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1.
Methods Enzymol ; 700: 127-159, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38971598

RESUMO

The natural asymmetry of the lipid bilayer in biological membranes is, in part, a testament to the complexity of the structure and function of this barrier limiting and protecting cells (or organelles). These lipid bilayers consist of two lipid leaflets with different lipid compositions, resulting in unique interactions within each leaflet. These interactions, combined with interactions between the two leaflets, determine the overall behavior of the membrane. Model membranes provide the most suitable option for investigating the fundamental interactions of lipids. This report describes a comprehensive method to make asymmetric giant unilamellar vesicles (aGUVs) using the technique of hemifusion. In this method, calcium ions induce the hemifusion of giant unilamellar vesicles (GUVs) with a supported lipid bilayer (SLB), both having different lipid compositions. During hemifusion, a stalk, or a more commonly seen hemifusion diaphragm, connects the outer leaflets of GUVs and the SLB. The lateral diffusion of lipids naturally promotes the lipid exchange between the connected outer leaflets. After calcium chelation to prevent further fusion, a mechanical shear detaches aGUVs from the SLB. A fluorescence quench assay is employed to test the extent of bilayer asymmetry. A fluorescence quenching assay tests bilayer asymmetry and verifies dye and lipid migration to a GUV's outer leaflet.


Assuntos
Cálcio , Bicamadas Lipídicas , Lipossomas Unilamelares , Lipossomas Unilamelares/química , Bicamadas Lipídicas/química , Cálcio/química , Cálcio/metabolismo , Fusão de Membrana
2.
Methods Enzymol ; 700: 329-348, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38971605

RESUMO

As the primary products of lipid oxidation, lipid hydroperoxides constitute an important class of lipids generated by aerobic metabolism. However, despite several years of effort, the structure of the hydroperoxidized bilayer has not yet been observed under electron microscopy. Here we use a 200 kV Cryo-TEM to image small unilamellar vesicles (SUVs) made (i) of pure POPC or SOPC, (ii) of their pure hydroperoxidized form, and (iii) of their equimolar mixtures. We show that the challenges posed by the determination of the thickness of the hydroperoxidized bilayers under these observation conditions can be addressed by an image analysis method that we developed and describe here.


Assuntos
Microscopia Crioeletrônica , Bicamadas Lipídicas , Fosfatidilcolinas , Lipossomas Unilamelares , Bicamadas Lipídicas/química , Bicamadas Lipídicas/metabolismo , Microscopia Crioeletrônica/métodos , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismo , Fosfatidilcolinas/química , Oxirredução , Processamento de Imagem Assistida por Computador/métodos , Peróxidos Lipídicos/química , Peróxidos Lipídicos/análise
3.
Methods Enzymol ; 700: 349-383, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38971607

RESUMO

Small-angle X-ray and neutron scattering (SAXS/SANS) techniques excel in unveiling intricate details of the internal structure of lipid membranes under physiologically relevant temperature and buffer conditions, all without the need to resort to bulky labels. By concurrently conducting and analyzing neutron and X-ray data, these methods harness the complete spectrum of contrast and resolution from various components constituting lipid membranes. Despite this, the literature exhibits only a sparse presence of applications compared to other techniques in membrane biophysics. This chapter serves as a primer for conducting joint SAXS/SANS analyses on symmetric and asymmetric large unilamellar vesicles, elucidating fundamental elements of the analysis process. Specifically, we introduce the basics of interactions of X-rays and neutrons with matter that lead to the scattering contrast and a description of membrane structure in terms of scattering length density profiles. These profiles allow fitting of the experimentally observed scattering intensity. We further integrate practical insights, unveiling strategies for successful data acquisition and providing a comprehensive assessment of the technique's advantages and drawbacks. By amalgamating theoretical underpinnings with practical considerations, this chapter aims to dismantle barriers hindering the adoption of joint SAXS/SANS approaches, thereby encouraging an influx of studies in this domain.


Assuntos
Difração de Nêutrons , Espalhamento a Baixo Ângulo , Difração de Raios X , Difração de Nêutrons/métodos , Difração de Raios X/métodos , Lipídeos de Membrana/química , Lipossomas Unilamelares/química , Bicamadas Lipídicas/química
4.
J Chem Phys ; 161(1)2024 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-38949588

RESUMO

Investigating the influence of the ambient chemical environment on molecular behaviors in liposomes is crucial for understanding and manipulating cellular vitality as well as the capabilities of lipid drug carriers in various environments. Here, we designed and synthesized a second harmonic generation (SHG) and fluorescence probe molecule called Pyr-Py+-N+ (PPN), which possesses membrane-targeting capability. We employed PPN to investigate the response of lipid vesicles composed of cardiolipin to the presence of exogenous salt. The kinetic behaviors, including the adsorption and embedding of PPN on the surface of small unilamellar vesicles (SUVs) composed of cardiolipin, were analyzed. The response of the SUVs to the addition of NaCl was also monitored. A rapid decrease in vesicle size can be evidenced through the rapid drop in SHG emission originating from PPN located on the vesicle surface.


Assuntos
Cardiolipinas , Corantes Fluorescentes , Lipossomas Unilamelares , Cardiolipinas/química , Corantes Fluorescentes/química , Lipossomas Unilamelares/química , Propriedades de Superfície , Lipossomos/química , Cloreto de Sódio/química , Tensoativos/química , Estrutura Molecular
5.
Commun Biol ; 7(1): 840, 2024 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-38987288

RESUMO

The architecture of the actin cortex determines the generation and transmission of stresses, during key events from cell division to migration. However, its impact on myosin-induced cell shape changes remains unclear. Here, we reconstitute a minimal model of the actomyosin cortex with branched or linear F-actin architecture within giant unilamellar vesicles (GUVs, liposomes). Upon light activation of myosin, neither the branched nor linear F-actin architecture alone induces significant liposome shape changes. The branched F-actin network forms an integrated, membrane-bound "no-slip boundary" -like cortex that attenuates actomyosin contractility. By contrast, the linear F-actin network forms an unintegrated "slip boundary" -like cortex, where actin asters form without inducing membrane deformations. Notably, liposomes undergo significant deformations at an optimized balance of branched and linear F-actin networks. Our findings highlight the pivotal roles of branched F-actin in force transmission and linear F-actin in force generation to yield membrane shape changes.


Assuntos
Actinas , Membrana Celular , Miosinas , Actinas/metabolismo , Membrana Celular/metabolismo , Miosinas/metabolismo , Forma Celular , Animais , Actomiosina/metabolismo , Lipossomas Unilamelares/metabolismo , Lipossomas Unilamelares/química , Biomimética , Lipossomos/metabolismo , Lipossomos/química , Modelos Biológicos , Citoesqueleto de Actina/metabolismo
6.
Methods Mol Biol ; 2839: 77-97, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39008249

RESUMO

Transmembrane transition metal transporter proteins are central gatekeepers in selectively controlling vectorial metal cargo uptake and extrusion across cellular membranes in all living organisms, thus playing key roles in essential and toxic metal homeostasis. Biochemical characterization of transporter-mediated translocation events and transport kinetics of redox-active metals, such as iron and copper, is challenged by the complexity in generating reconstituted systems in which vectorial metal transport can be studied in real time. We present fluorescence-based proteoliposome methods to monitor redox-active metal transmembrane translocation upon reconstitution of purified metal transporters in artificial lipid bilayers. By encapsulating turn-on/-off iron or copper-dependent sensors in the proteoliposome lumen and conducting real-time transport assays using small unilamellar vesicles (SUVs), in which selected purified Fe(II) and Cu(I) transmembrane importer and exporter proteins have been reconstituted, we provide a platform to monitor metal translocation events across lipid bilayers in real time. The strategy is modular and expandable toward the study of different transporter families featuring diverse metal substrate selectivity and promiscuity.


Assuntos
Bicamadas Lipídicas , Oxirredução , Proteolipídeos , Proteolipídeos/metabolismo , Proteolipídeos/química , Bicamadas Lipídicas/metabolismo , Bicamadas Lipídicas/química , Cobre/metabolismo , Cobre/química , Ferro/metabolismo , Metais/metabolismo , Metais/química , Transporte Biológico , Lipossomas Unilamelares/metabolismo , Lipossomas Unilamelares/química
7.
J Phys Chem B ; 128(28): 6940-6950, 2024 Jul 18.
Artigo em Inglês | MEDLINE | ID: mdl-38956449

RESUMO

Two ionic liquids (ILs) with amphiphilic properties composed of 1-butyl-3-methylimidazolium dioctylsulfosuccinate (bmim-AOT) and 1-hexyl-3-methylimidazolium dioctylsulfosuccinate (hmim-AOT) form unilamellar vesicles spontaneously simply by dissolving the IL-like surfactant in water. These novel vesicles were characterized using two different and highly sensitive fluorescent probes: 6-propionyl-2-(dimethylaminonaphthalene) (PRODAN) and trans-4-[4-(dimethylamino)-styryl]-1-methylpyridinium iodide (HC). These fluorescent probes provide information about the physicochemical properties of the bilayer, such as micropolarity, microviscosity, and electron-donor capacity. In addition, the biocompatibility of these vesicles with the blood medium was evaluated, and their toxicity was determined using Dictyostelium discoideum amoebas. First, using PRODAN and HC, it was found that the bilayer composition and the chemical structure of the ions at the interface produced differences between both amphiphiles, making the vesicles different. Thus, the bilayer of hmim-AOT vesicles is less polar, more rigid, and has a lower electron-donor capacity than those made by bmim-AOT. Finally, the results obtained from the hemolysis studies and the growth behavior of unicellular amoebas, particularly utilizing the D. discoideum assay, showed that both vesicular systems do not produce toxic effects up to a concentration of 0.02 mg/mL. This elegant assay, devoid of animal usage, highlights the potential of these newly organized systems for the delivery of drugs and bioactive molecules of different polarities.


Assuntos
Líquidos Iônicos , Tensoativos , Lipossomas Unilamelares , Líquidos Iônicos/química , Tensoativos/química , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismo , Nanomedicina , Corantes Fluorescentes/química , Compostos de Piridínio/química , Imidazóis/química , Bicamadas Lipídicas/química
8.
Proc Natl Acad Sci U S A ; 121(30): e2313609121, 2024 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-39012824

RESUMO

Mitofusins (Mfn1 and Mfn2) are the mitochondrial outer-membrane fusion proteins in mammals and belong to the dynamin superfamily of multidomain GTPases. Recent structural studies of truncated variants lacking alpha helical transmembrane domains suggested that Mfns dimerize to promote the approximation and the fusion of the mitochondrial outer membranes upon the hydrolysis of guanine 5'-triphosphate disodium salt (GTP). However, next to the presence of GTP, the fusion activity seems to require multiple regulatory factors that control the dynamics and kinetics of mitochondrial fusion through the formation of Mfn1-Mfn2 heterodimers. Here, we purified and reconstituted the full-length murine Mfn2 protein into giant unilamellar vesicles (GUVs) with different lipid compositions. The incubation with GTP resulted in the fusion of Mfn2-GUVs. High-speed video-microscopy showed that the Mfn2-dependent membrane fusion pathway progressed through a zipper mechanism where the formation and growth of an adhesion patch eventually led to the formation of a membrane opening at the rim of the septum. The presence of physiological concentration (up to 30 mol%) of dioleoyl-phosphatidylethanolamine (DOPE) was shown to be a requisite to observe GTP-induced Mfn2-dependent fusion. Our observations show that Mfn2 alone can promote the fusion of micron-sized DOPE-enriched vesicles without the requirement of regulatory cofactors, such as membrane curvature, or the assistance of other proteins.


Assuntos
GTP Fosfo-Hidrolases , Fusão de Membrana , Animais , GTP Fosfo-Hidrolases/metabolismo , GTP Fosfo-Hidrolases/genética , Camundongos , Fusão de Membrana/fisiologia , Lipossomas Unilamelares/metabolismo , Lipossomas Unilamelares/química , Guanosina Trifosfato/metabolismo , Fosfatidiletanolaminas/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Mitocôndrias/metabolismo
9.
Eur J Cell Biol ; 103(2): 151428, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38850712

RESUMO

Actin organization is crucial for establishing cell polarity, which influences processes such as directed cell motility and division. Despite its critical role in living organisms, achieving similar polarity in synthetic cells remains challenging. In this study, we employ a bottom-up approach to investigate how molecular crowders facilitate the formation of cortex-like actin networks and how these networks localize and organize based on membrane shape. Using giant unilamellar vesicles (GUVs) as models for cell membranes, we show that actin filaments can arrange along the membrane to form cortex-like structures. Notably, this organization is achieved using only actin and crowders as a minimal set of components. We utilize surface micropatterning to examine actin filament organization in deformed GUVs adhered to various pattern shapes. Our findings indicate that at the periphery of spherical GUVs, actin bundles align along the membrane. However, in highly curved regions of adhered GUVs, actin bundles avoid crossing the highly curved edges perpendicular to the adhesion site and instead remain in the lower curved regions by aligning parallel to the micropatterned surface. Furthermore, the actin bundles increase the stiffness of the GUVs, effectively counteracting strong deformations when GUVs adhere to micropatterns. This finding is corroborated by real-time deformability cytometry on GUVs with synthetic actin cortices. By precisely manipulating the shape of GUVs, our study provides a minimal system to investigate the interplay between actin structures and the membrane. Our findings provide insights into the spatial organization of actin structures within crowded environments, specifically inside GUVs that resemble the size and shape of cells. This study advances our understanding of actin network organization and functionality within cell-sized compartments.


Assuntos
Citoesqueleto de Actina , Membrana Celular , Lipossomas Unilamelares , Citoesqueleto de Actina/metabolismo , Lipossomas Unilamelares/metabolismo , Lipossomas Unilamelares/química , Membrana Celular/metabolismo , Actinas/metabolismo , Animais
10.
Sci Rep ; 14(1): 14071, 2024 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-38890456

RESUMO

In advanced drug delivery, versatile liposomal formulations are commonly employed for safer and more accurate therapies. Here we report a method that allows a straightforward production of synthetic monodisperse (~ 100 µm) giant unilamellar vesicles (GUVs) using a microfluidic system. The stability analysis based on the microscopy imaging showed that at ambient conditions the produced GUVs had a half-life of 61 ± 2 h. However, it was observed that ~ 90% of the calcein dye that was loaded into GUVs was transported into a surrounding medium in 24 h, thus indicating that the GUVs may release these small dye molecules without distinguishable membrane disruption. We further demonstrated the feasibility of our method by loading GUVs with larger and very different cargo objects; small soluble fluorescent proteins and larger magnetic microparticles in a suspension. Compared to previously reported microfluidics-based production techniques, the obtained results indicate that our simplified method could be equally harnessed in creating GUVs with less cost, effort and time, which could further benefit studying closed membrane systems.


Assuntos
Microfluídica , Lipossomas Unilamelares , Lipossomas Unilamelares/química , Microfluídica/métodos , Fluoresceínas/química , Corantes Fluorescentes/química , Técnicas Analíticas Microfluídicas/métodos
11.
PLoS One ; 19(6): e0304345, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38857287

RESUMO

Irreversible electroporation induces permanent permeabilization of lipid membranes of vesicles, resulting in vesicle rupture upon the application of a pulsed electric field. Electrofusion is a phenomenon wherein neighboring vesicles can be induced to fuse by exposing them to a pulsed electric field. We focus how the frequency of direct current (DC) pulses of electric field impacts rupture and electrofusion in cell-sized giant unilamellar vesicles (GUVs) prepared in a physiological buffer. The average time, probability, and kinetics of rupture and electrofusion in GUVs have been explored at frequency 500, 800, 1050, and 1250 Hz. The average time of rupture of many 'single GUVs' decreases with the increase in frequency, whereas electrofusion shows the opposite trend. At 500 Hz, the rupture probability stands at 0.45 ± 0.02, while the electrofusion probability is 0.71 ± 0.01. However, at 1250 Hz, the rupture probability increases to 0.69 ± 0.03, whereas the electrofusion probability decreases to 0.46 ± 0.03. Furthermore, when considering kinetics, at 500 Hz, the rate constant of rupture is (0.8 ± 0.1)×10-2 s-1, and the rate constant of fusion is (2.4 ± 0.1)×10-2 s-1. In contrast, at 1250 Hz, the rate constant of rupture is (2.3 ± 0.8)×10-2 s-1, and the rate constant of electrofusion is (1.0 ± 0.1)×10-2 s-1. These results are discussed by considering the electrical model of the lipid bilayer and the energy barrier of a prepore.


Assuntos
Eletroporação , Lipossomas Unilamelares , Lipossomas Unilamelares/química , Cinética , Eletroporação/métodos , Probabilidade , Fusão de Membrana
12.
Soft Matter ; 20(25): 4935-4949, 2024 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-38873752

RESUMO

Deformation of the cell membrane is well understood from the viewpoint of protein interactions and free energy balance. However, the various dynamic properties of the membrane, such as lipid packing and hydrophobicity, and their relationship with cell membrane deformation are unknown. Therefore, the deformation of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and oleic acid (OA) giant unilamellar vesicles (GUVs) was induced by heating and cooling cycles, and time-lapse analysis was conducted based on the membrane hydrophobicity and physical parameters of "single-parent" and "daughter" vesicles. Fluorescence ratiometric analysis by simultaneous dual-wavelength detection revealed the variation of different hydrophilic GUVs and enabled inferences of the "daughter" vesicle composition and the "parent" membrane's local composition during deformation; the "daughter" vesicle composition of OA was lower than that of the "parents", and lateral movement of OA was the primary contributor to the formation of the "daughter" vesicles. Thus, our findings and the newly developed methodology, named in situ quantitative membrane property-morphology relation (QmPMR) analysis, would provide new insights into cell deformation and accelerate research on both deformation and its related events, such as budding and birthing.


Assuntos
1,2-Dipalmitoilfosfatidilcolina , Membrana Celular , Interações Hidrofóbicas e Hidrofílicas , Ácido Oleico , Lipossomas Unilamelares , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismo , Ácido Oleico/química , 1,2-Dipalmitoilfosfatidilcolina/química , Membrana Celular/química
13.
Biophys J ; 123(13): 1857-1868, 2024 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-38822522

RESUMO

This study investigated the incorporation of triacylglycerol droplets in the bilayers of giant unilamellar vesicles (GUVs) using four triacylglycerols and four phosphatidylcholines by confocal laser scanning microscopy. The triacylglycerol droplets were incorporated between the monolayer leaflets of the GUVs. Among the spherical droplets protruding on only one side of the bilayers, the droplets bound to the outer leaflets outnumbered those bound to the inner leaflets. The more frequent droplet binding to the outer leaflet caused transbilayer asymmetry in the droplet surface density. A vesicle consisting of a single-bilayer spherical segment and a double-bilayer spherical segment was also observed. The yield of these vesicles was comparable with or higher than that of the droplet-incorporating GUVs for many of the phosphatidylcholine-triacylglycerol combinations. In a vesicle consisting of single-bilayer and double-bilayer segments, most of the triacylglycerol droplets were localized on the outermost membrane surface along the segment boundary and in the double-bilayer segment. To rationalize the formation of these vesicle structures, we propose that the transbilayer asymmetry in the droplet surface density induces spontaneous curvature of the bilayer, with the bilayer spontaneously bending away from the droplets. Energy calculations performed assuming the existence of spontaneous curvature of the bilayer corroborated the experimentally determined membrane shapes for the vesicles consisting of unilamellar and bilamellar regions.


Assuntos
Bicamadas Lipídicas , Fosfatidilcolinas , Triglicerídeos , Lipossomas Unilamelares , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismo , Bicamadas Lipídicas/química , Triglicerídeos/química , Triglicerídeos/metabolismo , Fosfatidilcolinas/química
14.
Analyst ; 149(12): 3317-3324, 2024 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-38742381

RESUMO

In this work, the release of giant liposome (∼100 µm in diameter) content was imaged by shadow electrochemiluminescence (ECL) microscopy. Giant unilamellar liposomes were pre-loaded with a sucrose solution and allowed to sediment at an ITO electrode surface immersed in a solution containing a luminophore ([Ru(bpy)3]2+) and a sacrificial co-reactant (tri-n-propylamine). Upon polarization, the electrode exhibited illumination over its entire surface thanks to the oxidation of ECL reagents. However, as soon as liposomes reached the electrode surface, dark spots appeared and then spread over time on the surface. This observation reflected a blockage of the electrode surface at the contact point between the liposome and the electrode surface, followed by the dilution of ECL reagents after the rupture of the liposome membrane and release of its internal ECL-inactive solution. Interestingly, ECL reappeared in areas where it initially faded, indicating back-diffusion of ECL reagents towards the previously diluted area and thus confirming liposome permeabilization. The whole process was analyzed qualitatively and quantitatively within the defined region of interest. Two mass transport regimes were identified: a gravity-driven spreading process when the liposome releases its content leading to ECL vanishing and a diffusive regime when ECL recovers. The reported shadow ECL microscopy should find promising applications for the imaging of transient events such as molecular species released by artificial or biological vesicles.


Assuntos
Eletrodos , Medições Luminescentes , Medições Luminescentes/métodos , Lipossomos/química , Técnicas Eletroquímicas/métodos , Técnicas Eletroquímicas/instrumentação , Propilaminas/química , Lipossomas Unilamelares/química , Sacarose/química , Compostos de Estanho
15.
Biomater Sci ; 12(13): 3423-3430, 2024 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-38809312

RESUMO

Micro-nanomaterials that can adopt different structures are powerful tools in the fields of biological and medical sciences. We previously developed a lipid membrane that can convert between 2D nanosheet and 3D vesicle forms using cationic copolymer polyallylamine-graft-polyethylene glycol and the anionic peptide E5. The properties of the membrane during conversion have been characterized only by confocal laser scan microscopy. Furthermore, due to the 2D symmetry of the lipid nanosheet, the random folding of the lipid bilayer into either the original or the reverse orientation occurs during sheet-to-vesicle conversion, compromising the structural consistency of the membrane. In this study, flow cytometry was applied to track the conversion of more than 5000 lipid membranes from 3D vesicles to 2D nanosheets and back to 3D vesicles, difficult with microscopies. The lipid nanosheets exhibited more side scattering intensity than 3D vesicles, presumably due to free fluctuation and spin of the sheets in the suspension. Furthermore, by immobilizing bovine serum albumin as one of the representative proteins on the outer leaflet of giant unilamellar vesicles at a relatively low coverage, complete restoration of lipid membranes to the original 3D orientation was obtained after sheet-to-vesicle conversion. This convertible membrane system should be applicable in a wide range of fields. Our findings also provide experimental evidence for future theoretical studies on membrane behavior.


Assuntos
Soroalbumina Bovina , Soroalbumina Bovina/química , Polietilenoglicóis/química , Animais , Bicamadas Lipídicas/química , Poliaminas/química , Bovinos , Nanoestruturas/química , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismo , Peptídeos/química
16.
Proc Natl Acad Sci U S A ; 121(22): e2317227121, 2024 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-38771870

RESUMO

The biophysical properties of lipid vesicles are important for their stability and integrity, key parameters that control the performance when these vesicles are used for drug delivery. The vesicle properties are determined by the composition of lipids used to form the vesicle. However, for a given lipid composition, they can also be tailored by tethering polymers to the membrane. Typically, synthetic polymers like polyethyleneglycol are used to increase vesicle stability, but the use of polysaccharides in this context is much less explored. Here, we report a general method for functionalizing lipid vesicles with polysaccharides by binding them to cholesterol. We incorporate the polysaccharides on the outer membrane leaflet of giant unilamellar vesicles (GUVs) and investigate their effect on membrane mechanics using micropipette aspiration. We find that the presence of the glycolipid functionalization produces an unexpected softening of GUVs with fluid-like membranes. By contrast, the functionalization of GUVs with polyethylene glycol does not reduce their stretching modulus. This work provides the potential means to study membrane-bound meshworks of polysaccharides similar to the cellular glycocalyx; moreover, it can be used for tuning the mechanical properties of drug delivery vehicles.


Assuntos
Polissacarídeos , Lipossomas Unilamelares , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismo , Polissacarídeos/química , Polissacarídeos/metabolismo , Polietilenoglicóis/química , Colesterol/química , Colesterol/metabolismo , Lipídeos/química
17.
J Colloid Interface Sci ; 669: 198-210, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38713958

RESUMO

A widespread strategy to increase the transport of therapeutic peptides across cellular membranes has been to attach lipid moieties to the peptide backbone (lipidation) to enhance their intrinsic membrane interaction. Efforts in vitro and in vivo investigating the correlation between lipidation characteristics and peptide membrane translocation efficiency have traditionally relied on end-point read-out assays and trial-and-error-based optimization strategies. Consequently, the molecular details of how therapeutic peptide lipidation affects it's membrane permeation and translocation mechanisms remain unresolved. Here we employed salmon calcitonin as a model therapeutic peptide and synthesized nine double lipidated analogs with varying lipid chain lengths. We used single giant unilamellar vesicle (GUV) calcein influx time-lapse fluorescence microscopy to determine how tuning the lipidation length can lead to an All-or-None GUV filling mechanism, indicative of a peptide mediated pore formation. Finally, we used a GUVs-containing-inner-GUVs assay to demonstrate that only peptide analogs capable of inducing pore formation show efficient membrane translocation. Our data provided the first mechanistic details on how therapeutic peptide lipidation affects their membrane perturbation mechanism and demonstrated that fine-tuning lipidation parameters could induce an intrinsic pore-forming capability. These insights and the microscopy based workflow introduced for investigating structure-function relations could be pivotal for optimizing future peptide design strategies.


Assuntos
Calcitonina , Lipossomas Unilamelares , Calcitonina/química , Calcitonina/metabolismo , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismo , Animais , Fluoresceínas/química , Membrana Celular/metabolismo , Membrana Celular/química
18.
J Phys Chem B ; 128(20): 4986-4995, 2024 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-38739415

RESUMO

Membrane fusion is considered the first step in the entry of enveloped viruses into the host cell. Several targeted strategies have been implemented to block viral entry by limiting the fusion protein to form a six-helix bundle, which is a prerequisite for fusion. Nonetheless, the development of broad-spectrum fusion inhibitors is essential to combat emerging and re-emerging viral infections. TG-23, a coronin 1, a tryptophan-aspartate-rich phagosomal protein-derived peptide, demonstrated inhibition of fusion between small unilamellar vesicles (SUVs) by modulating the membrane's physical properties. However, its inhibitory efficacy reduces with an increasing concentration of membrane cholesterol. The present work aims to develop a fusion inhibitor whose efficacy would be unaltered in the presence of membrane cholesterol. A stretch of the tryptophan-aspartic acid-containing peptide with a similar secondary structure and hydrophobicity profile of TG-23 from coronin 1 was synthesized, and its ability to inhibit SUV-SUV fusion with varying concentrations of membrane cholesterol was evaluated. Our results demonstrate that the GG-21 peptide inhibits fusion irrespective of the cholesterol content of the membrane. We have further evaluated the peptide-induced change in the membrane organization and dynamics utilizing arrays of steady-state and time-resolved fluorescence measurements and correlated these results with their effect on fusion. Interestingly, GG-21 displays inhibitory efficacy in a wide variety of lipid compositions despite having a secondary structure and physical properties similar to those of TG-23. Overall, our results advocate that the secondary structure and physical properties of the peptide may not be sufficient to predict its inhibitory efficacy.


Assuntos
Colesterol , Fusão de Membrana , Fusão de Membrana/efeitos dos fármacos , Colesterol/química , Peptídeos/química , Peptídeos/farmacologia , Lipossomas Unilamelares/química , Lipossomas Unilamelares/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/química
19.
Langmuir ; 40(20): 10477-10485, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38710504

RESUMO

Insertion of hydrophobic nanoparticles into phospholipid bilayers is limited to small particles that can incorporate into a hydrophobic membrane core between two lipid leaflets. Incorporation of nanoparticles above this size limit requires the development of challenging surface engineering methodologies. In principle, increasing the long-chain lipid component in the lipid mixture should facilitate incorporation of larger nanoparticles. Here, we explore the effect of incorporating very long phospholipids (C24:1) into small unilamellar vesicles on the membrane insertion efficiency of hydrophobic nanoparticles that are 5-11 nm in diameter. To this end, we improve an existing vesicle preparation protocol and utilized cryogenic electron microscopy imaging to examine the mode of interaction and evaluate the insertion efficiency of membrane-inserted nanoparticles. We also perform classical coarse-grained molecular dynamics simulations to identify changes in lipid membrane structural properties that may increase insertion efficiency. Our results indicate that long-chain lipids increase the insertion efficiency by preferentially accumulating near membrane-inserted nanoparticles to reduce the thermodynamically unfavorable disruption of the membrane.


Assuntos
Nanopartículas , Lipossomas Unilamelares , Nanopartículas/química , Lipossomas Unilamelares/química , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/química , Fosfolipídeos/química , Tamanho da Partícula
20.
J Vis Exp ; (206)2024 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-38682922

RESUMO

We present a method to incorporate into vesicles complex protein networks, involving integral membrane proteins, enzymes, and fluorescence-based sensors, using purified components. This method is relevant for the design and construction of bioreactors and the study of complex out-of-equilibrium metabolic reaction networks. We start by reconstituting (multiple) membrane proteins into large unilamellar vesicles (LUVs) according to a previously developed protocol. We then encapsulate a mixture of purified enzymes, metabolites, and fluorescence-based sensors (fluorescent proteins or dyes) via freeze-thaw-extrusion and remove non-incorporated components by centrifugation and/or size-exclusion chromatography. The performance of the metabolic networks is measured in real time by monitoring the ATP/ADP ratio, metabolite concentration, internal pH, or other parameters by fluorescence readout. Our membrane protein-containing vesicles of 100-400 nm diameter can be converted into giant-unilamellar vesicles (GUVs), using existing but optimized procedures. The approach enables the inclusion of soluble components (enzymes, metabolites, sensors) into micrometer-size vesicles, thus upscaling the volume of the bioreactors by orders of magnitude. The metabolic network containing GUVs are trapped in microfluidic devices for analysis by optical microscopy.


Assuntos
Lipossomas Unilamelares , Lipossomas Unilamelares/metabolismo , Lipossomas Unilamelares/química , Redes e Vias Metabólicas , Proteínas de Membrana/metabolismo , Proteínas de Membrana/química
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