared using a modified lipid film hydration method consisting of a lyophilization step for gene delivery applications. The obtained results demonstrated that the mean particle size had no significant change while the polydispersity (PDI) increased after lyophilization. The mean particle size slightly reduced after lyophilization (520±12nm to 464±25nm) while the PDI increased after lyophilization (0...
June 1, 2017: Materials Science & Engineering. C, Materials for Biological Applications
https://www.readbyqxmd.com/read/28408867/visualization-of-snare-mediated-hemifusion-between-giant-unilamellar-vesicles-arrested-by-myricetin
#5
Paul Heo, Joon-Bum Park, Yeon-Kyun Shin, Dae-Hyuk Kweon
Neurotransmitters are released within a millisecond after Ca(2+) arrives at an active zone. However, the vesicle fusion pathway underlying this synchronous release is yet to be understood. At the center of controversy is whether hemifusion, in which outer leaflets are merged while inner leaflets are still separated, is an on-pathway or off-pathway product of Ca(2+)-triggered exocytosis. Using the single vesicle fusion assay, we recently demonstrated that hemifusion is an on-pathway intermediate that immediately proceeds to full fusion upon Ca(2+) triggering...
2017: Frontiers in Molecular Neuroscience
https://www.readbyqxmd.com/read/28377464/stard3-mediates-endoplasmic-reticulum-to-endosome-cholesterol-transport-at-membrane-contact-sites
#6
Léa P Wilhelm, Corinne Wendling, Benoît Védie, Toshihide Kobayashi, Marie-Pierre Chenard, Catherine Tomasetto, Guillaume Drin, Fabien Alpy
StAR-related lipid transfer domain-3 (STARD3) is a sterol-binding protein that creates endoplasmic reticulum (ER)-endosome contact sites. How this protein, at the crossroad between sterol uptake and synthesis pathways, impacts the intracellular distribution of this lipid was ill-defined. Here, by using in situ cholesterol labeling and quantification, we demonstrated that STARD3 induces cholesterol accumulation in endosomes at the expense of the plasma membrane. STARD3-mediated cholesterol routing depends both on its lipid transfer activity and its ability to create ER-endosome contacts...
May 15, 2017: EMBO Journal
https://www.readbyqxmd.com/read/28356285/exosome-production-and-its-regulation-of-egfr-during-wound-healing-in-renal-tubular-cells
#7
Xiangjun Zhou, Wei Zhang, Qisheng Yao, Hao Zhang, Guie Dong, Ming Zhang, Yutao Liu, Jian-Kang Chen, Zheng Dong
Kidney repair following injury involves the reconstitution of a structurally and functionally intact tubular epithelium. Growth factors and their receptors, such as EGFR, are important in the repair of renal tubules. Exosomes are cell-produced small (~100nm in diameter) vesicles that contain and transfer proteins, lipids, RNAs and DNAs between cells. In this study, we examined the relationship between exosome production and EGFR activation, and the potential role of exosome in wound healing. EGFR activation occurred shortly after scratch wounding in renal tubular cells...
March 29, 2017: American Journal of Physiology. Renal Physiology
https://www.readbyqxmd.com/read/28320948/highly-oriented-photosynthetic-reaction-centers-generate-a-proton-gradient-in-synthetic-protocells
#8
Emiliano Altamura, Francesco Milano, Roberto R Tangorra, Massimo Trotta, Omar Hassan Omar, Pasquale Stano, Fabio Mavelli
Photosynthesis is responsible for the photochemical conversion of light into the chemical energy that fuels the planet Earth. The photochemical core of this process in all photosynthetic organisms is a transmembrane protein called the reaction center. In purple photosynthetic bacteria a simple version of this photoenzyme catalyzes the reduction of a quinone molecule, accompanied by the uptake of two protons from the cytoplasm. This results in the establishment of a proton concentration gradient across the lipid membrane, which can be ultimately harnessed to synthesize ATP...
April 11, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28301741/mechanisms-of-autophagy-initiation
#9
James H Hurley, Lindsey N Young
Autophagy is the process of cellular self-eating by a double-membrane organelle, the autophagosome. A range of signaling processes converge on two protein complexes to initiate autophagy: the ULK1 (unc51-like autophagy activating kinase 1) protein kinase complex and the PI3KC3- C1 (class III phosphatidylinositol 3-kinase complex I) lipid kinase complex. Some 90% of the mass of these large protein complexes consists of noncatalytic domains and subunits, and the ULK1 complex has essential noncatalytic activities...
March 15, 2017: Annual Review of Biochemistry
https://www.readbyqxmd.com/read/28274844/phosphatidylserine-lipids-and-membrane-order-precisely-regulate-the-activity-of-polybia-mp1-peptide
#10
Dayane S Alvares, João Ruggiero Neto, Ernesto E Ambroggio
Polybia-MP1 (IDWKKLLDAAKQIL-NH2) is a lytic peptide from the Brazilian wasp venom with known anti-cancer properties. Previous evidence indicates that phosphatidylserine (PS) lipids are relevant for the lytic activity of MP1. In agreement with this requirement, phosphatidylserine lipids are translocated to the outer leaflet of cells, and are available for MP1 binding, depending on the presence of liquid-ordered domains. Here, we investigated the effect of PS on MP1 activity when this lipid is reconstituted in membranes of giant or large liposomes with different lipid-phase states...
March 6, 2017: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/28255696/reconstitution-of-tcr-signaling-using-supported-lipid-bilayers
#11
Xiaolei Su, Jonathon A Ditlev, Michael K Rosen, Ronald D Vale
Biochemical reconstitution has served as an important tool for understanding the mechanisms of many cellular processes including DNA replication, transcription, translation, vesicle trafficking, and ubiquitin-mediated proteolysis. Here, we demonstrate that biochemical reconstitution can be applied to studying a complex signaling pathway involving as many as 12 proteins or protein complexes acting at the surface of model membranes. We show that a temporal sequence of events in activated T cells beginning with phosphorylation of the T cell receptor and culminating in the activation of actin polymerization can be replicated in vitro...
2017: Methods in Molecular Biology
https://www.readbyqxmd.com/read/28253967/in-vitro-reconstitution-of-atg8-conjugation-and-deconjugation
#12
D Fracchiolla, B Zens, S Martens
Macroautophagy, hereafter autophagy, is a major degradation pathway in eukaryotic systems that allows the removal of large intracellular structures such as entire organelles or protein aggregates, thus contributing to the homeostasis of cells and tissues. Autophagy entails the de novo formation of an organelle termed autophagosome, where a cup-shaped structure called isolation membrane nucleates in proximity of a cytoplasmic cargo material. Upon elongation and closure of isolation membranes, the mature autophagosome delivers the sequestered cargo into the lysosomal system for degradation...
2017: Methods in Enzymology
https://www.readbyqxmd.com/read/28202656/durable-vesicles-for-reconstitution-of-membrane-proteins-in-biotechnology
#13
REVIEW
Paul A Beales, Sanobar Khan, Stephen P Muench, Lars J C Jeuken
The application of membrane proteins in biotechnology requires robust, durable reconstitution systems that enhance their stability and support their functionality in a range of working environments. Vesicular architectures are highly desirable to provide the compartmentalisation to utilise the functional transmembrane transport and signalling properties of membrane proteins. Proteoliposomes provide a native-like membrane environment to support membrane protein function, but can lack the required chemical and physical stability...
February 8, 2017: Biochemical Society Transactions
https://www.readbyqxmd.com/read/27818274/phospholipids-and-calmodulin-modulate-the-inhibition-of-pmca-activity-by-tau
#14
María Berrocal, Isaac Corbacho, M Rosario Sepulveda, Carlos Gutierrez-Merino, Ana M Mata
The disruption of Ca(2+) signaling in neurons, together with a failure to keep optimal intracellular Ca(2+) concentrations, have been proposed as significant factors for neuronal dysfunction in the Ca(2+) hypothesis of Alzheimer's disease (AD). Tau is a protein that plays an essential role in axonal transport and can form abnormal structures such as neurofibrillary tangles that constitute one of the hallmarks of AD. We have recently shown that plasma membrane Ca(2+)-ATPase (PMCA), a key enzyme in the maintenance of optimal cytosolic Ca(2+) levels in cells, is inhibited by tau in membrane vesicles...
November 3, 2016: Biochimica et Biophysica Acta
https://www.readbyqxmd.com/read/27815070/direct-detection-of-serca-calcium-transport-and-small-molecule-inhibition-in-giant-unilamellar-vesicles
#15
Tengfei Bian, Joseph M Autry, Denise Casemore, Ji Li, David D Thomas, Gaohong He, Chengguo Xing
We have developed a charge-mediated fusion method to reconstitute the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) in giant unilamellar vesicles (GUV). Intracellular Ca(2+) transport by SERCA controls key processes in human cells such as proliferation, signaling, and contraction. Small-molecule effectors of SERCA are urgently needed as therapeutics for Ca(2+) dysregulation in human diseases including cancer, diabetes, and heart failure. Here we report the development of a method for efficiently reconstituting SERCA in GUV, and we describe a streamlined protocol based on optimized parameters (e...
December 9, 2016: Biochemical and Biophysical Research Communications
https://www.readbyqxmd.com/read/27803325/transport-efficiency-of-membrane-anchored-kinesin-1-motors-depends-on-motor-density-and-diffusivity
#16
Rahul Grover, Janine Fischer, Friedrich W Schwarz, Wilhelm J Walter, Petra Schwille, Stefan Diez
In eukaryotic cells, membranous vesicles and organelles are transported by ensembles of motor proteins. These motors, such as kinesin-1, have been well characterized in vitro as single molecules or as ensembles rigidly attached to nonbiological substrates. However, the collective transport by membrane-anchored motors, that is, motors attached to a fluid lipid bilayer, is poorly understood. Here, we investigate the influence of motors' anchorage to a lipid bilayer on the collective transport characteristics...
November 15, 2016: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/27711229/cell-free-phospholipid-biosynthesis-by-gene-encoded-enzymes-reconstituted-in-liposomes
#17
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
https://www.readbyqxmd.com/read/27693515/how-cellular-membrane-properties-are-affected-by-the-actin-cytoskeleton
#18
REVIEW
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...
November 2016: Biochimie
https://www.readbyqxmd.com/read/27554415/cell-sized-asymmetric-lipid-vesicles-facilitate-the-investigation-of-asymmetric-membranes
#19
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
https://www.readbyqxmd.com/read/27550794/asymmetry-in-inward-and-outward-affinity-constant-of-transport-explain-unidirectional-lysine-flux-in-saccharomyces-cerevisiae
#20
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
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