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In the past few decades great effort has been devoted to the study of water confined in hydrophobic geometries at the nanoscale (tubes and slit pores) due to the multiple technological applications of such systems, ranging from drug delivery to water desalination devices. To our knowledge, neither numerical/theoretical nor experimental approaches have so far reached a consensual understanding of structural and transport properties of water under these conditions. In this work, we present molecular dynamics simulations of TIP4P/2005 water under different nanoconfinements (slit pores or nanotubes, with two degrees of hydrophobicity) within a wide temperature range. It has been found that water is more structured near the less hydrophobic walls, independently of the confining geometries. Meanwhile, we observe an enhanced diffusion coefficient of water in both hydrophobic nanotubes. Finally, we propose a confined Stokes–Einstein relation to obtain the viscosity from diffusivity, whose result strongly differs from the Green–Kubo expression that has been used in previous works. While viscosity computed with the Green–Kubo formula (applied for anisotropic and confined systems) strongly differs from that of the bulk, viscosity computed with the confined Stokes–Einstein relation is not so much affected by the confinement, independently of its geometry. We discuss the shortcomings of both approaches, which could explain this discrepancy. [ABSTRACT FROM AUTHOR]

In a comment to our recent publication, Nicholls question our results and interpretation based on theoretical arguments that reveal a profound misunderstanding of our publication., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Eukaryotic cell function and survival rely on the use of a mitochondrial H + electrochemical gradient (Δp), which is composed of an inner mitochondrial membrane (IMM) potential (ΔΨmt) and a pH gradient (ΔpH). So far, ΔΨmt has been assumed to be composed exclusively of H + . Here, using a rainbow of mitochondrial and nuclear genetic models, we have discovered that a Na + gradient equates with the H + gradient and controls half of ΔΨmt in coupled-respiring mammalian mitochondria. This parallelism is controlled by the activity of the long-sought Na + -specific Na + /H + exchanger (mNHE), which we have identified as the P-module of complex I (CI). Deregulation of this mNHE function, without affecting the canonical enzymatic activity or the assembly of CI, occurs in Leber's hereditary optic neuropathy (LHON), which has profound consequences in ΔΨmt and mitochondrial Ca 2+ homeostasis and explains the previously unknown molecular pathogenesis of this neurodegenerative disease., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Competing interests statement:The authors declare no competing interest.

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., (© 2023 The Authors. Small published by Wiley‐VCH GmbH.)

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., Competing Interests: ZS, KD, YL, NV, DV, VA, JZ, KN, MC, JM, CC, AS, BS, IL, JL, JD, XZ No competing interests declared, (© 2024, Salloum et al.)

Expansion microscopy (ExM) is a new super-resolution technique based on embedding the biological sample within a hydrogel and its physical expansion after swelling. This allows increasing its size by several times while preserving its structural details. Applied to prokaryotic cells, ExM requires digestion steps for efficient expansion as bacteria are surrounded by a rigid cell wall. Furthermore, bacteria can live in social groups forming biofilms, where cells are protected from environmental stresses by a self-produced matrix. The extracellular matrix represents an additional impenetrable barrier for ExM. Here we optimize the current protocols of ExM and apply them to mono- and dual-species biofilms formed by clinical isolates of Limosilactobacillus reuteri, Enterococcus faecalis, Serratia marcescens and Staphylococcus aureus. Using scanning electron microscopy for comparison, our results demonstrate that embedded bacteria expanded 3-fold. Moreover, ExM allowed visualizing the three-dimensional architecture of the biofilm and identifying the distribution of different microbial species and their interactions. We also detected the presence of the extracellular matrix after expansion with a specific stain of the polysaccharide component. The potential applications of ExM in biofilms will improve our understanding of these complex communities and have far-reaching implications for industrial and clinical research., (© 2023. The Author(s).)

ATP synthases are proteins that catalyse the formation of ATP through the rotatory movement of their membrane-spanning subunit. In mitochondria, ATP synthases are found to arrange as dimers at the high-curved edges of cristae. Here, a direct link is explored between the rotatory movement of ATP synthases and their preference for curved membranes. An active curvature sorting of ATP synthases in lipid nanotubes pulled from giant vesicles is found. Coarse-grained simulations confirm the curvature-seeking behaviour of rotating ATP synthases, promoting reversible and frequent protein-protein contacts. The formation of transient protein dimers relies on the membrane-mediated attractive interaction of the order of 1.5 k B T produced by a hydrophobic mismatch upon protein rotation. Transient dimers are sustained by a conic-like arrangement characterized by a wedge angle of θ ≈ 50°, producing a dynamic coupling between protein shape and membrane curvature. The results suggest a new role of the rotational movement of ATP synthases for their dynamic self-assembly in biological membranes., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Adenosine-5-triphosphate (ATP) is the main energy vector in biological systems, thus its regeneration is an important issue for the application of many enzymes of interest in biocatalysis and synthetic biology. We have developed an electroenzymatic ATP regeneration system consisting in a gold electrode modified with a floating phospholipid bilayer that allows coupling the catalytic activity of two membrane-bound enzymes: NiFeSe hydrogenase from Desulfovibrio vulgaris and F 1 F o -ATP synthase from Escherichia coli. Thus, H 2 is used as a fuel for producing ATP. This electro-enzymatic assembly is studied as ATP regeneration system of phosphorylation reactions catalysed by kinases, such as hexokinase and NAD + -kinase for respectively producing glucose-6-phosphate and NADP + ., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Most antimicrobial peptides (AMPs) exert their microbicidal activity through membrane permeabilization. The designed AMP EcDBS1R4 has a cryptic mechanism of action involving the membrane hyperpolarization of Escherichia coli , suggesting that EcDBS1R4 may hinder processes involved in membrane potential dissipation. We show that EcDBS1R4 can sequester cardiolipin, a phospholipid that interacts with several respiratory complexes of E. coli . Among these, F 1 F O ATP synthase uses membrane potential to fuel ATP synthesis. We found that EcDBS1R4 can modulate the activity of ATP synthase upon partition to membranes containing cardiolipin. Molecular dynamics simulations suggest that EcDBS1R4 alters the membrane environment of the transmembrane F O motor, impairing cardiolipin interactions with the cytoplasmic face of the peripheral stalk that binds the catalytic F 1 domain to the F O domain. The proposed mechanism of action, targeting membrane protein function through lipid reorganization may open new venues of research on the mode of action and design of other AMPs., Competing Interests: Authors declare no competing interests., (© 2023 The Authors.)

A very simple, small and symmetric, but highly bright, photostable and functionalizable molecular probe for plasma membrane (PM) has been developed from an accessible, lipophilic and clickable organic dye based on BODIPY. To this aim, two lateral polar ammoniostyryl groups were easily linked to increase the amphiphilicity of the probe and thus its lipid membrane partitioning. Compared to the BODIPY precursor, the transversal diffusion across lipid bilayers of the ammoniostyryled BODIPY probe was highly reduced, as evidenced by fluorescence confocal microscopy on model membranes built up as giant unilamellar vesicles (GUVs). Moreover, the ammoniostyryl groups endow the new BODIPY probe with the ability to optically work (excitation and emission) in the bioimaging-useful red region, as shown by staining of the plasma membrane of living mouse embryonic fibroblasts (MEFs). Upon incubation, this fluorescent probe rapidly entered the cell through the endosomal pathway. By blocking the endocytic trafficking at 4 °C, the probe was confined within the PM of MEFs. Our experiments show the developed ammoniostyrylated BODIPY as a suitable PM fluorescent probe, and confirm the synthetic approach for advancing PM probes, imaging and science.

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.

The nasogastric enteral feeding tubes (NEFTs) used to feed preterm infants are commonly colonized by bacteria with the ability to form complex biofilms in their inner surfaces. Among them, staphylococci (mainly Staphylococcus epidermidis and Staphylococcus aureus ) and some species belonging to the Family Enterobacteriaceae are of special concern since they can cause nosocomial infections in this population. NETF-associated biofilms can also include lactic acid bacteria (LAB), with the ability to compete with pathogenic species for nutrients and space. Ecological interactions among the main colonizers of these devices have not been explored yet; however, such approach could guide future strategies involving the pre-coating of the inner surfaces of NEFTs with well adapted LAB strains in order to reduce the rates of nosocomial infections in neonatal intensive care units (NICUs). In this context, this work implied the formation of dual-species biofilms involving one LAB strain (either Ligilactobacillus salivarius 20SNG2 or Limosilactobacillus reuteri 7SNG3) and one nosocomial strain (either Klebsiella pneumoniae 9SNG3, Serratia marcescens 10SNG3, Staphylococcus aureus 45SNG3 or Staphylococcus epidermidis 46SNG3). The six strains used in this study had been isolated from the inner surface of NEFTs. Changes in adhesion ability of the pathogens were characterized using a culturomic approach. Species interactions and structural changes of the resulting biofilms were analyzed using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). No aggregation was observed in dual-species biofilms between any of the two LAB strains and either K. pneumoniae 9SNG3 or S. marcescens 10SNG3. In addition, biofilm thickness and volume were reduced, suggesting that both LAB strains can control the capacity to form biofilms of these enterobacteria. In contrast, a positive ecological relationship was observed in the combination L. reuteri 7SNG3- S. aureus 45SNG3. This relationship was accompanied by a stimulation of S. aureus matrix production when compared with its respective monospecies biofilm. The knowledge provided by this study may guide the selection of potentially probiotic strains that share the same niche with nosocomial pathogens, enabling the establishment of a healthier microbial community inside NEFTs., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer RC declared a past co-authorship with the author JR to the handling editor., (Copyright © 2022 Jara, Jurado, Almendro-Vedia, López-Montero, Fernández, Rodríguez and Orgaz.)

The mitochondrion is an essential organelle enclosed by two membranes whose functionalities depend on their very specific protein and lipid compositions. Proteins from the outer mitochondrial membrane (OMM) are specialized in mitochondrial dynamics and mitophagy, whereas proteins of the inner mitochondrial membrane (IMM) have dedicated functions in cellular respiration and apoptosis. As for lipids, the OMM is enriched in glycerophosphatidyl choline but cardiolipin is exclusively found within the IMM. Though the lipid topology and distribution of the OMM and IMM are known since more than four decades, little is known about the interfacial and dynamic properties of the IMM and OMM lipid extracts. Here we build monolayers, supported bilayers and giant unilamellar vesicles (GUVs) of native OMM and IMM lipids extracts from porcine heart. Additionally, we perform a comparative analysis on the interfacial, phase immiscibility and mechanical properties of both types of extract. Our results show that IMM lipids form more expanded and softer membranes than OMM lipids, allowing a better understanding of the physicochemical and biophysical properties of mitochondrial membranes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Schiaffarino, Valdivieso González, García-Pérez, Peñalva, Almendro-Vedia, Natale and López-Montero.)

Nonyl acridine orange (NAO) is a lipophilic and positively charged molecule widely used as a mitochondrial fluorescent probe. NAO is cytotoxic at micromolar concentration and might be potentially used as a mitochondria-targeted drug for cancer therapy. However, the use of NAO under in vivo conditions would be compromised by the unspecific interactions with off-target cells and negatively charged proteins present in the bloodstream. To tackle this limitation, we have synthesized NAO analogues carrying an imidazole group for their specific binding to nitrilotriacetic (NTA) functionalized gold nanorods (AuNRs). We demonstrate that AuNRs provide 10 4 binding sites and a controlled delivery under acidic conditions. Upon incubation with mouse embryonic fibroblasts, the endosomal acidic environment releases the NAO analogues from AuNRs, as visualized through the staining of the mitochondrial network. The addition of the monoclonal antibody Cetuximab to the conjugates enhanced their uptake within lung cancer cells and the conjugates were cytotoxic at subnanomolar concentrations ( c 50 ≈ 0.06 nM). Moreover, the specific interactions of Cetuximab with the epidermal growth factor receptor (EGFR) provided a specific targeting of EGFR-expressing lung cancer cells. After intravenous administration in patient-derived xenografts (PDX) mouse models, the conjugates reduced the progression of EGFR-positive tumors. Overall, the NAO-AuNRs provide a promising strategy to realize membrane mitochondria-targeted conjugates for lung cancer therapy.

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 C 16 alkyl chains and two imidazolium polar head-groups linked with a polyoxyethylene spacer, (C 16 Im) 2 (C 4 O). 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 (C 16 Im) 2 (C 4 O)-based lipoplexes as valuable therapeutic agents in the field of gene delivery., (© 2021. The Author(s).)

F 1 F o -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., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

In some conditions, bacteria self-organize into biofilms, supracellular structures made of a self-produced embedding matrix, mainly composed of polysaccharides, DNA, proteins, and lipids. It is known that bacteria change their colony/matrix ratio in the presence of external stimuli such as hydrodynamic stress. However, little is still known about the molecular mechanisms driving this self-adaptation. In this work, we monitor structural features of Pseudomonas fluorescens biofilms grown with and without hydrodynamic stress. Our measurements show that the hydrodynamic stress concomitantly increases the cell density population and the matrix production. At short growth timescales, the matrix mediates a weak cell-cell attractive interaction due to the depletion forces originated by the polymer constituents. Using a population dynamics model, we conclude that hydrodynamic stress causes a faster diffusion of nutrients and a higher incorporation of planktonic bacteria to the already formed microcolonies. This results in the formation of more mechanically stable biofilms due to an increase of the number of crosslinks, as shown by computer simulations. The mechanical stability also relies on a change in the chemical compositions of the matrix, which becomes enriched in carbohydrates, known to display adhering properties. Overall, we demonstrate that bacteria are capable of self-adapting to hostile hydrodynamic stress by tailoring the biofilm chemical composition, thus affecting both the mesoscale structure of the matrix and its viscoelastic properties that ultimately regulate the bacteria-polymer interactions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Jara, Alarcón, Monnappa, Santos, Bianco, Nie, Ciamarra, Canales, Dinis, López-Montero, Valeriani and Orgaz.)

All metazoans depend on the consumption of O 2 by the mitochondrial oxidative phosphorylation system (OXPHOS) to produce energy. In addition, the OXPHOS uses O 2 to produce reactive oxygen species that can drive cell adaptations 1-4 , a phenomenon that occurs in hypoxia 4-8 and whose precise mechanism remains unknown. Ca 2+ is the best known ion that acts as a second messenger 9 , yet the role ascribed to Na + is to serve as a mere mediator of membrane potential 10 . 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 hypoxia 11 drives acidification of the matrix and the release of free Ca 2+ from calcium phosphate (CaP) precipitates. The concomitant activation of the mitochondrial Na + /Ca 2+ 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 + /Ca 2+ 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.

Mitochondria are double-membrane organelles that continuously undergo fission and fusion. Outer mitochondrial membrane fusion is mediated by the membrane proteins mitofusin 1 (Mfn1) and mitofusin 2 (Mfn2), carrying a GTP hydrolyzing domain (GTPase) and two coiled-coil repeats. The detailed mechanism on how the GTP hydrolysis allows Mfns to approach adjacent membranes into proximity and promote their fusion is currently under debate. Using model membranes built up as giant unilamellar vesicles (GUVs), we show here that Mfn1 promotes membrane adhesion of apposing lipid vesicles. The adhesion forces were sustained by the GDP-bound state of Mfn1 after GTP hydrolysis. In contrast, the incubation with the GDP:AlF 4 - , which mimics the GTP transition state, did not induce membrane adhesion. Due to the flexible nature of lipid membranes, the adhesion strength depended on the surface concentration of Mfn1 through a cooperative binding mechanism. We discuss a possible scenario for the outer mitochondrial membrane fusion based on the modulated action of Mfn1.

Adenosine triphosphate (ATP) is a key molecule as energy vector for living organisms, therefore its detection reveals the presence of microbial colonies. Environments where the existence of microbial pathogens suppose a health hazard can benefit from real time monitoring of such molecule. We report a potentiometric biosensor based on ATP-synthase from Escherichia coli reconstituted in a floating phospholipid bilayer over gold electrodes modified with a 4-aminothiophenol self-assembled monolayer. The use of a pH-dependent redox probe on the electrode surface allows a simple, specific and reliable on site determination of ATP concentration from 1 μM to 1 mM. The broad range ATP biosensor can offer an alternative way of measuring in a few minutes the presence of microbial contamination., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Golgins are an abundant class of peripheral membrane proteins of the Golgi. These very long (50-400 nm) rod-like proteins initially capture cognate transport vesicles, thus enabling subsequent SNARE-mediated membrane fusion. Here, we explore the hypothesis that in addition to serving as vesicle tethers, Golgins may also possess the capacity to phase separate and, thereby, contribute to the internal organization of the Golgi. GM130 is the most abundant Golgin at the cis Golgi. Remarkably, overexpressed GM130 forms liquid droplets in cells analogous to those described for numerous intrinsically disordered proteins with low complexity sequences, even though GM130 is neither low in complexity nor intrinsically disordered. Virtually pure recombinant GM130 also phase-separates into dynamic, liquid-like droplets in close to physiological buffers and at concentrations similar to its estimated local concentration at the cis Golgi., (© 2019 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Efficient plasmonic photothermal therapies (PPTTs) using non-harmful pulse laser irradiation at the near-infrared (NIR) are a highly sought goal in nanomedicine. These therapies rely on the use of plasmonic nanostructures to kill cancer cells while minimizing the applied laser power density. Cancer cells have an unsettled capacity to uptake, retain, release, and re-uptake gold nanoparticles, thus offering enormous versatility for research. In this work, we have studied such cell capabilities for nanoparticle trafficking and its impact on the effect of photothermal treatments. As our model system, we chose uveal (eye) melanoma cells, since laser-assisted eye surgery is routinely used to treat glaucoma and cataracts, or vision correction in refractive surgery. As nanostructure, we selected gold nanostars (Au NSs) due to their high photothermal efficiency at the near-infrared (NIR) region of the electromagnetic spectrum. We first investigated the photothermal effect on the basis of the dilution of Au NSs induced by cell division. Using this approach, we obtained high PPTT efficiency after several cell division cycles at an initial low Au NS concentration (pM regime). Subsequently, we evaluated the photothermal effect on account of cell division upon mixing Au NS-loaded and non-loaded cells. Upon such mixing, we observed trafficking of Au NSs between loaded and non-loaded cells, thus achieving effective PPTT after several division cycles under low irradiation conditions (below the maximum permissible exposure threshold of skin). Our study reveals the ability of uveal melanoma cells to release and re-uptake Au NSs that maintain their plasmonic photothermal properties throughout several cell division cycles and re-uptake. This approach may be readily extrapolated to real tissue and even to treat in situ the eye tumor itself. We believe that our method can potentially be used as co-therapy to disperse plasmonic gold nanostructures across affected tissues, thus increasing the effectiveness of classic PPTT., Competing Interests: The authors declare no conflict of interest.

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.

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.

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.

Background: The fluorescent dye 10-N-nonyl acridine orange (NAO) is widely used as a mitochondrial marker. NAO was reported to have cytotoxic effects in cultured eukaryotic cells when incubated at high concentrations. Although the biochemical response of NAO-induced toxicity has been well identified, the underlying molecular mechanism has not yet been explored in detail., Methods: We use optical techniques, including fluorescence confocal microscopy and lifetime imaging microscopy (FLIM) both in model membranes built up as giant unilamellar vesicles (GUVs) and cultured cells. These experiments are complemented with computational studies to unravel the molecular mechanism that makes NAO cytotoxic., Results: We have obtained direct evidence that NAO promotes strong membrane adhesion of negatively charged vesicles. The attractive forces are derived from van der Waals interactions between anti-parallel H-dimers of NAO molecules from opposing bilayers. Semi-empirical calculations have confirmed the supramolecular scenario by which anti-parallel NAO molecules form a zipper of bonds at the contact region. The membrane remodeling effect of NAO, as well as the formation of H-dimers, was also confirmed in cultured fibroblasts, as shown by the ultrastructure alteration of the mitochondrial cristae., Conclusions: We conclude that membrane adhesion induced by NAO stacking accounts for the supramolecular basis of its cytotoxicity., General Significance: Mitochondria are a potential target for cancer and gene therapies. The alteration of the mitochondrial structure by membrane remodeling agents able to form supramolecular assemblies via adhesion properties could be envisaged as a new therapeutic strategy., (Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.)

Biocompatible soft materials have recently found applications in interventional endoscopy to facilitate resection of mucosal tumors. When neoplastic lesions are in organs that can be easily damaged by perforation, such as stomach, intestine, and esophagus, the formation of a submucosal fluid cushion (SFC) is needed to lift the tumor from the underlying muscle during the resection of neoplasias. Such procedure is called endoscopic submucosal dissection (ESD). We describe an injectable, biodegradable, hybrid hydrogel able to form a SFC and to facilitate ESD. The hydrogel, based on polyamidoamines, contains breakable silica nanocapsules covalently bound to its network and able to release biomolecules. To promote degradation, the hydrogel is composed of cleavable disulfide moieties that are reduced by the cells through secretion of glutathione. The same stimulus triggers the breaking of the silica nanocapsules; therefore, the entire hybrid material can be completely degraded and its decomposition depends entirely on the presence of cells. Interestingly, the hydrogel precursor solution showed rapid gelation when injected in vivo and afforded a long-lasting high mucosal elevation, keeping the cushion volume constant during the dissection. This novel material can provide a solution to ESD limitations and promote healing of tissues after surgery.

Cancer cell mitochondria represent an attractive target for oncological treatment as they have unique hallmarks that differ from their healthy counterparts, as the presence of a stronger membrane potential that can be exploited to specifically accumulate cytotoxic cationic molecules. Here, we explore the selective cytotoxic effect of 10- N -nonyl acridine orange (NAO) on human lung carcinoma H520 cells and compare them with healthy human lung primary fibroblasts. NAO is a lipophilic and positively charged molecule that promotes mitochondrial membrane adhesion that eventually leads to apoptosis when incubated at high micromolar concentration. We found an enhanced cytotoxicity of NAO in H520 cancer cells. By means Fluorescence lifetime imaging microscopy (FLIM) we also confirmed the formation of H-dimeric aggregates originating from opposing adjacent membranes that interfere with the mitochondrial membrane structure. Based on our results, we suggest the mitochondrial membrane as a potential target in cancer therapy to mechanically control the cell proliferation of cancer cells.

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., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: The authors declare no conflict of interest.

Cholesterol is an intriguing component of fluid lipid membranes: It makes them stiffer but also more fluid. Despite the enormous biological significance of this complex dynamical behavior, which blends aspects of membrane elasticity with viscous friction, their mechanical bases remain however poorly understood. Here, we show that the incorporation of physiologically relevant contents of cholesterol in model fluid membranes produces a fourfold increase in the membrane bending modulus. However, the increase in the compression rigidity that we measure is only twofold; this indicates that cholesterol increases coupling between the two membrane leaflets. In addition, we show that although cholesterol makes each membrane leaflet more fluid, it increases the friction between the membrane leaflets. This dissipative dynamics causes opposite but advantageous effects over different membrane motions: It allows the membrane to rearrange quickly in the lateral dimension, and to simultaneously dissipate out-of-plane stresses through friction between the two membrane leaflets. Moreover, our results provide a clear correlation between coupling and friction of membrane leaflets. Furthermore, we show that these rigid membranes are optimal to resist slow deformations with minimum energy dissipation; their optimized stability might be exploited to design soft technological microsystems with an encoded mechanics, vesicles or capsules for instance, useful beyond classical applications as model biophysical systems., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Whereas most of lipids have viscous properties and they do not have significant elastic features, ceramides behave as very rigid solid assemblies, displaying viscoelastic behaviour at physiological temperatures. The present review addresses the surface rheology of lipid binary mixtures made of sphingomyelin and ceramide. However, ceramide is formed by the enzymatic cleavage of sphingomyelin in cell plasma membranes. The consequences of the enzymatically-driven ceramide formation involve mechanical alterations of the embedding membrane. Here, an increase on surface shear viscosity was evidenced upon enzymatic incubation of sphingomyelin monolayers. The overall rheological data are discussed in terms of the current knowledge of the thermotropic behaviour of ceramide-containing model membranes., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

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 (K b = 5.0 × 10 5 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.

Erythrocyte membranes have been particularly useful as a model for studies of membrane structure and mechanics. Native erythroid membranes can be electroformed as giant unilamellar vesicles (eGUVs). In the presence of ATP, the erythroid membrane proteins of eGUVs rearrange into protein networks at the microscale. Here, we present a detailed nanomechanical study of individual protein microfilaments forming the protein networks of eGUVs when spread on supporting surfaces. Using Peak Force tapping Atomic Force Microscopy (PF-AFM) in liquid environment we have obtained the mechanical maps of the composite lipid-protein networks supported on solid surface. In the absence of ATP, the protein pool was characterized by a Young's Modulus E pool ≈5-15MPa whereas the complex filaments were found softer after protein supramolecular rearrangement; E fil ≈0.4MPa. The observed protein softening and reassembling could be relevant for understanding the mechanisms of cytoskeleton reorganization found in pathological erythrocytes or erythrocytes that are affected by biological agents., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

The search for efficient plasmonic photothermal therapies using nonharmful pulse laser irradiation at the near-infrared (NIR) is fundamental for biomedical cancer research. Therefore, the development of novel assembled plasmonic gold nanostructures with the aim of reducing the applied laser power density to a minimum through hot-spot-mediated cell photothermolysis is an ongoing challenge. We demonstrate that gold nanorods (Au NRs) functionalized at their tips with a pH-sensitive ligand assemble into oligomers within cell lysosomes through hydrogen-bonding attractive interactions. The unique intracellular features of the plasmonic oligomers allow us to significantly reduce the femtosecond laser power density and Au NR dose while still achieving excellent cell killing rates. The formation of gold tip-to-tip oligomers with longitudinal localized surface plasmon resonance bands at the NIR, obtained from low-aspect-ratio Au NRs close in resonance with 800 nm Ti:sapphire 90 fs laser pulses, was found to be the key parameter for realizing the enhanced plasmonic photothermal therapy.

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., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Ceramides have unique biophysical properties. Their high melting temperature and their ability to form lateral domains have converted ceramides into the paradigm of rigid lipids. Here, using shear surface rheology of egg-ceramide Langmuir monolayers, a solid to fluid transition was evidenced as a vanishing shear rigidity at lower temperatures than the lipid melting temperature. Such a mechanical transition, which depends on the lipid lateral pressure, was found in a broad range temperature (40-50 °C). The solid to fluid transition was correlated to a LC to LC+LE phase transition, as confirmed by BAM experiments. Interestingly, together with the softening transition, a supercooling process compatible with a glassy behavior was found upon freezing. A new phase scenario is then depicted that broadens the mechanical behavior of natural ceramides. The phase diversity of ceramides might have important implications in their physiological roles.

The presence of coupled modes of membrane motion in closed shells is extensively predicted by theory. The bilayer structure inherent to lipid vesicles is suitable to support hybrid modes of curvature motion coupling membrane bending with the local reorganization of the bilayer material through relaxation of the dilatational stresses. Previous experiments evidenced the existence of such hybrid modes facilitating membrane bending at high curvatures in lipid vesicles [Rodríguez-García, R., Arriaga, L.R., Mell, M., Moleiro, L.H., López-Montero, I., Monroy, F., 2009. Phys. Rev. Lett. 102, 128201.]. For lipid bilayers that are able to undergo intermonolayer sliding, the experimental fluctuation spectra are found compatible with a bimodal schema. The usual tension/bending fluctuations couple with the hybrid modes in a mechanical interplay, which becomes progressively efficient with increasing vesicle radius, to saturate at infinity radius into the behavior expected for a flat membrane. Grounded on the theory of closed shells, we propose an approximated expression of the bimodal spectrum, which predicts the observed dependencies on the vesicle radius. The dynamical features obtained from the autocorrelation functions of the vesicle fluctuations are found in quantitative agreement with the proposed theory., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

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.

Large vesicles obtained by the extrusion method represent adequate membrane models to probe membrane dynamics with neutron radiation. Particularly, the shape fluctuations around the spherical average topology can be recorded by neutron spin echo (NSE). In this paper we report on the applicable theories describing the scattering contributions from bending-dominated shape fluctuations in diluted vesicle dispersions, with a focus on the relative relevance of the master translational mode with respect to the internal fluctuations. Different vesicle systems, including bilayer and non-bilayer membranes, have been scrutinized. We describe the practical ranges where the exact theory of bending fluctuations is applicable to obtain the values of the bending modulus from experiments, and we discuss about the possible internal modes that could be alternatively contributing to shape fluctuations.

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.

The portal connector of bacteriophage viruses constitutes a robust molecular machine for DNA translocation. In this paper we propose an optimized reconstitution method for efficient orthogonal integration of native viral connectors into lipid bilayers, particularly of giant unilamellar vesicles. Our nanoengineering plan considers the hydrophilic connector protein of the bacteriophage virus ϕ29 integrated into a specifically engineered bilayer made of "hydrophylized" lipids. From the precise knowledge of the connector structure, the membrane chemistry was designed by tuning reactivity in the bilayer using specific functional lipids. We show details on the reconstitution methods and experimental evidence about the integration of the portal protein in the engineered membrane. The proposed route provides an efficient method for orthogonal integration of native viral connectors into lipid bilayers in conditions adequate for functional DNA translocation. This concept could be potentially exploited in advanced nanotechnological realizations, particularly for the integration of these powerful machines into giant lipid vesicles with the aim of building a cargo-device useful for gene delivery applications.

In the experimental approach to a synthetic minimal cell, the membrane compartment is a main component. Lipid vesicles represent the natural host for the artificial reconstruction of a cytomimetic membrane skeleton able to support mechanical function. Using the membrane component of human erythroid cells, we have reconstructed a membrane shell composed of a spectrin skeleton and fed by ATP. The structural and mechanical analysis reveals this spectrin skeleton as topological network supporting mechanical rigidity. Such an artificial shell would define a membrane compartment mechanically stable under physiological conditions.