Statin-mediated reduction in mitochondrial cholesterol primes an anti-inflammatory response in macrophages by upregulating Jmjd3.

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.

Statin-mediated reduction in mitochondrial cholesterol primes an anti-inflammatory response in macrophages by upregulating JMJD3.

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.

Expansion microscopy applied to mono- and dual-species biofilms.

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.

Rotation of the c-Ring Promotes the Curvature Sorting of Monomeric ATP Synthases.

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 kB 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.

Activity modulation of the Escherichia coli F1FO ATP synthase by a designed antimicrobial peptide via cardiolipin sequestering.

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, F1FO 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 FO motor, impairing cardiolipin interactions with the cytoplasmic face of the peripheral stalk that binds the catalytic F1 domain to the FO 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.

Polar ammoniostyryls easily converting a clickable lipophilic BODIPY in an advanced plasma membrane probe.

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.

Interspecies relationships between nosocomial pathogens associated to preterm infants and lactic acid bacteria in dual-species biofilms.

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.

Mitochondrial membrane models built from native lipid extracts: Interfacial and transport properties.

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.

Tunable gold nanorod/NAO conjugates for selective drug delivery in mitochondria-targeted cancer therapy.

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 104 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 (c50 ≈ 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.

Membrane rigidity regulates E. coli proliferation rates.

Combining single cell experiments, population dynamics and theoretical methods of membrane mechanics, we put forward that the rate of cell proliferation in E. coli colonies can be regulated by modifiers of the mechanical properties of the bacterial membrane. Bacterial proliferation was modelled as mediated by cell division through a membrane constriction divisome based on FtsZ, a mechanically competent protein at elastic interaction against membrane rigidity. Using membrane fluctuation spectroscopy in the single cells, we revealed either membrane stiffening when considering hydrophobic long chain fatty substances, or membrane softening if short-chained hydrophilic molecules are used. Membrane stiffeners caused hindered growth under normal division in the microbial cultures, as expected for membrane rigidification. Membrane softeners, however, altered regular cell division causing persistent microbes that abnormally grow as long filamentous cells proliferating apparently faster. We invoke the concept of effective growth rate under the assumption of a heterogeneous population structure composed by distinguishable individuals with different FtsZ-content leading the possible forms of cell proliferation, from regular division in two normal daughters to continuous growing filamentation and budding. The results settle altogether into a master plot that captures a universal scaling between membrane rigidity and the divisional instability mediated by FtsZ at the onset of membrane constriction.

How rotating ATP synthases can modulate membrane structure.

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.

The GDP-Bound State of Mitochondrial Mfn1 Induces Membrane Adhesion of Apposing Lipid Vesicles through a Cooperative Binding Mechanism.

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.

pH-triggered endosomal escape of pore-forming Listeriolysin O toxin-coated gold nanoparticles.

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.

Enhanced Cytotoxic Activity of Mitochondrial Mechanical Effectors in Human Lung Carcinoma H520 Cells: Pharmaceutical Implications for Cancer Therapy.

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.

Supramolecular zippers elicit interbilayer adhesion of membranes producing cell death.

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.

Nonequilibrium fluctuations of lipid membranes by the rotating motor protein F1F0-ATP synthase.

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.

Rhodamine-based sensor for real-time imaging of mitochondrial ATP in living fibroblasts.

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.

Modeling the Mechanics of Cell Division: Influence of Spontaneous Membrane Curvature, Surface Tension, and Osmotic Pressure.

Many cell division processes have been conserved throughout evolution and are being revealed by studies on model organisms such as bacteria, yeasts, and protozoa. Cellular membrane constriction is one of these processes, observed almost universally during cell division. It happens similarly in all organisms through a mechanical pathway synchronized with the sequence of cytokinetic events in the cell interior. Arguably, such a mechanical process is mastered by the coordinated action of a constriction machinery fueled by biochemical energy in conjunction with the passive mechanics of the cellular membrane. Independently of the details of the constriction engine, the membrane component responds against deformation by minimizing the elastic energy at every constriction state following a pathway still unknown. In this paper, we address a theoretical study of the mechanics of membrane constriction in a simplified model that describes a homogeneous membrane vesicle in the regime where mechanical work due to osmotic pressure, surface tension, and bending energy are comparable. We develop a general method to find approximate analytical expressions for the main descriptors of a symmetrically constricted vesicle. Analytical solutions are obtained by combining a perturbative expansion for small deformations with a variational approach that was previously demonstrated valid at the reference state of an initially spherical vesicle at isotonic conditions. The analytic approximate results are compared with the exact solution obtained from numerical computations, getting a good agreement for all the computed quantities (energy, area, volume, constriction force). We analyze the effects of the spontaneous curvature, the surface tension and the osmotic pressure in these quantities, focusing especially on the constriction force. The more favorable conditions for vesicle constriction are determined, obtaining that smaller constriction forces are required for positive spontaneous curvatures, low or negative membrane tension and hypertonic media. Conditions for spontaneous constriction at a given constriction force are also determined. The implications of these results for biological cell division are discussed. This work contributes to a better quantitative understanding of the mechanical pathway of cellular division, and could assist the design of artificial divisomes in vesicle-based self-actuated microsystems obtained from synthetic biology approaches.

Analytical results for cell constriction dominated by bending energy.

Analytical expressions are obtained for the main magnitudes of a symmetrically constricted vesicle. These equations provide an easy and compact way to predict minimal requirements for successful constriction and its main magnitudes. Thus, they can be useful for the design of synthetic divisomes and give good predictions for magnitudes including constriction energy, length of the constriction zone, volume and area of the vesicle, and the stability coefficient for symmetric constriction. The analytical expressions are derived combining a perturbative expansion in the Lagrangian for small deformations with a cosine ansatz in the constriction region. Already the simple fourth-order (or sixth-order) approximation provides a good approximation to the values of the main physical magnitudes during constriction, as we show through comparison with numerical results. Results are for vesicles with negligible effects from spontaneous curvature, surface tension, and pressure differences. This is the case when membrane components generating spontaneous curvature are scarce, membrane trafficking is present with low energetic cost, and the external medium is isotonic.