RESUMEN
Avian eggshells exhibit excellent antimicrobial properties. In this study, we conducted simulation experiments to explore the defense mechanisms of eggshell membranes with regards to their physical features. We developed a mathematical model for the movement of microorganisms and estimated their penetration ratio into eggshell membranes based on several factors, including membrane thickness, microbial size, directional drift, and attachment probability to membrane fibers. These results not only suggest that an eggshell membrane with multiple layers and low porosity indicates high antimicrobial performance, but also imply that the fibrous network structure of the membrane might contribute to effective defense. Our simulation results aligned with experimental findings, specifically in measuring the penetration time of Escherichia coli through the eggshell membrane. We briefly discuss the significance and limitations of this pilot study, as well as the potential for these results, to serve as a foundation for the development of antimicrobial materials.
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Cáscara de Huevo , Escherichia coli , Cáscara de Huevo/microbiología , Animales , Escherichia coli/fisiología , Simulación por Computador , Modelos Biológicos , Membranas/metabolismo , Aves , Modelos TeóricosRESUMEN
Liquid protein condensates play important roles in orchestrating subcellular organization and as biochemical reaction hubs. Recent studies have linked lipid membranes to proteins capable of forming liquid condensates, and shown that biophysical parameters, like protein enrichment and restricted diffusion at membranes, regulate condensate formation and size. However, the impact of membrane topography on liquid condensates remains poorly understood. Here, we devised a cell-free system to reconstitute liquid condensates on lipid membranes with microstructured topographies and demonstrated that lipid membrane topography is a significant biophysical regulator. Using membrane surfaces designed with microwells, we observed ordered condensate patterns. Furthermore, we demonstrate that membrane topographies influence the shape of liquid condensates. Finally, we show that capillary forces, mediated by membrane topographies, lead to the directed fusion of liquid condensates. Our results demonstrate that membrane topography is a potent biophysical regulator for the localization and shape of mesoscale liquid protein condensates.
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Lípidos , Membranas , Transporte Biológico , Biofisica , Sistema Libre de CélulasRESUMEN
The plasma membrane forms the boundary between a living entity and its environment and acts as a barrier to permeation and flow of substances. Several computational means of calculating permeability have been implemented for molecular dynamics (MD) simulations-based approaches. Except for double bilayer systems, most permeability studies have been performed under equilibrium conditions, in large part due to the challenges associated with creating concentration gradients in simulations utilizing periodic boundary conditions. To enhance the scientific understanding of permeation and complement the existing computational means of characterizing membrane permeability, we developed a non-equilibrium method that enables the generation and maintenance of steady-state gradients in MD simulations. We utilize PBCs advantageously by imposing a directional bias to the motion of permeants so that their crossing of the boundary replenishes the gradient, like a previous study on ions. Under these conditions, a net flow of permeants across membranes may be observed to determine bulk permeability by a direct application of J=PΔc. In the present study, we explore the results of its application to an exemplary O2 and POPC bilayer system, demonstrating accurate and precise permeability measurements. In addition, we illustrate the impact of permeant concentration and the choice of thermostat on the permeability. Moreover, we demonstrate that energetics of permeation can be closely examined by the dissipation of the gradient across the membrane to gain nuanced insights into the thermodynamics of permeability.
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Fiebre Mediterránea Familiar , Simulación de Dinámica Molecular , Humanos , Membranas , Membrana Celular , Permeabilidad de la Membrana CelularRESUMEN
Glycerophospholipids are synthesized primarily in the cytosolic leaflet of the endoplasmic reticulum (ER) membrane and must be equilibrated between bilayer leaflets to allow the ER and membranes derived from it to grow. Lipid equilibration is facilitated by integral membrane proteins called "scramblases." These proteins feature a hydrophilic groove allowing the polar heads of lipids to traverse the hydrophobic membrane interior, similar to a credit card moving through a reader. Nevertheless, despite their fundamental role in membrane expansion and dynamics, the identity of most scramblases has remained elusive. Here, combining biochemical reconstitution and molecular dynamics simulations, we show that lipid scrambling is a general feature of protein insertases, integral membrane proteins which insert polypeptide chains into membranes of the ER and organelles disconnected from vesicle trafficking. Our data indicate that lipid scrambling occurs in the same hydrophilic channel through which protein insertion takes place and that scrambling is abolished in the presence of nascent polypeptide chains. We propose that protein insertases could have a so-far-overlooked role in membrane dynamics as scramblases.
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Proteínas de la Membrana , Péptidos , Membrana Celular/metabolismo , Proteínas de la Membrana/metabolismo , Péptidos/metabolismo , Membranas/metabolismo , Lípidos , Membrana Dobles de Lípidos/químicaRESUMEN
Metabolic abnormalities are at the center of many diseases, and the capability to film and quantify the metabolic activities of a single cell is important for understanding the heterogeneities in these abnormalities. In this paper, a functional plasmonic microscope (FPM) is used to image and measure metabolic activities without fluorescent labels at a single-cell level. The FPM can accurately image and quantify the subnanometer membrane fluctuations with a spatial resolution of 0.5 µm in real time. These active cell membrane fluctuations are caused by metabolic activities across the cell membrane. A three-dimensional (3D) morphology of the bottom cell membrane was imaged and reconstructed with FPM to illustrate the capability of the microscope for cell membrane characterization. Then, the subnanometer cell membrane fluctuations of single cells were imaged and quantified with the FPM using HeLa cells. Cell metabolic heterogeneity is analyzed based on membrane fluctuations of each individual cell that is exposed to similar environmental conditions. In addition, we demonstrated that the FPM could be used to evaluate the therapeutic responses of metabolic inhibitors (glycolysis pathway inhibitor STF 31) on a single-cell level. The result showed that the metabolic activities significantly decrease over time, but the nature of this response varies, depicting cell heterogeneity. A low-concentration dose showed a reduced fluctuation frequency with consistent fluctuation amplitudes, while the high-concentration dose showcased a decreasing trend in both cases. These results have demonstrated the capabilities of the functional plasmonic microscope to measure and quantify metabolic activities for drug discovery.
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Colorantes , Microscopía , Humanos , Células HeLa , Membrana Celular , MembranasRESUMEN
Mapping the N-glycome of porcine sperm before and after sperm capacitation is important for understanding the rearrangement of glycoconjugates during capacitation. In this work, we characterized the N-glycome on the membranes of 18 pairs of fresh porcine sperm before capacitation and porcine sperm after capacitation by MALDI-MS (Matrix-assisted laser desorption/ionization-mass spectrometry). A total of 377 N-glycans were detected and a comprehensive N-glycome map of porcine sperm membranes before and after capacitation was generated, which presents the largest N-glycome dataset of porcine sperm cell membranes. Statistical analysis revealed a significantly higher level of high mannose glycosylation and a significantly lower level of fucosylation, galactosylation, and α-2,6-NeuAc after capacitation, which is further verified by flow cytometry and lectin blotting. This research reveals new insights into the relationship between N-glycosylation variations and sperm capacitation, including the underlying mechanisms of the capacitation process.
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Peróxido de Benzoílo , Semen , Masculino , Porcinos , Animales , Membranas , Membrana Celular , EspermatozoidesRESUMEN
Membrane-based sensors (MePSs) exhibit remarkable precision and sensitivity in detecting pressure changes. MePSs are commonly used to monitor catalytic reactions in solution, generating gas products crucial for signal amplification in bioassays. They also allow for catalyst quantification by indirectly measuring the pressure generated by the gaseous products. This is particularly interesting for detecting enzymes in biofluids associated with disease onset. To enhance the performance of a MePS, various structural factors influence membrane flexibility and response time, ultimately dictating the device's pressure sensitivity. In this study, we fabricated MePSs using polydimethylsiloxane (PDMS) and investigated how structural modifications affect the Young's modulus (E) and residual stress (σ0) of the membranes. These modifications have a direct impact on the sensors' sensitivity to pressure variations, observed as a function of the volume of the chamber (Σ) or of the mechanical properties of the membrane itself (S). MePSs exhibiting the highest sensitivities were then employed to detect catalyst quantities inducing the dismutation of hydrogen peroxide, producing dioxygen as a gaseous product. As a result, a catalase enzyme was successfully detected using these optimized MePSs, achieving a remarkable sensitivity of (22.7 ± 1.2) µm/nM and a limit of detection (LoD) of 396 pM.
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Bioensayo , Gases , Catalasa , Membranas , Catálisis , Módulo de ElasticidadRESUMEN
Endocytosis is a common process observed in most eukaryotic cells, although its complexity varies among different organisms. In Trypanosoma brucei, the endocytic machinery is under special selective pressure because rapid membrane recycling is essential for immune evasion. This unicellular parasite effectively removes host antibodies from its cell surface through hydrodynamic drag and fast endocytic internalization. The entire process of membrane recycling occurs exclusively through the flagellar pocket, an extracellular organelle situated at the posterior pole of the spindle-shaped cell. The high-speed dynamics of membrane flux in trypanosomes do not seem compatible with the conventional concept of distinct compartments for early endosomes (EE), late endosomes (LE), and recycling endosomes (RE). To investigate the underlying structural basis for the remarkably fast membrane traffic in trypanosomes, we employed advanced techniques in light and electron microscopy to examine the three-dimensional architecture of the endosomal system. Our findings reveal that the endosomal system in trypanosomes exhibits a remarkably intricate structure. Instead of being compartmentalized, it constitutes a continuous membrane system, with specific functions of the endosome segregated into membrane subdomains enriched with classical markers for EE, LE, and RE. These membrane subdomains can partly overlap or are interspersed with areas that are negative for endosomal markers. This continuous endosome allows fast membrane flux by facilitated diffusion that is not slowed by multiple fission and fusion events.
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Endosomas , Trypanosoma , Membranas , Membrana Celular , Vesículas TransportadorasRESUMEN
Salmonella utilizes a type 3 secretion system to translocate virulence proteins (effectors) into host cells during infection1. The effectors modulate host cell machinery to drive uptake of the bacteria into vacuoles, where they can establish an intracellular replicative niche. A remarkable feature of Salmonella invasion is the formation of actin-rich protuberances (ruffles) on the host cell surface that contribute to bacterial uptake. However, the membrane source for ruffle formation and how these bacteria regulate membrane mobilization within host cells remains unclear. Here, we show that Salmonella exploits membrane reservoirs for the generation of invasion ruffles. The reservoirs are pre-existing tubular compartments associated with the plasma membrane (PM) and are formed through the activity of RAB10 GTPase. Under normal growth conditions, membrane reservoirs contribute to PM homeostasis and are preloaded with the exocyst subunit EXOC2. During Salmonella invasion, the bacterial effectors SipC, SopE2, and SopB recruit exocyst subunits from membrane reservoirs and other cellular compartments, thereby allowing exocyst complex assembly and membrane delivery required for bacterial uptake. Our findings reveal an important role for RAB10 in the establishment of membrane reservoirs and the mechanisms by which Salmonella can exploit these compartments during host cell invasion.
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Infecciones por Salmonella , Salmonella typhimurium , Humanos , Salmonella typhimurium/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Infecciones por Salmonella/microbiología , Membrana Celular/metabolismo , Membranas/metabolismo , Células HeLaRESUMEN
The organization of membrane proteins between and within membrane-bound compartments is critical to cellular function. Yet we lack approaches to regulate this organization in a range of membrane-based materials, such as engineered cells, exosomes, and liposomes. Uncovering and leveraging biophysical drivers of membrane protein organization to design membrane systems could greatly enhance the functionality of these materials. Towards this goal, we use de novo protein design, molecular dynamic simulations, and cell-free systems to explore how membrane-protein hydrophobic mismatch could be used to tune protein cotranslational integration and organization in synthetic lipid membranes. We find that membranes must deform to accommodate membrane-protein hydrophobic mismatch, which reduces the expression and co-translational insertion of membrane proteins into synthetic membranes. We use this principle to sort proteins both between and within membranes, thereby achieving one-pot assembly of vesicles with distinct functions and controlled split-protein assembly, respectively. Our results shed light on protein organization in biological membranes and provide a framework to design self-organizing membrane-based materials with applications such as artificial cells, biosensors, and therapeutic nanoparticles.
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Células Artificiales , Proteínas de la Membrana , Membrana Celular/metabolismo , Membranas/metabolismo , Proteínas de la Membrana/metabolismo , Liposomas , Membrana Dobles de Lípidos/químicaRESUMEN
The formation of pores over lipid membranes by the application of electric fields, termed membrane electroporation, is widely used in biotechnology and medicine to deliver drugs, vaccines, or genes into living cells. Continuum models for describing the free energy landscape of membrane electroporation were proposed decades ago, but they have never been tested against spatially detailed atomistic models. Using molecular dynamics (MD) simulations with a recently proposed reaction coordinate, we computed potentials of mean force of pore nucleation and pore expansion in lipid membranes at various transmembrane potentials. Whereas the free energies of pore expansion are compatible with previous continuum models, the experimentally important free energy barrier of pore nucleation is at variance with established models. The discrepancy originates from different geometries of the transition state; previous continuum models assumed the presence of a membrane-spanning defect throughout the process, whereas, according to the MD simulations, the transition state of pore nucleation is typically passed before a transmembrane defect has formed. A modified continuum model is presented that qualitatively agrees with the MD simulations. Using kinetics of pore opening together with transition state theory, our free energies of pore nucleation are in excellent agreement with previous experimental data.
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Electroporación , Membrana Dobles de Lípidos , Simulación de Dinámica Molecular , Membranas , Potenciales de la MembranaRESUMEN
Intercellular membrane-membrane interfaces are compartments with specialized functions and unique biophysical properties that are essential in numerous cellular processes including cell signaling, development, and immunity. Using synthetic biology to engineer or to create novel cellular functions in the intercellular regions has led to an increasing need for a platform that allows generation of functionalized intercellular membrane-membrane interfaces. Here, we present a synthetic biology platform to engineer functional membrane-membrane interfaces using a pair of dimerizing proteins in both cell-free and cellular environments. We envisage this platform to be a helpful tool for synthetic biologists who wish to engineer novel intercellular signaling and communication systems.
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Transducción de Señal , Biología Sintética , Animales , Membranas , Biofisica , Dimerización , MamíferosRESUMEN
Diatoms such as Phaeodactylum tricornutum arose through a process termed secondary endosymbiosis, in which red alga-derived plastids are surrounded by a complicated membrane system. Subcellular marker proteins provide defined localizations on the compartmental and even sub-compartmental levels in the complex plastids of diatoms. Here we introduce how to use subcellular marker proteins and in vivo co-localization in the diatom P. tricornutum by presenting a step-by-step method allowing the determination of subcellular localization of proteins in different membranes of the secondary plastid. This chapter describes the materials required and the procedures of transformation and microscopic observation.
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Diatomeas , Diatomeas/metabolismo , Proteínas/metabolismo , Membranas , Simbiosis , Plastidios/metabolismoRESUMEN
Plasma membrane damage occurs in healthy cells and more frequently in cancer cells where high growth rates and metastasis result in frequent membrane damage. The annexin family of proteins plays a key role in membrane repair. Annexins are recruited at the membrane injury site by Ca+2 and repair the damaged membrane in concert with several other proteins. Annexin A4 (ANXA4) and ANXA5 form trimers at the bilayer surface, and previous simulations show that the trimers induce high local negative membrane curvature on a flat bilayer. The membrane-curvature-inducing property of ANXA5 is presumed to be vital to the membrane repair mechanism. A previously proposed descriptive model hypothesizes that ANXA5-mediated curvature force is utilized at the free edge of the membrane at a wound site to pull the wound edges together, resulting in the formation of a "neck"-shaped structure, which, when combined with a constriction force exerted by ANXA6, leads to membrane repair. The molecular details and mechanisms of repair remain unknown, in part because the membrane edge is a transient structure that is difficult to investigate both experimentally and computationally. For the first time, we investigate the impact of ANXA5 near a membrane edge, which is modeled by a bicelle under periodic boundary conditions. ANXA5 trimers induce local curvature on the membrane leading to global bending of the bicelle. The global curvature depends on the density of annexins on the bicelle, and the curvature increases with the ANXA5 concentration until it reaches a plateau. The simulations suggest that not only do annexins induce local membrane curvature, but they can change the overall shape of a free-standing membrane. We also demonstrate that ANXA5 trimers reduce the rate of phosphatidylserine lipid diffusion from the cytoplasmic to the exoplasmic leaflet along the edge of the bicelle. In this way, membrane-bound annexins can potentially delay the apoptotic signal triggered by the presence of phosphatidylserine lipids in the outer leaflet, thus biding time for repair of the membrane hole. Our findings provide new insights into the role of ANXA5 at the edges of the membrane (the injury site) and support the curvature-constriction model of membrane repair.
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Anexinas , Fosfatidilserinas , Anexina A5/análisis , Anexina A5/metabolismo , Fosfatidilserinas/metabolismo , Membrana Celular/metabolismo , Anexinas/análisis , Anexinas/química , Anexinas/metabolismo , Membranas/metabolismoRESUMEN
The cellular endocytosis of nanoparticles (NPs) is a fundamental biological process with significant potential in biomedical applications. However, a comprehensive understanding of the mechanistic aspects of endocytosis and the impact of particle properties on this process remains elusive. In this study, we investigated the membrane-wrapping behavior of soft NPs (SNPs) with varying rigidities using theoretical calculations. Our findings reveal that the membrane-wrapping process of SNPs involves a complex energy change including the possible existence of an energy barrier; moreover, it is found that the location and height of this barrier strongly depend on the mechanistic properties of the NPs and membranes. Additionally, by considering force balance in the membrane-wrapping process, we calculated the speed at which NP is internalized by the membrane, showing a nonmonotonic dependence on particle rigidity and/or wrapping degree. These phenomena can be attributed to competition between different energy components associated with NP-membrane binding, membrane tension, and deformations occurring during SNP-membrane interaction processes. Our results contribute to a deeper understanding of cellular-level endocytosis mechanisms and offer potential applications for soft NPs in biomedicine.
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Nanopartículas , Membrana Celular/química , Nanopartículas/química , Membranas , Endocitosis , Fenómenos FísicosRESUMEN
Cell-free expression (CFE) systems are powerful tools in synthetic biology that allow biomimicry of cellular functions like biosensing and energy regeneration in synthetic cells. Reconstruction of a wide range of cellular processes, however, requires successful reconstitution of membrane proteins into the membrane of synthetic cells. While the expression of soluble proteins is usually successful in common CFE systems, the reconstitution of membrane proteins in lipid bilayers of synthetic cells has proven to be challenging. Here, a method for reconstitution of a model membrane protein, bacterial glutamate receptor (GluR0), in giant unilamellar vesicles (GUVs) as model synthetic cells based on encapsulation and incubation of the CFE reaction inside synthetic cells is demonstrated. Utilizing this platform, the effect of substituting the N-terminal signal peptide of GluR0 with proteorhodopsin signal peptide on successful cotranslational translocation of GluR0 into membranes of hybrid GUVs is demonstrated. This method provides a robust procedure that will allow cell-free reconstitution of various membrane proteins in synthetic cells.
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Membrana Dobles de Lípidos , Proteínas de la Membrana , Proteínas de la Membrana/metabolismo , Liposomas Unilamelares/metabolismo , Membranas/metabolismo , Señales de Clasificación de ProteínaRESUMEN
It is well-known that aqueous dispersions of phospholipids spontaneously assemble into bilayer structures. These structures have numerous applications across chemistry and materials science and form the fundamental structural unit of the biological membrane. The particular environment of the lipid bilayer, with a water-poor low dielectric core surrounded by a more polar and better hydrated interfacial region, gives the membrane particular biophysical and physicochemical properties and presents a unique environment for chemical reactions to occur. Many different types of molecule spanning a range of sizes, from dissolved gases through small organics to proteins, are able to interact with membranes and promote chemical changes to lipids that subsequently affect the physicochemical properties of the bilayer. This Review describes the chemical reactivity exhibited by lipids in their membrane form, with an emphasis on conditions where the lipids are well hydrated in the form of bilayers. Key topics include the following: lytic reactions of glyceryl esters, including hydrolysis, aminolysis, and transesterification; oxidation reactions of alkenes in unsaturated fatty acids and sterols, including autoxidation and oxidation by singlet oxygen; reactivity of headgroups, particularly with reactive carbonyl species; and E/Z isomerization of alkenes. The consequences of reactivity for biological activity and biophysical properties are also discussed.
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Membrana Dobles de Lípidos , Lípidos de la Membrana , Lípidos de la Membrana/química , Membrana Dobles de Lípidos/química , Membrana Celular/metabolismo , Membranas/metabolismo , Fosfolípidos/metabolismo , Alquenos/metabolismoRESUMEN
Voltage measurement via small-molecule fluorescent indicators is a valuable approach in deciphering complex dynamics in electrically excitable cells. However, our understanding of various physicochemical properties governing the performance of fluorescent voltage sensors based on the photoinduced electron transfer (PeT) mechanism remains incomplete. Here, through extensive molecular dynamics and free energy calculations, we systematically examine the orientation and membrane partition of three PeT-based voltage-sensing VoltageFluor (VF) dyes in different lipid environment. We show that the symmetry of the molecular scaffold and the net charge of the hydrophilic headgroup of a given VF dye dominate its orientation and membrane partition, respectively. Our work provides a mechanistic understanding of the physical properties contributing to the voltage sensitivity, signal-to-noise ratio, as well as membrane distribution of VF dyes and sheds light onto rational design principles of PeT-based fluorescent probes in general.
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Colorantes Fluorescentes , Simulación de Dinámica Molecular , Colorantes Fluorescentes/química , Potenciales de la Membrana , Transporte de Electrón , MembranasRESUMEN
OBJECTIVE: This study analyzed and compared the biomechanical properties of maxillary sinus floor mucosa with implants at three different maxillary sinus angles during a modified internal sinus floor elevation procedure. METHODS: 3D reconstruction of the implant, maxillary sinus bone, and membrane were performed. The maxillary sinus model was set at three different angles. Two internal maxillary sinus elevation models were established, and finite element analysis was used to simulate the modified maxillary sinus elevation process. The implant was elevated to 10 mm at three maxillary sinus angles when the maxillary sinus floor membrane was separated by 0 and 4 mm. The stress of the maxillary sinus floor membrane was analyzed and compared. RESULTS: When the maxillary sinus floor membrane was separated by 0 mm and elevated to 10 mm, the peak stress values of the implant on the maxillary sinus floor membrane at three different angles were as follows: maxillary sinus I: 5.14-78.32 MPa; maxillary sinus II: 2.81-73.89 MPa; and maxillary sinus III: 2.82-51.87 MPa. When the maxillary sinus floor membrane was separated by 4 mm and elevated to 10 mm, the corresponding values were as follows: maxillary sinus I: 0.50-7.25 MPa; maxillary sinus II: 0.81-16.55 MPa; and maxillary sinus III: 0.49-22.74 MPa. CONCLUSION: The risk of sinus floor membrane rupture is greatly reduced after adequate dissection of the maxillary sinus floor membrane when performing modified internal sinus elevation in a narrow maxillary sinus. In a wide maxillary sinus, the risk of rupture or perforation of the wider maxillary sinus floor is reduced, regardless of whether traditional or modified internal sinus elevation is performed at the same height.
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Implantes Dentales , Elevación del Piso del Seno Maxilar , Implantación Dental Endoósea/métodos , Elevación del Piso del Seno Maxilar/métodos , Seno Maxilar/cirugía , Membranas/cirugíaRESUMEN
Extracellular vesicles (EVs) are lipid bilayers derived from cell membranes, released by both eukaryotic cells and bacteria into the extracellular environment. During production, EVs carry proteins, nucleic acids, and various compounds, which are then released. While Gram-positive bacteria were traditionally thought incapable of producing EVs due to their thick peptidoglycan cell walls, recent studies on membrane vesicles (MVs) in Gram-positive bacteria have revealed their significant role in bacterial physiology and disease progression. This review explores the current understanding of MVs in Gram-positive bacteria, including the characterization of their content and functions, as well as their interactions with host and bacterial cells. It offers a fresh perspective to enhance our comprehension of Gram-positive bacterial EVs.