RESUMEN
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.
Asunto(s)
GTP Fosfohidrolasas , Fusión de Membrana , Animales , GTP Fosfohidrolasas/metabolismo , GTP Fosfohidrolasas/genética , Ratones , Fusión de Membrana/fisiología , Liposomas Unilamelares/metabolismo , Liposomas Unilamelares/química , Guanosina Trifosfato/metabolismo , Fosfatidiletanolaminas/metabolismo , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , Mitocondrias/metabolismoRESUMEN
All metazoans depend on the consumption of O2 by the mitochondrial oxidative phosphorylation system (OXPHOS) to produce energy. In addition, the OXPHOS uses O2 to produce reactive oxygen species that can drive cell adaptations1-4, a phenomenon that occurs in hypoxia4-8 and whose precise mechanism remains unknown. Ca2+ is the best known ion that acts as a second messenger9, yet the role ascribed to Na+ is to serve as a mere mediator of membrane potential10. Here we show that Na+ acts as a second messenger that regulates OXPHOS function and the production of reactive oxygen species by modulating the fluidity of the inner mitochondrial membrane. A conformational shift in mitochondrial complex I during acute hypoxia11 drives acidification of the matrix and the release of free Ca2+ from calcium phosphate (CaP) precipitates. The concomitant activation of the mitochondrial Na+/Ca2+ exchanger promotes the import of Na+ into the matrix. Na+ interacts with phospholipids, reducing inner mitochondrial membrane fluidity and the mobility of free ubiquinone between complex II and complex III, but not inside supercomplexes. As a consequence, superoxide is produced at complex III. The inhibition of Na+ import through the Na+/Ca2+ exchanger is sufficient to block this pathway, preventing adaptation to hypoxia. These results reveal that Na+ controls OXPHOS function and redox signalling through an unexpected interaction with phospholipids, with profound consequences for cellular metabolism.
Asunto(s)
Transporte de Electrón , Hipoxia/metabolismo , Mitocondrias/metabolismo , Sistemas de Mensajero Secundario , Sodio/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Fosfatos de Calcio/metabolismo , Línea Celular Tumoral , Precipitación Química , Humanos , Masculino , Fluidez de la Membrana , Ratones Endogámicos C57BL , Membranas Mitocondriales/química , Membranas Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , Fosforilación Oxidativa , Ratas , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Intercambiador de Sodio-Calcio/metabolismoRESUMEN
Since WHO has declared the COVID-19 outbreak a global pandemic, nearly seven million deaths have been reported. This efficient spread of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is facilitated by the ability of the spike glycoprotein to bind multiple cell membrane receptors. Although ACE2 is identified as the main receptor for SARS-CoV-2, other receptors could play a role in viral entry. Among others, C-type lectins such as DC-SIGN are identified as efficient trans-receptor for SARS-CoV-2 infection, so the use of glycomimetics to inhibit the infection through the DC-SIGN blockade is an encouraging approach. In this regard, multivalent nanostructures based on glycosylated [60]fullerenes linked to a central porphyrin scaffold have been designed and tested against DC-SIGN-mediated SARS-CoV-2 infection. First results show an outstanding inhibition of the trans-infection up to 90%. In addition, a deeper understanding of nanostructure-receptor binding is achieved through microscopy techniques, high-resolution NMR experiments, Quartz Crystal Microbalance experiments, and molecular dynamic simulations.
Asunto(s)
Moléculas de Adhesión Celular , Fulerenos , Lectinas Tipo C , Porfirinas , Receptores de Superficie Celular , SARS-CoV-2 , Humanos , Moléculas de Adhesión Celular/metabolismo , Moléculas de Adhesión Celular/antagonistas & inhibidores , COVID-19/virología , Tratamiento Farmacológico de COVID-19 , Fulerenos/química , Fulerenos/farmacología , Lectinas Tipo C/metabolismo , Lectinas Tipo C/antagonistas & inhibidores , Simulación de Dinámica Molecular , Porfirinas/química , Porfirinas/farmacología , Unión Proteica , Receptores de Superficie Celular/metabolismo , Receptores de Superficie Celular/antagonistas & inhibidores , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Glicoproteína de la Espiga del Coronavirus/químicaRESUMEN
Bacterial biofilms mechanically behave as viscoelastic media consisting of micron-sized bacteria cross-linked to a self-produced network of extracellular polymeric substances (EPSs) embedded in water. Structural principles for numerical modeling aim at describing mesoscopic viscoelasticity without losing details on the underlying interactions existing in wide regimes of deformation under hydrodynamic stress. Here, we approach the computational challenge to model bacterial biofilms for predictive mechanics in silico under variable stress conditions. Up-to-date models are not entirely satisfactory due to the plethora of parameters required to make them functioning under the effects of stress. As guided by the structural depiction gained in a previous work with Pseudomonas fluorescens [Jara et al., Front. Microbiol. 11, 588884 (2021)], we propose a mechanical modeling by means of Dissipative Particle Dynamics (DPD), which captures the essentials of topological and compositional interactions between bacterial particles and cross-linked EPS-embedding under imposed shear. The P. fluorescens biofilms have been modeled under mechanical stress mimicking shear stresses as undergone in vitro. The predictive capacity for mechanical features in DPD-simulated biofilms has been investigated by varying the externally imposed field of shear strain at variable amplitude and frequency. The parametric map of essential biofilm ingredients has been explored by making the rheological responses to emerge among conservative mesoscopic interactions and frictional dissipation in the underlying microscale. The proposed coarse grained DPD simulation qualitatively catches the rheology of the P. fluorescens biofilm over several decades of dynamic scaling.
Asunto(s)
Pseudomonas fluorescens , Pseudomonas fluorescens/fisiología , Biopelículas , Reología , Simulación por Computador , HidrodinámicaRESUMEN
F1Fo-ATP synthase (ATP synthase) is a central membrane protein that synthetizes most of the ATP in the cell through a rotational movement driven by a proton gradient across the hosting membrane. In mitochondria, ATP synthases can form dimers through specific interactions between some subunits of the protein. The dimeric form of ATP synthase provides the protein with a spontaneous curvature that sustain their arrangement at the rim of the high-curvature edges of mitochondrial membrane (cristae). Also, a direct interaction with cardiolipin, a lipid present in the inner mitochondrial membrane, induces the dimerization of ATP synthase molecules along cristae. The deletion of those biochemical interactions abolishes the protein dimerization producing an altered mitochondrial function and morphology. Mechanically, membrane bending is one of the key deformation modes by which mitochondrial membranes can be shaped. In particular, bending rigidity and spontaneous curvature are important physical factors for membrane remodelling. Here, we discuss a complementary mechanism whereby the rotatory movement of the ATP synthase might modify the mechanical properties of lipid bilayers and contribute to the formation and regulation of the membrane invaginations.
Asunto(s)
Membrana Celular/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Rotación , Membrana Celular/química , HumanosRESUMEN
BACKGROUND: Lipoplexes are non-viral vectors based on cationic lipids used to deliver DNA into cells, also known as lipofection. The positively charge of the hydrophilic head-group provides the cationic lipids the ability to condensate the negatively charged DNA into structured complexes. The polar head can carry a large variety of chemical groups including amines as well as guanidino or imidazole groups. In particular, gemini cationic lipids consist of two positive polar heads linked by a spacer with different length. As for the hydrophobic aliphatic chains, they can be unsaturated or saturated and are connected to the polar head-groups. Many other chemical components can be included in the formulation of lipoplexes to improve their transfection efficiency, which often relies on their structural features. Varying these components can drastically change the arrangement of DNA molecules within the lamellar, hexagonal or cubic phases that are provided by the lipid matrix. Lipofection is widely used to deliver genetic material in cell culture experiments but the simpler formulations exhibit major drawbacks related to low transfection, low specificity, low circulation half-life and toxicity when scaled up to in vivo experiments. RESULTS: So far, we have explored in cell cultures the transfection ability of lipoplexes based on gemini cationic lipids that consist of two C16 alkyl chains and two imidazolium polar head-groups linked with a polyoxyethylene spacer, (C16Im)2(C4O). Here, PEGylated lipids have been introduced to the lipoplex formulation and the transgene expression of the Opa1 mitochondrial transmembrane protein in mice was assessed. The addition of PEG on the surface of the lipid mixed resulted in the formation of Ia3d bicontinuous cubic phases as determined by small angle X-ray scattering. After a single intramuscular administration, the cubic lipoplexes were accumulated in tissues with tight endothelial barriers such as brain, heart, and lungs for at least 48 h. The transgene expression of Opa1 in those organs was identified by western blotting or RNA expression analysis through quantitative polymerase chain reaction. CONCLUSIONS: The expression reported here is sufficient in magnitude, duration and toxicity to consolidate the bicontinuous cubic structures formed by (C16Im)2(C4O)-based lipoplexes as valuable therapeutic agents in the field of gene delivery.
Asunto(s)
GTP Fosfohidrolasas/genética , Imidazoles/química , Liposomas/química , Tensoactivos/química , Transfección/métodos , Animales , Encéfalo/metabolismo , Cationes/química , Línea Celular , Supervivencia Celular/efectos de los fármacos , GTP Fosfohidrolasas/deficiencia , GTP Fosfohidrolasas/metabolismo , Riñón/metabolismo , Liposomas/farmacocinética , Liposomas/farmacología , Ratones , Plásmidos/química , Plásmidos/genética , Plásmidos/metabolismo , Polietilenglicoles/química , Distribución TisularRESUMEN
ATP synthase is a rotating membrane protein that synthesizes ATP through proton-pumping activity across the membrane. To unveil the mechanical impact of this molecular active pump on the bending properties of its lipid environment, we have functionally reconstituted the ATP synthase in giant unilamellar vesicles and tracked the membrane fluctuations by means of flickering spectroscopy. We find that ATP synthase rotates at a frequency of about 20 Hz, promoting large nonequilibrium deformations at discrete hot spots in lipid vesicles and thus inducing an overall membrane softening. The enhanced nonequilibrium fluctuations are compatible with an accumulation of active proteins at highly curved membrane sites through a curvature-protein coupling mechanism that supports the emergence of collective effects of rotating ATP synthases in lipid membranes.
Asunto(s)
ATPasas de Translocación de Protón Bacterianas/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Proteínas de Escherichia coli/metabolismo , Adenosina Trifosfato/biosíntesis , ATPasas de Translocación de Protón Bacterianas/química , ATPasas de Translocación de Protón Bacterianas/genética , Membrana Celular/efectos de los fármacos , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Colorantes Fluorescentes/química , Concentración de Iones de Hidrógeno , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Microscopía por Video , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rodamina 123/química , Liposomas Unilamelares/química , Liposomas Unilamelares/metabolismo , Valinomicina/farmacologíaRESUMEN
Mitochondria form a dynamic network of constantly dividing and fusing organelles. The balance between these antagonistic processes is crucial for normal cellular function and requires the action of specialized proteins. The mitochondrial membrane proteins mitofusin 1 (Mfn1) and mitofusin 2 (Mfn2) are responsible for the fusion of the outer membrane of adjacent mitochondria. Mutations within Mfn1 or Mfn2 impair mitochondrial fusion and lead to some severe mitochondrial dysfunctions and mitochondrial diseases (MDs). A characteristic phenotype of cells carrying defective Mfn1 or Mfn2 is the presence of a highly fragmented mitochondrial network. Here, we use a biocompatible mixture of lipids, consisting on synthetic gemini cationic lipids (GCLs) and the zwitterionic phospholipid (DOPE), to complex, transport, and deliver intact copies of MFN1 gene into MFN1-Knockout mouse embryonic fibroblasts (MFN1-KO MEFs). We demonstrate that the GCL/DOPE-DNA lipoplexes are able to introduce the intact MFN1 gene into the cells and ectopically produce functional Mfn1. A four-fold increase of the Mfn1 levels is necessary to revert the MFN1-KO phenotype and to partially restore a mitochondrial network. This phenotype complementation was correlated with the transfection of GCL/DOPE-MFN1 lipoplexes that exhibited a high proportion of highly packaged hexagonal phase. GCL/DOPE-DNA lipoplexes are formulated as efficient therapeutic agents against MDs.
Asunto(s)
Fibroblastos/metabolismo , GTP Fosfohidrolasas/metabolismo , Lípidos/química , Mitocondrias/metabolismo , Animales , Supervivencia Celular/genética , Supervivencia Celular/fisiología , GTP Fosfohidrolasas/genética , Terapia Genética/métodos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Mutación/genéticaRESUMEN
BACKGROUND: The design of efficient drug delivery vectors requires versatile formulations able to simultaneously direct a multitude of molecular targets and to bypass the endosomal recycling pathway of cells. Liposomal-based vectors need the decoration of the lipid surface with specific peptides to fulfill the functional requirements. The unspecific binding of peptides to the lipid surface is often accompanied with uncontrolled formulations and thus preventing the molecular mechanisms of a successful therapy. RESULTS: We present a simple synthesis pathway to anchor cysteine-terminal peptides to thiol-reactive lipids for adequate and quantitative liposomal formulations. As a proof of concept, we have synthesized two different lipopeptides based on (a) the truncated Fibroblast Growth Factor (tbFGF) for cell targeting and (b) the pH sensitive and fusogenic GALA peptide for endosomal scape. CONCLUSIONS: The incorporation of these two lipopeptides in the liposomal formulation improves the fibroblast cell targeting and promotes the direct delivery of cargo molecules to the cytoplasm of the cell.
Asunto(s)
Disulfuros/química , Lípidos/química , Péptidos/química , Piridinas/química , Animales , Supervivencia Celular/efectos de los fármacos , Cisteína/química , Composición de Medicamentos/métodos , Sistemas de Liberación de Medicamentos , Endosomas/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Liposomas/química , Ratones , Estructura Molecular , Imagen Óptica/métodos , Prueba de Estudio ConceptualRESUMEN
BACKGROUND: A major bottleneck in drug delivery is the breakdown and degradation of the delivery system through the endosomal/lysosomal network of the host cell, hampering the correct delivery of the drug of interest. In nature, the bacterial pathogen Listeria monocytogenes has developed a strategy to secrete Listeriolysin O (LLO) toxin as a tool to escape the eukaryotic lysosomal system upon infection, allowing it to grow and proliferate unharmed inside the host cell. RESULTS: As a "proof of concept", we present here the use of purified His-LLO H311A mutant protein and its conjugation on the surface of gold nanoparticles to promote the lysosomal escape of 40 nm-sized nanoparticles in mouse embryonic fibroblasts. Surface immobilization of LLO was achieved after specific functionalization of the nanoparticles with nitrile acetic acid, enabling the specific binding of histidine-tagged proteins. CONCLUSIONS: Endosomal acidification leads to release of the LLO protein from the nanoparticle surface and its self-assembly into a 300 Å pore that perforates the endosomal/lysosomal membrane, enabling the escape of nanoparticles.
Asunto(s)
Toxinas Bacterianas/metabolismo , Portadores de Fármacos/metabolismo , Endosomas/metabolismo , Oro/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas Hemolisinas/metabolismo , Nanopartículas/metabolismo , Animales , Línea Celular , Fibroblastos/metabolismo , Concentración de Iones de Hidrógeno , Listeria monocytogenes/metabolismo , Lisosomas/metabolismo , Ratones , Modelos MolecularesRESUMEN
Mitochondria are essential for the production and maintenance of ATP in the eukaryotic cell. To image and monitor intracellular ATP level without cell breakage, biological and chemical sensors were developed in the last years. Here, we have internalized a rhodamine-based sensor RSL+ into living cells and monitored the mitochondrial ATP levels in cultured mouse embryonic fibroblasts. To evaluate the robustness of the sensor we imaged the changes of the mitochondrial ATP levels under non-physiological conditions upon incubation with FCCP, oligomycin, azide, deoxyglucose or phosphoenolpyruvate; all compounds that interfere with ATP homeostasis of the cell. The ATP sensor allowed us to determine the mitochondrial ATP levels in human skin fibroblasts where we observe a similar amount of ATP compared to mouse embryonic fibroblasts. We propose the RSL+ to be a valuable tool for the assessment of mitochondrial dysfunction in human cells derived from mitochondrial OXPHOS patients and for basic studies on bioenergetics metabolism.
Asunto(s)
Adenosina Trifosfato/aislamiento & purificación , Técnicas Biosensibles/métodos , Fibroblastos/metabolismo , Mitocondrias/química , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Animales , Azidas/farmacología , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Desoxiglucosa/farmacología , Fibroblastos/química , Fibroblastos/efectos de los fármacos , Humanos , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Oligomicinas/farmacología , Fosforilación Oxidativa/efectos de los fármacos , Rodaminas/químicaRESUMEN
The ability to respond toward mechanical stimuli is a fundamental property of biological organisms at both the macroscopic and cellular levels, yet it has been considerably less observed in artificial supramolecular and colloidal homologues. An archetypal example in this regard is cellular mechanosensation, a process by which mechanical forces applied on the cell membrane are converted into biochemical or electrical signals through nanometer-scale changes in molecular conformations. In this article, we report an artificial gold nanoparticle (Au NP)-discrete π-conjugated molecule hybrid system that mimics the mechanical behavior of biological membranes and is able to self-assemble into colloidal gold nanoclusters or membranes in a controlled and reversible fashion by changing the concentration or the mechanical force (pressure) applied. This has been achieved by rational design of a small π-conjugated thiolated molecule that controls, to a great extent, the hierarchy levels involved in Au NP clustering by enabling reversible, cooperative non-covalent (π-π, solvophobic, and hydrogen bonding) interactions. In addition, the Au NP membranes have the ability to entrap and release aromatic guest molecules reversibly (Kb = 5.0 × 105 M-1) for several cycles when subjected to compression-expansion experiments, in close analogy to the behavior of cellular mechanosensitive channels. Not only does our hybrid system represent the first example of a reversible colloidal membrane, but it also can be controlled by a dynamic mechanical stimulus using a new supramolecular surface-pressure-controlled strategy. This approach holds great potential for the development of multiple colloidal assemblies within different research fields.
Asunto(s)
Oro/química , Nanopartículas del Metal/química , Coloides/química , Sustancias Macromoleculares/síntesis química , Sustancias Macromoleculares/química , Conformación Molecular , Tamaño de la Partícula , Propiedades de SuperficieRESUMEN
The challenge of mimicking the extracellular matrix with artificial scaffolds that are able to reduce immunoresponse is still unmet. Recent findings have shown that mesenchymal stem cells (MSC) infiltrating into the implanted scaffold have effects on the implant integration by improving the healing process. Toward this aim, a novel polyamidoamine-based nanocomposite hydrogel is synthesized, cross-linked with porous nanomaterials (i.e., mesoporous silica nanoparticles), able to release chemokine proteins. A comprehensive viscoelasticity study confirms that the hydrogel provides optimal structural support for MSC infiltration and proliferation. The efficiency of this hydrogel, containing the chemoattractant stromal cell-derived factor 1α (SDF-1α), in promoting MSC migration in vitro is demonstrated. Finally, subcutaneous implantation of SDF-1α-releasing hydrogels in mice results in a modulation of the inflammatory reaction. Overall, the proposed SDF-1α-nanocomposite hydrogel proves to have potential for applications in tissue engineering.
Asunto(s)
Quimiotaxis , Hidrogeles/química , Células Madre Mesenquimatosas/citología , Nanocompuestos/química , Animales , Materiales Biocompatibles/química , Células de la Médula Ósea/citología , Proliferación Celular/efectos de los fármacos , Quimiocina CXCL12/farmacología , Quimiotaxis/efectos de los fármacos , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos BALB C , Nanopartículas/química , Nanopartículas/ultraestructura , Porosidad , Reología , Dióxido de Silicio/químicaRESUMEN
ATP, the molecule used by living organisms to supply energy to many different metabolic processes, is synthesized mostly by the ATPase synthase using a proton or sodium gradient generated across a lipid membrane. We present evidence that a modified electrode surface integrating a NiFeSe hydrogenase and a F1 F0 -ATPase in a lipid membrane can couple the electrochemical oxidation of H2 to the synthesis of ATP. This electrode-assisted conversion of H2 gas into ATP could serve to generate this biochemical fuel locally when required in biomedical devices or enzymatic synthesis of valuable products.
Asunto(s)
Adenosina Trifosfato/metabolismo , Hidrógeno/química , Técnicas Electroquímicas , Electrodos , Hidrógeno/metabolismo , Hidrogenasas/química , Hidrogenasas/metabolismo , Membrana Dobles de Lípidos/química , Microscopía de Fuerza Atómica , ATPasas de Translocación de Protón Mitocondriales/química , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Oxidación-Reducción , Tecnicas de Microbalanza del Cristal de CuarzoRESUMEN
Erythrocytes are flexible cells specialized in the systemic transport of oxygen in vertebrates. This physiological function is connected to their outstanding ability to deform in passing through narrow capillaries. In recent years, there has been an influx of experimental evidence of enhanced cell-shape fluctuations related to metabolically driven activity of the erythroid membrane skeleton. However, no direct observation of the active cytoskeleton forces has yet been reported to our knowledge. Here, we show experimental evidence of the presence of temporally correlated forces superposed over the thermal fluctuations of the erythrocyte membrane. These forces are ATP-dependent and drive enhanced flickering motions in human erythrocytes. Theoretical analyses provide support for a direct force exerted on the membrane by the cytoskeleton nodes as pulses of well-defined average duration. In addition, such metabolically regulated active forces cause global membrane softening, a mechanical attribute related to the functional erythroid deformability.
Asunto(s)
Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Eritrocitos/metabolismo , Estrés Mecánico , Adenosina Trifosfato/metabolismo , Fenómenos Biomecánicos , Células Cultivadas , HumanosRESUMEN
The full-length ZipA protein from Escherichia coli, one of the essential elements of the cell division machinery, was studied in a surface model built as adsorbed monolayers. The interplay between lateral packing and molecular conformation was probed using a combined methodology based on the scaling analysis of the surface pressure isotherms and ellipsometry measurements of the monolayer thickness. The observed behavior is compatible with the one expected for an intrinsically disordered and highly flexible protein that is preferentially structured in a random coil conformation. At low grafting densities, ZipA coils organize in a mushroom-like regime, whereas a coil-to-brush transition occurs on increasing lateral packing. The structural results suggest a functional scenario in which ZipA acts as a flexible tether anchoring bacterial proto-ring elements to the membrane during the earlier stages of division.
Asunto(s)
Proteínas Portadoras/química , Proteínas de Ciclo Celular/química , Proteínas de Escherichia coli/química , Conformación Proteica , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , División Celular/genética , Membrana Celular/metabolismo , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Elasticidad , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Cinética , Modelos Moleculares , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Propiedades de Superficie , TermodinámicaRESUMEN
The concept of membrane fluidity usually refers to a high molecular mobility inside the lipid bilayer which enables lateral diffusion of embedded proteins. Fluids have the ability to flow under an applied shear stress whereas solids resist shear deformations. Biological membranes require both properties for their function: high lateral fluidity and structural rigidity. Consequently, an adequate account must include, in addition to viscosity, the possibility for a nonzero shear modulus. This knowledge is still lacking as measurements of membrane shear properties have remained incomplete so far. In the present contribution we report a surface shear rheology study of different lipid monolayers that model distinct biologically relevant situations. The results evidence a large variety of mechanical behavior under lateral shear flow.
Asunto(s)
Membrana Celular/química , Membrana Dobles de Lípidos/química , Fluidez de la Membrana , Microdominios de Membrana/química , Reología , ViscosidadRESUMEN
Statins are known to be anti-inflammatory, but the mechanism remains poorly understood. Here, we show that macrophages, either treated with statin in vitro or from statin-treated mice, have reduced cholesterol levels and higher expression of Jmjd3, a H3K27me3 demethylase. We provide evidence that lowering cholesterol levels in macrophages suppresses the adenosine triphosphate (ATP) synthase in the inner mitochondrial membrane and changes the proton gradient in the mitochondria. This activates nuclear factor kappa-B (NF-κB) and Jmjd3 expression, which removes the repressive marker H3K27me3. Accordingly, the epigenome is altered by the cholesterol reduction. When subsequently challenged by the inflammatory stimulus lipopolysaccharide (M1), macrophages, either treated with statins in vitro or isolated from statin-fed mice, express lower levels proinflammatory cytokines than controls, while augmenting anti-inflammatory Il10 expression. On the other hand, when macrophages are alternatively activated by IL-4 (M2), statins promote the expression of Arg1, Ym1, and Mrc1. The enhanced expression is correlated with the statin-induced removal of H3K27me3 from these genes prior to activation. In addition, Jmjd3 and its demethylase activity are necessary for cholesterol to modulate both M1 and M2 activation. We conclude that upregulation of Jmjd3 is a key event for the anti-inflammatory function of statins on macrophages.
Asunto(s)
Colesterol , Inhibidores de Hidroximetilglutaril-CoA Reductasas , Histona Demetilasas con Dominio de Jumonji , Macrófagos , Mitocondrias , Regulación hacia Arriba , Histona Demetilasas con Dominio de Jumonji/metabolismo , Histona Demetilasas con Dominio de Jumonji/genética , Animales , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Colesterol/metabolismo , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Antiinflamatorios/farmacología , Ratones Endogámicos C57BL , MasculinoRESUMEN
Stains are known to be anti-inflammatory, but the mechanism remains poorly understood. Here we show that macrophages, either treated with statin in vitro or from statin-treated mice, have reduced cholesterol levels and higher expression of Jmjd3, a H3K27me3 demethylase. We provide evidence that lowering cholesterol levels in macrophages suppresses the ATP synthase in the inner mitochondrial membrane (IMM) and changes the proton gradient in the mitochondria. This activates NFkB and Jmjd3 expression to remove the repressive marker H3K27me3. Accordingly, the epigenome is altered by the cholesterol reduction. When subsequently challenged by the inflammatory stimulus LPS (M1), both macrophages treated with statins in vitro or isolated from statin-treated mice in vivo, express lower levels pro-inflammatory cytokines than controls, while augmenting anti-inflammatory Il10 expression. On the other hand, when macrophages are alternatively activated by IL4 (M2), statins promote the expression of Arg1, Ym1, and Mrc1. The enhanced expression is correlated with the statin-induced removal of H3K27me3 from these genes prior to activation. In addition, Jmjd3 and its demethylase activity are necessary for cholesterol to modulate both M1 and M2 activation. We conclude that upregulation of Jmjd3 is a key event for the anti-inflammatory function of statins on macrophages.
RESUMEN
The present work addresses the fundamental question of membrane elasticity of ceramide layers with a special focus on the plastic regime. The compression and shear viscoelasticity of egg-ceramide Langmuir monolayers were investigated using oscillatory surface rheology in the linear regime and beyond. High compression and shear moduli were measured at room temperature-a clear signature for a solid behavior. At deformations larger than one per mill, egg-ceramide monolayers display plastic features characterized by a decrease of the storage modulus followed by a viscous regime typical of fluid lipids. This behavior is accompanied by a marked decrease of the loss modulus with increasing stress above a yield point. The results permit to univocally classify ceramide monolayers as 2D solids able to undergo plastic deformations, at the difference of typical fluid lipid monolayers. These unusual features are likely to have consequences in the mechanical behavior of ceramide-rich emplacements in biological membranes.