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

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.

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.

C60-based Multivalent Glycoporphyrins Inhibit SARS-CoV-2 Specific Interaction with the DC-SIGN Transmembrane Receptor.

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.

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.

Electro-enzymatic ATP regeneration coupled to biocatalytic phosphorylation reactions.

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 F1Fo-ATP synthase from Escherichia coli. Thus, H2 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+.

Rheology of Pseudomonas fluorescens biofilms: From experiments to predictive DPD mesoscopic modeling.

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.

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.

Transgene expression in mice of the Opa1 mitochondrial transmembrane protein through bicontinuous cubic lipoplexes containing gemini imidazolium surfactants.

BACKGROUND: Lipoplexes are non-viral vectors based on cationic lipids used to deliver DNA into cells, also known as lipofection. The positively charge of the hydrophilic head-group provides the cationic lipids the ability to condensate the negatively charged DNA into structured complexes. The polar head can carry a large variety of chemical groups including amines as well as guanidino or imidazole groups. In particular, gemini cationic lipids consist of two positive polar heads linked by a spacer with different length. As for the hydrophobic aliphatic chains, they can be unsaturated or saturated and are connected to the polar head-groups. Many other chemical components can be included in the formulation of lipoplexes to improve their transfection efficiency, which often relies on their structural features. Varying these components can drastically change the arrangement of DNA molecules within the lamellar, hexagonal or cubic phases that are provided by the lipid matrix. Lipofection is widely used to deliver genetic material in cell culture experiments but the simpler formulations exhibit major drawbacks related to low transfection, low specificity, low circulation half-life and toxicity when scaled up to in vivo experiments. RESULTS: So far, we have explored in cell cultures the transfection ability of lipoplexes based on gemini cationic lipids that consist of two C16 alkyl chains and two imidazolium polar head-groups linked with a polyoxyethylene spacer, (C16Im)2(C4O). Here, PEGylated lipids have been introduced to the lipoplex formulation and the transgene expression of the Opa1 mitochondrial transmembrane protein in mice was assessed. The addition of PEG on the surface of the lipid mixed resulted in the formation of Ia3d bicontinuous cubic phases as determined by small angle X-ray scattering. After a single intramuscular administration, the cubic lipoplexes were accumulated in tissues with tight endothelial barriers such as brain, heart, and lungs for at least 48 h. The transgene expression of Opa1 in those organs was identified by western blotting or RNA expression analysis through quantitative polymerase chain reaction. CONCLUSIONS: The expression reported here is sufficient in magnitude, duration and toxicity to consolidate the bicontinuous cubic structures formed by (C16Im)2(C4O)-based lipoplexes as valuable therapeutic agents in the field of gene delivery.

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.

Na+ controls hypoxic signalling by the mitochondrial respiratory chain.

All metazoans depend on the consumption of O2 by the mitochondrial oxidative phosphorylation system (OXPHOS) to produce energy. In addition, the OXPHOS uses O2 to produce reactive oxygen species that can drive cell adaptations1-4, a phenomenon that occurs in hypoxia4-8 and whose precise mechanism remains unknown. Ca2+ is the best known ion that acts as a second messenger9, yet the role ascribed to Na+ is to serve as a mere mediator of membrane potential10. Here we show that Na+ acts as a second messenger that regulates OXPHOS function and the production of reactive oxygen species by modulating the fluidity of the inner mitochondrial membrane. A conformational shift in mitochondrial complex I during acute hypoxia11 drives acidification of the matrix and the release of free Ca2+ from calcium phosphate (CaP) precipitates. The concomitant activation of the mitochondrial Na+/Ca2+ exchanger promotes the import of Na+ into the matrix. Na+ interacts with phospholipids, reducing inner mitochondrial membrane fluidity and the mobility of free ubiquinone between complex II and complex III, but not inside supercomplexes. As a consequence, superoxide is produced at complex III. The inhibition of Na+ import through the Na+/Ca2+ exchanger is sufficient to block this pathway, preventing adaptation to hypoxia. These results reveal that Na+ controls OXPHOS function and redox signalling through an unexpected interaction with phospholipids, with profound consequences for cellular metabolism.

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.

Potentiometric detection of ATP based on the transmembrane proton gradient generated by ATPase reconstituted on a gold electrode.

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.

Intercellular Trafficking of Gold Nanostars in Uveal Melanoma Cells for Plasmonic Photothermal Therapy.

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.

Self-Adaptation of Pseudomonas fluorescens Biofilms to Hydrodynamic Stress.

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.

Liquid-liquid phase separation of the Golgi matrix protein GM130.

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.