Bibliografie tematiche / Giant unilamellar vesicle (GUVs)

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Letteratura scientifica selezionata sul tema "Giant unilamellar vesicle (GUVs)"

Autore: Grafiati

Pubblicato: 25 gennaio 2025

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Articoli di riviste sul tema "Giant unilamellar vesicle (GUVs)"

Litschel, Thomas, e Petra Schwille. "Protein Reconstitution Inside Giant Unilamellar Vesicles". Annual Review of Biophysics 50, n. 1 (6 maggio 2021): 525–48. http://dx.doi.org/10.1146/annurev-biophys-100620-114132.

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Giant unilamellar vesicles (GUVs) have gained great popularity as mimicries for cellular membranes. As their sizes are comfortably above the optical resolution limit, and their lipid composition is easily controlled, they are ideal for quantitative light microscopic investigation of dynamic processes in and on membranes. However, reconstitution of functional proteins into the lumen or the GUV membrane itself has proven technically challenging. In recent years, a selection of techniques has been introduced that tremendously improve GUV-assay development and enable the precise investigation of protein–membrane interactions under well-controlled conditions. Moreover, due to these methodological advances, GUVs are considered important candidates as protocells in bottom-up synthetic biology. In this review, we discuss the state of the art of the most important vesicle production and protein encapsulation methods and highlight some key protein systems whose functional reconstitution has advanced the field.

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Wu, Hao, Zhong-Can Ou-Yang e Rudolf Podgornik. "A Note on Vestigial Osmotic Pressure". Membranes 13, n. 3 (14 marzo 2023): 332. http://dx.doi.org/10.3390/membranes13030332.

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Recent experiments have indicated that at least a part of the osmotic pressure across the giant unilamellar vesicle (GUV) membrane was balanced by the rapid formation of the monodisperse daughter vesicles inside the GUVs through an endocytosis-like process. Therefore, we investigated a possible osmotic role played by these daughter vesicles for the maintenance of osmotic regulation in the GUVs and, by extension, in living cells. We highlighted a mechanism whereby the daughter vesicles acted as osmotically active solutes (osmoticants), contributing an extra vestigial osmotic pressure component across the membrane of the parent vesicle, and we showed that the consequences were consistent with experimental observations. Our results highlight the significance of osmotic regulation in cellular processes, such as fission/fusion, endocytosis, and exocytosis.

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Mardešić, Ivan, Zvonimir Boban e Marija Raguz. "Electroformation of Giant Unilamellar Vesicles from Damp Lipid Films with a Focus on Vesicles with High Cholesterol Content". Membranes 14, n. 4 (27 marzo 2024): 79. http://dx.doi.org/10.3390/membranes14040079.

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Giant unilamellar vesicles (GUVs) are membrane models used to study membrane properties. Electroformation is one of the methods used to produce GUVs. During electroformation protocol, dry lipid film is formed. The drying of the lipid film induces the cholesterol (Chol) demixing artifact, in which Chol forms anhydrous crystals which do not participate in the formation of vesicles. This leads to a lower Chol concentration in the vesicle bilayers compared to the Chol concentration in the initial lipid solution. To address this problem, we propose a novel electroformation protocol that includes rapid solvent exchange (RSE), plasma cleaning, and spin-coating methods to produce GUVs. We tested the protocol, focusing on vesicles with a high Chol content using different spin-coating durations and vesicle type deposition. Additionally, we compared the novel protocol using completely dry lipid film. The optimal spin-coating duration for vesicles created from the phosphatidylcholine/Chol mixture was 30 s. Multilamellar vesicles (MLVs), large unilamellar vesicles (LUVs) obtained by the extrusion of MLVs through 100 nm membrane pores and LUVs obtained by extrusion of previously obtained LUVs through 50 nm membrane pores, were deposited on an electrode for 1.5/1 Chol/phosphatidylcholine (POPC) lipid mixture, and the results were compared. Electroformation using all three deposited vesicle types resulted in a high GUV yield, but the deposition of LUVs obtained by the extrusion of MLVs through 100 nm membrane pores provided the most reproducible results. Using the deposition of these LUVs, we produced high yield GUVs for six different Chol concentrations (from 0% to 71.4%). Using a protocol that included dry lipid film GUVs resulted in lower yields and induced the Chol demixing artifact, proving that the lipid film should never be subjected to drying when the Chol content is high.

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Boban, Zvonimir, Ivan Mardešić, Sanja Perinović Jozić, Josipa Šumanovac, Witold Karol Subczynski e Marija Raguz. "Electroformation of Giant Unilamellar Vesicles from Damp Lipid Films Formed by Vesicle Fusion". Membranes 13, n. 3 (18 marzo 2023): 352. http://dx.doi.org/10.3390/membranes13030352.

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Abstract (sommario):

Giant unilamellar vesicles (GUVs) are artificial membrane models which are of special interest to researchers because of their similarity in size to eukaryotic cells. The most commonly used method for GUVs production is electroformation. However, the traditional electroformation protocol involves a step in which the organic solvent is completely evaporated, leaving behind a dry lipid film. This leads to artifactual demixing of cholesterol (Chol) in the form of anhydrous crystals. These crystals do not participate in the formation of the lipid bilayer, resulting in a decrease of Chol concentration in the bilayer compared to the initial lipid solution. We propose a novel electroformation protocol which addresses this issue by combining the rapid solvent exchange, plasma cleaning and spin-coating techniques to produce GUVs from damp lipid films in a fast and reproducible manner. We have tested the protocol efficiency using 1/1 phosphatidylcholine/Chol and 1/1/1 phosphatidylcholine/sphingomyelin/Chol lipid mixtures and managed to produce a GUV population of an average diameter around 40 µm, with many GUVs being larger than 100 µm. Additionally, compared to protocols that include the dry film step, the sizes and quality of vesicles determined from fluorescence microscopy images were similar or better, confirming the benefits of our protocol in that regard as well.

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Tan, Cherng-Wen Darren, Magdalena Schöller e Eva-Kathrin Ehmoser. "Rapid Multi-Well Evaluation of Assorted Materials for Hydrogel-Assisted Giant Unilamellar Vesicle Production: Empowering Bottom-Up Synthetic Biology". Gels 11, n. 1 (2 gennaio 2025): 29. https://doi.org/10.3390/gels11010029.

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Giant unilamellar vesicles (GUVs) are versatile cell models in biomedical and environmental research. Of the various GUV production methods, hydrogel-assisted GUV production is most easily implemented in a typical biological laboratory. To date, agarose, polyvinyl alcohol, cross-linked dextran-PEG, polyacrylamide, and starch hydrogels have been used to produce GUVs. Some leach and contaminate the GUVs, while others require handling toxic material or specialised chemistry, thus limiting their use by novices. Alternative hydrogel materials could address these issues or even offer novel advantages. To facilitate discovery, we replaced the manual spreading of reagents with controlled drop-casting in glass Petri dishes and polystyrene multi-well plates, allowing us to rapidly screen up to 96 GUV-production formulations simultaneously. Exploiting this, we rapidly evaluated assorted biomedical hydrogels, including PEG-DA, cross-linked hyaluronic acid, Matrigel, and cross-linked DNA. All of these alternatives successfully produced GUVs. In the process, we also developed a treatment for recycling agarose and polyvinyl alcohol hydrogels for GUV production, and successfully encapsulated porcine liver esterase (PLE-GUVs). PLE-GUVs offer a novel method of GUV labelling and tracing, which emulates the calcein-AM staining behaviour of cells. Our results highlight the utility of our protocol for potentiating substrate material discovery, as well as protocol and product development.

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Sirbu, Dumitru, Lingli Zeng, Paul G. Waddell, Nikolai V. Tkachenko, Stanley W. Botchway e Andrew C. Benniston. "Voltage-induced fluorescence lifetime imaging of a BODIPY derivative in giant unilamellar vesicles as potential neuron membrane mimics". Chemical Communications 57, n. 94 (2021): 12631–34. http://dx.doi.org/10.1039/d1cc03756k.

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Voltage induced lifetime imaging (VILI) is described using a BODIPY dye and Giant Unilamellar Vesicles (GUVs) as neuron membrane mimics. Fluorescence lifetime modulation was shown to map to a change in voltage across the GUV.

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Boban, Zvonimir, Ivan Mardešić, Witold Karol Subczynski, Dražan Jozić e Marija Raguz. "Optimization of Giant Unilamellar Vesicle Electroformation for Phosphatidylcholine/Sphingomyelin/Cholesterol Ternary Mixtures". Membranes 12, n. 5 (16 maggio 2022): 525. http://dx.doi.org/10.3390/membranes12050525.

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Artificial vesicles are important tools in membrane research because they enable studying membrane properties in controlled conditions. Giant unilamellar vesicles (GUVs) are specially interesting due to their similarity in size to eukaryotic cells. We focus on optimization of GUV production from phosphatidylcholine/sphingomyelin/cholesterol mixtures using the electroformation method. This mixture has been extensively researched lately due to its relevance for the formation of lipid rafts. We measured the effect of voltage, frequency, lipid film thickness, and cholesterol (Chol) concentration on electroformation successfulness using spin-coating for reproducible lipid film deposition. Special attention is given to the effect of Chol concentrations above the phospholipid bilayer saturation threshold. Such high concentrations are of interest to groups studying the role of Chol in the fiber cell plasma membranes of the eye lens or development of atherosclerosis. Utilizing atomic force and fluorescence microscopy, we found the optimal lipid film thickness to be around 30 nm, and the best frequency–voltage combinations in the range of 2–6 V and 10–100 Hz. Increasing the Chol content, we observed a decrease in GUV yield and size. However, the effect was much less pronounced when the optimal lipid film thickness was used. The results underline the need for simultaneous optimization of both electrical parameters and thickness in order to produce high-quality GUVs for experimental research.

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Yandrapalli, Naresh, Tina Seemann e Tom Robinson. "On-Chip Inverted Emulsion Method for Fast Giant Vesicle Production, Handling, and Analysis". Micromachines 11, n. 3 (10 marzo 2020): 285. http://dx.doi.org/10.3390/mi11030285.

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Abstract (sommario):

Liposomes and giant unilamellar vesicles (GUVs) in particular are excellent compartments for constructing artificial cells. Traditionally, their use requires bench-top vesicle growth, followed by experimentation under a microscope. Such steps are time-consuming and can lead to loss of vesicles when they are transferred to an observation chamber. To overcome these issues, we present an integrated microfluidic chip which combines GUV formation, trapping, and multiple separate experiments in the same device. First, we optimized the buffer conditions to maximize both the yield and the subsequent trapping of the vesicles in micro-posts. Captured GUVs were monodisperse with specific size of 18 ± 4 µm in diameter. Next, we introduce a two-layer design with integrated valves which allows fast solution exchange in less than 20 s and on separate sub-populations of the trapped vesicles. We demonstrate that multiple experiments can be performed in a single chip with both membrane transport and permeabilization assays. In conclusion, we have developed a versatile all-in-one microfluidic chip with capabilities to produce and perform multiple experiments on a single batch of vesicles using low sample volumes. We expect this device will be highly advantageous for bottom-up synthetic biology where rapid encapsulation and visualization is required for enzymatic reactions.

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Billah, Md Masum, Samiron Kumar Saha, Md Mamun Or Rashid, Farzana Hossain e Masahito Yamazaki. "Effect of osmotic pressure on pore formation in lipid bilayers by the antimicrobial peptide magainin 2". Physical Chemistry Chemical Physics 24, n. 11 (2022): 6716–31. http://dx.doi.org/10.1039/d1cp05764b.

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Osmotic pressure (Π) induces membrane tension σ in giant unilamellar vesicles (GUVs), which elevates rate constant kp for antimicrobial peptide magainin 2-induced pore formation in GUVs. The process of its pore evolution in GUVs with σ was revealed.

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Jennings, Christopher S., Jeremy S. Rossman, Braeden A. Hourihan, Ross J. Marshall, Ross S. Forgan e Barry A. Blight. "Immobilising giant unilamellar vesicles with zirconium metal–organic framework anchors". Soft Matter 17, n. 8 (2021): 2024–27. http://dx.doi.org/10.1039/d0sm02188a.

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A straightforward procedure for immobilising giant unilamellar vesicles (GUVs) using zircomium metal-organic frameworks as the anchroing medium is presented. Using this method GUVs can be immoblised and visualised for hours.

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Più fonti

Tesi sul tema "Giant unilamellar vesicle (GUVs)"

Karamdad, Kaiser. "Development of microfluidic platforms to construct giant unilamellar vesicles (GUVs) for the biophysical study of lipid membranes". Thesis, Imperial College London, 2016. http://hdl.handle.net/10044/1/56915.

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This thesis presents the design, development and application of several platforms through which to generate giant vesicles for biophysical and mechanical membrane studies. There has been a growing focus on manufacturing model membrane systems with improved throughput and structural properties in recent years. GUVs are a popular model membrane system for studying lipid membrane-associated phenomena due to their inherent similarity to biological cells. Traditional methods to construct vesicles offer little control over nuanced membrane properties such as asymmetry and patterning, which has paved the way for more refined techniques to be developed. This thesis details the development of a microfluidic platform technology that addresses this chasm in sophisticated GUV fabrication strategies. The technique presented offers control over key structural features such as vesicle size dispersity, internal content, membrane composition and asymmetry. Vesicles were investigated using contour detection and fluctuation analysis in order to quantify the bending rigidity in membranes constructed by microfluidics for the very first time. Furthermore, the emulsion phase transfer (EPT) method was refined for the construction of GUVs with phase separated membranes across three of compositions. This is the first investigation concerning domain formation in membranes constructed from emulsion precursors at a range of a compositions. The progress made in advancing platform technologies opens up various avenues through which to further explore biophysical phenomena such as a lipid flip-flop dynamics, as well as for the high-throughput generation of artificial cell systems, with potential relevance for therapeutic applications such as smart drug delivery.

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Aimon, Sophie. "Study of a voltage-gated ion channel reconstituted in Giant Unilamellar Vesicles". Phd thesis, Université Pierre et Marie Curie - Paris VI, 2011. http://tel.archives-ouvertes.fr/tel-00736743.

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Il est difficile d'étudier in vivo le rôle de la membrane dans l'excitabilité des cellules car les paramètres pertinents (composition et état mécanique de la membrane, densité de canaux...) sont activement régulés par la cellule elle-même et donc difficilement ajustables expérimentalement. J'ai donc développé une méthode pour reconstituer un canal voltage-dépendant dans une membrane où ces paramètres peuvent être contrôlés. Pour cela j'ai exprimé, purifié et marqué KvAP, un canal potassique voltage-dépendant. J'ai ensuite adapté une méthode existante pour le reconstituer dans des Vésicules Unilamellaires Géantes (GUVs). J'ai mesuré la densité des canaux dans les GUVs grâce à la microscopie confocale. Des expériences d'électrophysiologie ont, de plus, montré que le canal reste fonctionnel après reconstitution. Ce système m'a permis d'étudier tout d'abord l'affinité du canal pour les membranes courbées. Pour cela, j'ai tiré des nanotubes de rayon contrôlé à partir de ces GUVs et j'ai mesuré la distribution du canal entre la vésicule et le tube par microscopie confocale. J'ai montré que le canal est enrichi dans le tube proportionnellement à sa courbure. Ce résultat est en accord avec une théorie basée sur l'élasticité de la membrane. Nous avons également étudié l'effet du confinement de la membrane sur la diffusion de KvAP. Par des expériences de suivi de particule unique, nous avons montré que le coefficient de diffusion le long du tube diminue d'un facteur 3 lorsque le rayon du tube décroît de 250 à 10 nm. Ce résultat est en accord avec le modèle hydrodynamique de Saffman et Delbrück appliqué à la géométrie cylindrique.

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Checchia, Robert Garcia. "Estudo de propriedades biofísicas de membrana sob estresse oxidativo e a interação com proteínas formadoras de poros". Universidade de São Paulo, 2019. http://www.teses.usp.br/teses/disponiveis/43/43134/tde-21032019-171115/.

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Neste trabalho investigamos efeitos de fotoirradiação e toxinas sob membranas celulares miméticas. Foram utilizadas, como modelo de membranas lipídicas, vesiculas unilamelares gigantes (GUVs) compostas pro lipídeos oxidados e não oxidados observadas por microscopia ótica de contraste de fase. Inicialmente estudamos a foto-resposta de membranas compostas por POPC e POPG dispersas em solução contendo azul de metileno (MB). Na sequência, estudamos o efeito de toxinas formadoras de poros, Esticolisina I (ST I) e Esticolisina II (ST II), em membranas contendo lipídeos oxidados e não oxidados. Os resultados de MB (10 µM) disperso em solução de membranas compostas por POPC e o lipídeo aniônico POPG indicaram que o aumento da densidade de carga negativa nas membranas das GUVs, que favorece a ligação da moléculas positivamente carregadas como MB nas membranas, tem como consequência um aumento de permeabilidade da membrana muito mais rápído em relação a membranas compostas apenas por POPC. Isto se deve ao fato que a localização preferencial do MB na membrana de POPC:POPG favorece a formação de oxigênio singlete próximo a dupla ligação da cadeia alquílica, dando início a reação de peroxidação lipídica de maneira mais efetiva que em membrana de POPC. Os resultados da ação das toxinas STI e STII (21 nM) em GUVs contendo lipídeos não oxidados PC e esfingomielina evidenciam que apenas STII é capaz de permear estas membranas a esta concentração. Mais ainda, nossos resultados sugerem que a existência de separação de fases fluida-gel na bicamada lipidica composta por PC:SM (razão molar 1:1) favorece a ação da toxina StII. Ao analisarmos membranas contendo lipídeos hidroperoxidados (POPC-OOH) dispersas em solução contendo STII (21 nM) observamos um aumento de permeabilidade na membrana num conjunto de GUVs, associado a formação de poros, apenas em bicamadas lipídicas formadas por misturas de lipídeos oxidados (POPC) e não oxidados (POPC-OOH). Quanto maior a concentração de lipídeos oxidados na membrana mais rapidamente ocorre o aumento de permeabilidade.
In this work we investigate the effects of photoirradiation and toxins on mimetic cell membranes. As a model of lipid membranes, giant unilamellar vesicles (GUVs) composed of oxidized and oxidized pro-lipids were observed by optical phase contrast microscopy. Initially we studied the photo-response of membranes composed of POPC and POPG dispersed in solution containing methylene blue (MB). Following, we studied the effect of pore-forming toxins, Sticolysin I (ST I) and Sticolysin II (ST II), on membranes containing oxidized and non-oxidized lipids. The results of MB (10 M) dispersed in solution of membranes composed of POPC and the anionic lipid POPG indicated that the increase in the negative charge density in the membranes of GUVs, which favors the binding of positively charged molecules as MB in the membranes, consequently increases membrane permeability in regard to membranes composed only of POPC. This is due to the fact that the preferred location of the MB in the POPC: POPG membrane favors the formation of singlet oxygen near the double bond of the alkyl chain, initiating the lipid peroxidation reaction more effectively than in the POPC membrane. The results of the action of the STI and STII toxins (21 nM) on GUVs containing non oxidised lipids PC and sphingomyelin show that only STII is able to permeate these membranes at this concentration. Moreover, our results suggest that the existence of fluid-gel phase separation in the lipid bilayer composed of PC:SM (molar ratio 1:1) favors the action of the StII toxin. When analyzing membranes containing hydroperoxidized lipids (POPC-OOH) dispersed in solution containing STII (21 nM) we observed an increase in membrane permeability in a set of GUVs, associated with pore formation, only in lipid bilayers formed by mixtures of oxidized lipids (POPC-OOH) and non-oxidized ones. The higher the concentration of oxidized lipids in the membrane, the faster the permeability increases.

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Equy, Eloïse. "Polymersomes Janus : conception rationnelle, préparation et fonctionnalisation asymétrique pour le développement de systèmes auto-propulsés de délivrance ciblée de médicaments". Electronic Thesis or Diss., Bordeaux, 2024. http://www.theses.fr/2024BORD0465.

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Mimer les propriétés des cellules vivantes dans des protocellules artificielles suscite un intérêt considérable, notamment pour reproduire la motilité et le mouvement directionnel dans des applications de thérapies « intelligentes ». En raison de leur morphologie vésiculaire et de leur stabilité, les polymersomes présentent un grand potentiel pour la délivrance de médicaments, et l'introduction d'une asymétrie est essentielle pour permettre leur auto-propulsion. Bien que plusieurs approches, telles que la séparation de phase au sein de la membrane, aient été utilisées pour créer des polymersomes asymétriques, le choix des polymères appropriés reste un défi. Cette thèse de doctorat vise à concevoir des polymersomes asymétriques, de type Janus, capables de s'auto-propulser grâce à la décomposition enzymatique du glucose. Nous décrivons le développement de vésicules géantes unilamellaires de type Janus (JGUVs) par séparation de phase au sein de la membrane de deux copolymères à blocs distincts composés de blocs hydrophobes chimiquement incompatibles. En utilisant la théorie de Flory-Huggins, nous démontrons que les copolymères peuvent être rationnellement sélectionnés et conçus pour s'auto-assembler en polymersomes asymétriques, avec une séparation de phase modulable selon des paramètres tels que la composition, la masse molaire et la température. Notre méthode prédictive s'est avérée efficace pour les techniques d'auto-assemblage avec et sans solvant, permettant l'élaboration de diagrammes de phase génériques corrélant l'énergie libre de mélange à la morphologie des polymersomes, fournissant ainsi des indications clés pour la conception de JGUVs. Nous montrons également que la présence de solvant lors de la formation des vésicules permet d'étendre la gamme des polymères incompatibles pouvant être utilisés. De plus, nous avons réussi à contrôler, grâce à l'extrusion, la taille des vésicules tout en préservant leur morphologie Janus et avons montré que les JGUVs ainsi obtenus pouvaient être stables pendant plusieurs mois. Enfin, nous avons fonctionnalisé asymétriquement les JGUVs avec l'enzyme glucose oxydase par chimie click, et une étude préliminaire sur leur dynamique en présence de glucose est présentée, fournissant des indications pour leur utilisation comme micromoteurs
Mimicking the properties of living cells in artificial protocells has attracted significant interest, particularly for replicating motility and directional swimming for applications in smart therapeutics. Due to their vesicular and stable morphology, polymersomes hold great promise for drug delivery, and the introduction of asymmetry is crucial to enable self-propulsion. While several approaches, such as phase separation within the membrane, have been used to create asymmetric polymersomes, the selection of appropriate polymers remains a challenge. This PhD thesis aims at designing asymmetric, Janus-like polymersomes capable of self-propulsion, and powered by enzymatic glucose decomposition. We describe the development of Janus Giant Unilamellar Vesicles (JGUVs) through phase separation within the membrane of two distinct block copolymers comprising chemically incompatible hydrophobic blocks. We demonstrate, using the Flory-Huggins theory, that copolymers can be rationally selected and designed to self-assemble into asymmetric polymersomes, with tunable phase separation driven by parameters such as composition, molecular weight, and temperature. Our predictive method proves to be effective for both solvent-free and solvent-switch self-assembly processes, enabling the elaboration of generic phase diagrams correlating mixing free energy with polymersome morphology, providing valuable insights for JGUVs design. We also evidence that the presence of solvent during the vesicle formation broadens the range of incompatible polymers that can be used. Additionally, we successfully control, thanks to extrusion, the vesicle size while preserving their Janus morphology and evidence that the resulting JGUVs could be stable for several months. Furthermore, we asymmetrically functionalized JGUVs with glucose oxidase enzymes via click-chemistry, and a preliminary study on their dynamic behavior in the presence of glucose is presented, looking forward to their potential use as micromotors

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Stöckl, Martin Thomas. "Giant vesicles". Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2009. http://dx.doi.org/10.18452/15869.

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In der vorliegenden Arbeit wird ein neuer Ansatz vorgestellt, um Lipiddomänen, die Bindungsorte peripherer und integraler Membranproteine darstellen können, zu charakterisieren. Insbesondere wurde die Analyse der Fluoreszenzlebenszeiten von NBD-markierten Lipidanaloga benutzt, um Lipiddomänen in Giant unilamellar vesicles (GUV) und darauf aufbauend, in der Plasmamembran von Säugerzellen zu untersuchen. Das typische Zeitfenster von Fluoreszenzlebenszeiten im Bereich von Nanosekunden ermöglicht es, auch sehr kurzlebige Lipiddomänen nachzuweisen. Mit Hilfe des Fluorescence lifetime imaging (FLIM) wurden für die liquid disordered (ld) und liquid ordered (lo) Domänen in GUV jeweils spezifische Werte für das Abklingen der Fluoreszenz gemessen. Sogar die Existenz von submikroskopischen Domänen in GUV konnte nachgewiesen werden. Die Fluoreszenzlebenszeit des Lipidanalogs C6-NBD-PC zeigte in der Plasmamembran von Säugerzellen eine breite Verteilung. Dies legt in Übereinstimmung mit FLIM-Experimenten an aus der Plasmamembran von HeLa-Zellen gewonnenen Giant vesicles nahe, dass in der Plasmamembran von Zellen eine Vielzahl verschiedener submikroskopischer Lipiddomänen existiert. Darauf aufbauend wurde die Fluoreszenzmikroskopie an GUV angewendet, um die Bindung von fluoreszenzmarkiertem alpha-Synuclein an mittels FLIM charakterisierte Lipiddomänen zu untersuchen. Die Experimente zeigten, dass das Protein mit hoher Affinität an negativ geladene Phospholipide unter der Vorraussetzung bindet, dass diese sich in ld Domänen befinden. Im Gegensatz dazu erfolgt keine Bindung wenn diese Lipide in lo Domänen lokalisiert sind. Im Vergleich zum wildtypischen alpha-Synuclein zeigte die Variante A30P eine geringere Affinität zur Membran, während die E46K-Variante eine stärkere Bindung zeigte. Dies deutet darauf hin, dass bei den erblichen Formen des Morbus Parkinson eine veränderte Assoziation des alpha-Synucleins mit der Membran eine Rolle spielen kann.
In the present study a novel approach to characterize lipid domains, which may provide binding sites for peripheral or integral membrane proteins, is demonstrated. In particular, analysis of fluorescence lifetimes of NBD-labeled lipid analogues was used to study lipid domains in Giant unilamellar vesicles (GUV) and – based on the GUV results – in the plasma membrane of mammalian cells. As fluorescence decays in a few nanoseconds it is possible to to detect also very short-lived lipid domains. Fluorescence Lifetime Imaging (FLIM) revealed that the fluorescence decay of NBD-lipid analogues showed domain dependent decay times in the liquid disordered (ld) and the liquid ordered (lo) phase of GUV. Even the existence of submicroscopic domains in lipid membranes could be detected by FLIM. A broad distribution of the fluorescence lifetime was found for C6-NBD-PC inserted in the plasma membrane of mammalian cells. In agreement with FLIM studies on lipid domain forming Giant vesicles derived from the plasma membrane of HeLa-cells this may suggest that a variety of submicroscopic lipid domains exists in the plasma membrane of intact mammalian cells. Based on that, fluorescence microscopy was used on GUV to study the binding of fluorescently labeled alpha-synuclein at lipid domains previously characterized by FLIM. The experiments suggested that alpha-synuclein binds with high affinity to negatively charged phospholipids, when they are embedded in a ld as opposed to a lo environment. When compared with wildtype alpha-synuclein, the disease-causing alpha-synuclein variant A30P bound less efficiently to anionic phospholipids, while the variant E46K showed enhanced binding. This suggests that an altered association of alpha-synuclein with membranes may play a role in the inherited forms of Parkinson’s disease.

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Nikolaus, Jörg. "Hemifusion and lateral lipid domain partition in lipid membranes of different complexity". Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2011. http://dx.doi.org/10.18452/16437.

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Die Fusion von Membranen erfordert die Verschmelzung von zwei Phospholipiddoppel-schichten, wobei dies über dieselben Zwischenschritte abzulaufen scheint. Eine lokale Störung (‚Stalk’) stellt eine erste Verbindung der äußeren Membranhälften dar, die anschließend lateral expandiert und ein Hemifusionsdiaphragma (HD) bildet. Das Öffnen einer Fusionspore im HD führt zur vollständigen Fusion. Mittels konfokaler Mikroskopie wurde die Fusion von Giant unilamellar vesicles (GUVs) mit negativ geladenen Lipiden und transmembranen (TM) Peptiden in Anwesenheit von zweiwertigen Kationen beobachtet, wobei die Peptide bei der HD Entstehung völlig verdrängt wurden. Eine detaillierte Analyse zeigte, dass es sich bei diesem Mikrometer-großen Bereich um ein HD handelt, dessen Größe von der Lipidzusammensetzung und Peptidkonzentration in den GUVs abhängt. Laterale Lipiddomänen gelten als entscheidend für Signal- und Sortierungsprozesse in der Zelle. Liquid ordered (Lo) Domänen in Modellsystemen wie GUVs ähneln den mit Sphingo-lipiden und Cholesterol angereicherten biologischen Raft-Domänen, allerdings scheinen Membraneigenschaften wie die Lipidpackung sich von biologischen Membranen zu unterscheiden. In diesem Zusammenhang wird die Sortierung des TM-verankerten Hemag-glutinin (HA) des Influenzavirus und von lipidverankerten Ras-Proteinen in GUVs wie auch in abgelösten Plasmamembran-Ausstülpungen (GPMVs) untersucht. HA Protein und TM-Pepitde von HA wurden ausschließlich (GUVs) bzw. vorwiegend (GPMVs) in der liquid disordered (Ld) Domäne gefunden. K-Ras wurde inmitten der Ld detektiert, während N-Ras zur Lo/Ld Grenzlinie diffundierte. Diese Ergebnisse werden im Zusammenhang mit den Unterschieden der Lipidpackung innerhalb der verschiedenen membranverankerten Systeme diskutiert. Es ist wahrscheinlich, dass die Bildung, Größe und Stabilität sowie die physikalischen Eigenschaften der Lipiddomänen in biologischen Membranen stark von Protein-Lipid-Wechsel-wirkungen beeinflusst werden.
Membrane fusion is ubiquitous in life and requires remodelling of two phospholipid bilayers. Fusion likely proceeds through similar sequential intermediates. A stalk between the contacting leaflets forms and radially expands into a hemifusion diaphragm (HD) wherein finally a fusion pore opens up. Direct experimental verification of this key structure is difficult due to its transient nature. Confocal microscopy was used to visualize the fusion of giant unilamellar vesicles (GUVs) comprising negatively charged phosphatidylserine and fluorescent transmembrane (TM) entities in the presence of divalent cations. A complete displacement of TM peptides preceded full fusion. This is consistent with HD formation. Detailed analysis provided proof that the micrometer sized structures are in fact HDs. HD size is dependent on lipid composition and peptide concentration. Lateral lipid domain formation is believed to be essential for sorting and signalling processes in the cell. Liquid ordered (Lo) domains in model systems like GUVs resemble biological rafts enriched in sphingolipids and cholesterol, but their physical properties seem distinct from biological membranes as judged by e.g. lipid order and packing. In this context the sorting of TM anchored influenza virus hemagglutinin (HA) and different lipid anchored Ras proteins is studied in GUVs and giant plasma membrane derived vesicles (GPMVs). Authentic HA or the TM domain peptides were sorted exclusively (GUVs) or predominantly (GPMVs) to the liquid disordered (Ld) domains. Whereas K-Ras was found in the bulk Ld domains, N-Ras diffuses to the Lo/Ld interface. These results are discussed with respect to differences in lipid packing in the different membrane systems and regarding the membrane anchors and their hydrophobic matching. The results suggest that the formation, size and stability as well as the physical properties of lipid domains in biological membranes are tightly regulated by protein-lipid interactions.

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Ma, Yaning. "Characterization of Membrane Permeability and Polymer-Stabilized Model Membranes". Thesis, The University of Arizona, 2007. http://hdl.handle.net/10150/193347.

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The permeability of lipid bilayer membranes to glucose and carboxyfluorescein has been studied in model membranes. Using an enzyme assay, the permeability of glucose was monitored spectrometrically with both large and giant unilamellar vesicles (LUVs and GUVs). The permeability of carboxyfluorescein was studied by entrapping the dye and monitoring its leakage over time from a single GUV. Permeability study using GUVs may provide new information that cannot be obtained from LUVs.The stability of lipid membranes was enhanced by incorporating polymer scaffold. LUVs were prepared with hydrophobic monomers partitioned and then polymerized inside the hydrophobic interior of the lipid bilayers. The sizes of the formed polymers were characterized using gel permeation chromatography and mass spectrometry. This study suggests that large molecular weight polymers were formed inside the lipid bilayers and that the stability of the membranes is related to the size of the polymers.

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Ho, Chian Sing. "Inquiry of Lipid Membranes Interacting with Functional Peptides and Polyphenol Drug Molecules". Scholar Commons, 2016. http://scholarcommons.usf.edu/etd/6255.

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Cellular membranes are important targets for many membrane-active peptides and drug compounds. Here we are interested in deciphering how lipid membranes are perturbed by several membrane-active molecules, including the transmembrane domain of the influenza M2 protein (M2TM), aggregates formed by a synthetic polyglutamine peptide, and three polyphenol compounds (i.e., tamoxifen, genistein, and verapamil). We employ phase-separated ternary lipid model membranes in the form of giant unilamellar vesicles (GUVs) to simulate raft-like structures that have been proposed to govern many important processes in plasma membranes (e.g., intracellular singling and trafficking). Specifically, we use fluorescent microscopy to interrogate how those membrane additives modulate the phase behavior of free-standing GUVs, as well as the miscibility transition temperature (Tm). We find that M2TM increases Tm and causes vesicle budding; polyglutamine aggregates disrupt lipid membranes; and the three polyphenol compounds exert disparate effects on GUV Tm.

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GRISANTI, GIULIA. "Microfluidic systems for artificial biological barriers". Doctoral thesis, 2022. http://hdl.handle.net/11573/1637919.

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The concept of biological barriers, albeit intuitive, implies a certain extent of complexity, related to the diversity of their structure, organisation and size throughout the living organisms. For this reason, reliable experimental models are necessary to reproduce and characterise these biological structures in a relatively simple and isolated environment. In this PhD project, microfluidics has been employed for the realisation of experimental models resembling two different biological barriers: the endothelial barrier and the cell plasma membrane. In the first case, endothelial cells (ECs) were grown in the microchannels of a microfluidic device under physiological levels of shear stress, obtained by perfusing the system with a continuous constant flow. This allowed cells to form a mature physiological-like barrier, whose integrity is guaranteed by the formation of adherens junctions, containing the adhesion protein vascular endothelial (VE )-cadherin. This model was exploited for enhanced drug delivery studies, with the aim of increasing endothelial permeability through microbubble-enhanced ultrasound-induced (USMB) cavitation. Particular attention was paid to characterise the dynamics of permeabilisation events, with the employment of genetically engineered ECs expressing constitutively fluorescent VE-cadherin. Another microfluidic platform was developed and realised for the high-throughput and reproducible production of giant unilamellar vesicles (GUVs), a vesicular model of phospholipid bilayer that reproduces some of the features of cell plasma membrane. The produced GUVs were exploited for the measurement of important biomechanical parameters, such as the bending rigidity modulus, playing a role in cell membrane response to mechanical stimuli, like cavitation. The geometry of the device was designed to produce GUVs through the double emulsion method, including in the protocol the removal of the oil phase residues from the bilayer, to prevent them from affecting the bilayer biomechanical properties. The collected GUVs are intended for biomechanical studies through thermal fluctuation analysis.

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Chiu, Ping-Chen, e 邱平晨. "Dynamics of transient pores on single giant unilamellar vesicle probed with laser confocal fluorescence detection". Thesis, 2019. http://ndltd.ncl.edu.tw/handle/rn4t86.

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碩士
國立交通大學
應用化學系碩博士班
108
Transmembrane transport is not only an important subject in biophysics but also intimately related to varied practical applications such as drug delivery, gene therapy, et al. While a large body of recent studies have shown that transient pores can form on lipid membranes either spontaneously or subject to stress, our understanding of transient pores remains elusive mainly due to the difficulty to characterize their fundamental properties. Here we report a novel experimental approach to determine the opening time and the dimension of transient pores formed spontaneously on giant unilamellar vesicles (GUV). GUV made of phospholipids (POPC) were immersed in a solution of fluorescent probes (carboxyfluorescein), and a sensitive lock-in amplified confocal detection scheme was employed to measure the fluorescence change at the center of the GUV that arises from the leakage of fluorescent probes through a transient pore. Notably, the temporal trace of the fluorescence intensity comprised numerous intermittent fluorescent bursts, and each of which generally has a rapid rise (~0.2 s) and a slow decay (a few seconds). The rapid rise of individual fluorescent burst was attributed to the influx of fluorescent probes through a transient pore, and the slow decay to the subsequent photo bleaching of fluorescent probes that were enclosed in the GUV, respectively. We then adopted Berg and Purcell’s approach to analyze the temporal profile of these bursts, and found that these transient pores generally have an opening time about 0.27 s and have two prominent dimensions (radius = 12.8 nm and 34.4 nm) respectively. Further studies show that transient pores found on GUV comprising cholesterol (3 mol% and 10 mol%) exhibit comparable characteristics. Nevertheless, the edge tension of small pores on GUV comprising cholesterol seems slightly larger relative to that of small pores on POPC GUV (~1 pN vs. 0.85 pN). Our approach makes possible the determination of the essential characteristics of transient pores formed spontaneously on lipid membranes, and should open a new route to study fundamental aspects of transient pores formed either spontaneously or by external stress.

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Libri sul tema "Giant unilamellar vesicle (GUVs)"

Franzin, Carla Maria Mirella. Solid state deuterium nuclear magnetic resonance detection of transmembrane-potential-driven tetraphenylphosphonium redistribution across giant unilamellar vesicle bilayers. Ottawa: National Library of Canada, 1995.

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Capitoli di libri sul tema "Giant unilamellar vesicle (GUVs)"

Rols, Marie-Pierre. "Molecular Transmembrane Transport with Giant Unilamellar Vesicles (GUVs)". In Handbook of Electroporation, 1–17. Cham: Springer International Publishing, 2016. http://dx.doi.org/10.1007/978-3-319-26779-1_84-1.

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Rols, Marie-Pierre. "Molecular Transmembrane Transport with Giant Unilamellar Vesicles (GUVs)". In Handbook of Electroporation, 95–111. Cham: Springer International Publishing, 2017. http://dx.doi.org/10.1007/978-3-319-32886-7_84.

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Sengupta, Kheya, e Ana-Sunčana Smith. "Measuring giant unilamellar vesicle adhesion". In The Giant Vesicle Book, 381–99. Boca Raton, FL : CRC Press, Taylor & Francis Group, [2020]: CRC Press, 2019. http://dx.doi.org/10.1201/9781315152516-17.

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Härtel, Tobias, e Petra Schwille. "Giant unilamellar vesicles with cytoskeleton". In The Giant Vesicle Book, 53–70. Boca Raton, FL : CRC Press, Taylor & Francis Group, [2020]: CRC Press, 2019. http://dx.doi.org/10.1201/9781315152516-4.

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Dimova, Rumiana, Pasquale Stano, Carlos M. Marques e Peter Walde. "Preparation methods for giant unilamellar vesicles". In The Giant Vesicle Book, 3–20. Boca Raton, FL : CRC Press, Taylor & Francis Group, [2020]: CRC Press, 2019. http://dx.doi.org/10.1201/9781315152516-1.

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Janshoff, Andreas. "Atomic force microscopy of giant unilamellar vesicles". In The Giant Vesicle Book, 305–17. Boca Raton, FL : CRC Press, Taylor & Francis Group, [2020]: CRC Press, 2019. http://dx.doi.org/10.1201/9781315152516-12.

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Prévost, Coline, Mijo Simunovic e Patricia Bassereau. "Creating membrane nanotubes from giant unilamellar vesicles". In The Giant Vesicle Book, 365–79. Boca Raton, FL : CRC Press, Taylor & Francis Group, [2020]: CRC Press, 2019. http://dx.doi.org/10.1201/9781315152516-16.

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Ugarte-Uribe, Begoña, Kushal Kumar Das e Ana J. García-Sáez. "Lipid and protein mobility in giant unilamellar vesicles". In The Giant Vesicle Book, 455–71. Boca Raton, FL : CRC Press, Taylor & Francis Group, [2020]: CRC Press, 2019. http://dx.doi.org/10.1201/9781315152516-21.

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Bagatolli, Luis A. "Application of optical microscopy techniques on giant unilamellar vesicles". In The Giant Vesicle Book, 265–81. Boca Raton, FL : CRC Press, Taylor & Francis Group, [2020]: CRC Press, 2019. http://dx.doi.org/10.1201/9781315152516-10.

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Blosser, Matthew C., Caitlin E. Cornell, Scott P. Rayermann e Sarah L. Keller. "Phase diagrams and tie lines in giant unilamellar vesicles". In The Giant Vesicle Book, 401–16. Boca Raton, FL : CRC Press, Taylor & Francis Group, [2020]: CRC Press, 2019. http://dx.doi.org/10.1201/9781315152516-18.

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Atti di convegni sul tema "Giant unilamellar vesicle (GUVs)"

Jiang, Yanfei, Guy M. Genin, Srikanth Singamaneni e Elliot L. Elson. "Interfacial Phases on Giant Unilamellar Vesicles". In ASME 2012 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2012. http://dx.doi.org/10.1115/sbc2012-80942.

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Lipid nanodomains in cell membranes are believed to play a significant role in a number of critical cellular processes (Elson, et al., 2010). These include, for example, replication processes in enveloped viruses such as bird flu and HIV and signaling mechanisms underlying pathological conditions such as cancer. Due to the potential for developing new disease treatments through the control of these membrane rafts, the biophysics underlying their formation has been the subject of intense study, much of this focused on domain formation in giant unilamellar lipid vesicles (GUVs), a simplified model system.

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Rong, Xi, Kenneth M. Pryse, Jordan A. Whisler, Yanfei Jiang, William B. McConnaughey, Artem Melnykov, Guy M. Genin e Elliot L. Elson. "Confidence Intervals for Estimation of the Concentration and Brightness of Multiple Diffusing Species". In ASME 2012 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2012. http://dx.doi.org/10.1115/sbc2012-80921.

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Lipid nanodomains in cell membranes are believed to play a significant role in replication of enveloped viruses such as bird flu and HIV and signaling mechanisms underlying pathological conditions such as cancer. However, the forces that govern the formation and availability of these “membrane rafts” are uncertain, and even their existence is questioned. The central challenge is that no suitable imaging modalities exist (Elson, et al., 2010). We are developing tools to characterize and visualize dynamics of lipid nanodomains on idealized systems called giant unilamellar vesicles (GUVs) using fluorescence correlation spectroscopy (FCS) (Figure 1).

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Saha, Shovon, Md Saif Ishtiaque, Md Imran Hossain e Md Khorshed Alam. "Simulation and Numerical Scheme of Molecular Transport Due to Dielectrophoresis and Electroporation in a Giant Unilamellar Vesicle". In 2023 International Conference on Electrical, Computer and Communication Engineering (ECCE). IEEE, 2023. http://dx.doi.org/10.1109/ecce57851.2023.10101507.

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Saha, Shovon, Md Saif Ishtiaque, Mohammad Abu Sayem Karal e Md Khorshed Alam. "Simulation and Intelligent Data Mining of Molecular Transport Through Multiple Nanopores in an Electroporated Giant Unilamellar Vesicle". In 2023 International Conference on Information and Communication Technology for Sustainable Development (ICICT4SD). IEEE, 2023. http://dx.doi.org/10.1109/icict4sd59951.2023.10303300.

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Tamba, Yukihiro, Takuya Yoshitani e Masahito Yamazaki. "Single Giant Unilamellar Vesicle Method Reveals Effect of Antimicrobial Peptide, Magainin 2, and Antibacterial Substance, Tea Catechin, on Membrane Permeability and Membrane Structure". In 2006 IEEE International Symposium on Micro-NanoMechatronics and Human Science. IEEE, 2006. http://dx.doi.org/10.1109/mhs.2006.320282.

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