Vesicle Formation Research Articles

High-Speed Imaging of Giant Unilamellar Vesicle Formation in cDICE.

Giant unilamellar vesicles (GUVs) are widely used as in vitro model membranes in biophysics and as cell-sized containers in synthetic biology. Despite their ubiquitous use, there is no one-size-fits-all method for their production. Numerous methods have been developed to meet the demanding requirements of reproducibility, reliability, and high yield while simultaneously achieving robust encapsulation. Emulsion-based methods are often praised for their apparent simplicity and good yields; hence, methods like continuous droplet interface crossing encapsulation (cDICE), which make use of this principle, have gained popularity. However, the underlying physical principles governing the formation of GUVs in cDICE and related methods remain poorly understood. To this end, we have developed a high-speed microscopy setup that allows us to visualize GUV formation in real time. Our experiments reveal a complex droplet formation process occurring at the capillary orifice, generating >30 μm-sized droplets and only in some cases GUV-sized (∼15 μm) satellite droplets. According to existing theoretical models, the oil-water interface should allow for the crossing of all droplets, but based on our observations and scaling arguments on the fluid dynamics within the system, we find a size-selective crossing of GUV-sized droplets only. The origin of these droplets remains partly unclear; we hypothesize that some small GUVs might be formed from large droplets sitting at the second interface. Finally, we demonstrate that proteins in the inner solution affect GUV formation by increasing the viscosity and altering the lipid adsorption kinetics. These results will not only contribute to a better understanding of GUV formation processes in cDICE but ultimately also aid in the development of more reliable and efficient methods for GUV production.

S1P regulates intervertebral disc aging by mediating endoplasmic reticulum - mitochondrial calcium ion homeostasis.

Mechanistically, S1P deficiency impeded COP II-mediated transport vesicles formation, which leads to proteins retention in the endoplasmic reticulum (ER) and subsequently ER distension. ER distension increased the contact between the ER and mitochondria, disrupting ER-to-mitochondria calcium flow, resulting in mitochondrial dysfunction and energy metabolism disturbance. Finally, using 2-APB to inhibit calcium ion channels and the senolytic drug dasatinib and quercetin (D + Q) partially rescued the aging and degenerative phenotypes caused by S1P deficiency. In conclusion, our findings suggest that S1P is a critical factor in causing IVDD in the process of aging and highlight the potential of targeting S1P as a therapeutic approach for age-related IVDD.

Ablating the glutaredoxin-2 (Glrx2) gene protects male mice against non-alcoholic fatty liver disease (NAFLD) by limiting oxidative distress

In the present study, we investigated the consequences of deleting the glutaredoxin-2 gene (Glrx2−/−) on the development of non-alcoholic fatty liver disease (NAFLD) in male and female C57BL6N mice fed a control (CD) or high-fat diet (HFD). We report that the HFD induced a significant increase in body mass in the wild-type (Wt) and Glrx2−/− male, but not female, mice, which was associated with the hypertrophying of the abdominal fat. Interestingly, while the Wt male mice fed the HFD developed NAFLD, the deletion of the Glrx2 gene mitigated vesicle formation, intrahepatic lipid accumulation, and fibrosis in the males. The protective effect associated with ablating the Glrx2 gene in male mice was due to enhancement of mitochondrial redox buffering capacity. Specifically, liver mitochondria from male Glrx2−/− fed a CD or HFD produced significantly less hydrogen peroxide (mtH2O2), had lower malondialdehyde levels, greater activities for glutathione peroxidase and thioredoxin reductase, and less protein glutathione mixed disulfides (PSSG) when compared to the Wt male mice fed the HFD. These effects correlated with the S-glutathionylation of α-ketoglutarate dehydrogenase (KGDH), a potent mtH2O2 source and key redox sensor in hepatic mitochondria. In comparison to the male mice, both Wt and Glrx2−/− female mice displayed almost complete resistance to HFD-induced body mass increases and the development of NAFLD, which was attributed to the superior redox buffering capacity of the liver mitochondria. Together, our findings show that modulation of mitochondrial S-glutathionylation signaling through Glrx2 augments resistance of male mice towards the development of NAFLD through preservation of mitochondrial redox buffering capacity. Additionally, our findings demonstrate the sex dimorphisms associated with the manifestation of NAFLD is related to the superior redox buffering capacity and modulation of the S-glutathionylome in hepatic mitochondria from female mice.

Interaction between ESCRT-III proteins and the yeast SERINC homolog Tms1.

The endosomal sorting complex required for transport (ESCRT)-III is involved in membrane remodeling and abscission during intraluminal vesicle (ILV) formation at endosomes. Our data now suggest that ESCRT-III function could be connected to lipid remodeling of the endosomal membrane. This notion is based on our finding that ESCRT-III proteins bind to the yeast serine incorporator (SERINC) homolog Tms1. Human SERINC3 and SERINC5 are HIV-1 restriction factors and have been shown to act as scramblases, flipping phospholipids between membrane leaflets. Due to the extraordinarily high sequence conservation between Tms1 and human SERINCs, it is likely that Tms1 is also a scramblase. While deletion of TMS1 had only a moderate effect on the sorting of multivesicular body (MVB) cargo proteins, the simultaneous deletion of a component of the Vps55/Vps68 complex led to a strong synergistic phenotype. This pronounced synergism suggests that Tms1 and Vps55/Vps68 perform a parallel function at endosomes. Vps55/Vps68 loosely resembles Tms1 in its overall structure. Thus, it is possible that Vps55/Vps68 is also a scramblase. Since both Vps55 and Tms1 physically interact with ESCRT-III proteins, we propose that the recruitment of a scramblase plays a crucial role in ESCRT-III-dependent membrane remodeling at endosomes.

Stretch triggers microtubule stabilization and MARCKS-dependent membrane incorporation in the shaft of embryonic axons

Neurons have a unique polarized nature that must adapt to environmental changes throughout their lifespan. During embryonic development, axon elongation is led by the growth cone,1 culminating in the formation of a presynaptic terminal. After synapses are formed, axons elongate in a growth cone-independent manner to accompany body growth while maintaining their ultrastructure and function.2,3,4,5,6 To further understand mechanical strains on the axon shaft, we developed a computer-controlled stretchable microfluidic platform compatible with multi-omics and live imaging. Our data show that sensory embryonic dorsal root ganglia (DRGs) neurons have high plasticity, with axon shaft microtubules decreasing polymerization rates, aligning with the direction of tension, and undergoing stabilization. Moreover, in embryonic DRGs, stretch triggers yes-associated protein (YAP) nuclear translocation, supporting its participation in the regulatory network that enables tension-driven axon growth. Other than cytoskeleton remodeling, stretch prompted MARCKS-dependent formation of plasmalemmal precursor vesicles (PPVs), resulting in new membrane incorporation throughout the axon shaft. In contrast, adolescent DRGs showed a less robust adaptation, with axonal microtubules being less responsive to stretch. Also, while adolescent DRGs were still amenable to strain-induced PPV formation at higher stretch rates, new membrane incorporation in the axon shaft failed to occur. In summary, we developed a new resource to study the biology of axon stretch growth. By unraveling cytoskeleton adaptation and membrane remodeling in the axon shaft of stretched neurons, we are moving forward in understanding axon growth.

How coat proteins shape autophagy in plant cells.

Autophagy is a membrane trafficking pathway through which eukaryotic cells target their own cytoplasmic constituents for degradation in the lytic compartment. Proper biogenesis of autophagic organelles requires a conserved set of autophagy-related (ATG) proteins and their interacting factors, such as signalling phospholipid phosphatidylinositol 3-phosphate (PI3P) and coat complex II (COPII). The COPII machinery, which was originally identified as a membrane coat involved in the formation of vesicles budding from the endoplasmic reticulum, contributes to the initiation of autophagic membrane formation in yeast, metazoan, and plant cells; however, the exact mechanisms remain elusive. Recent studies using the plant model species Arabidopsis thaliana have revealed that plant-specific PI3P effectors are involved in autophagy. The PI3P effector FYVE2 interacts with the conserved PI3P effector ATG18 and with COPII components, indicating an additional role for the COPII machinery in the later stages of autophagosome biogenesis. In this Update, we examined recent research on plant autophagosome biogenesis and proposed working models on the functions of the COPII machinery in autophagy, including its potential roles in stabilizing membrane curvature and sealing the phagophore.

Biomolecular condensation of ERC1 recruits ATG8 and NBR1 to drive autophagosome formation for plant heat tolerance.

Macroautophagy (hereafter autophagy) is essential for cells to respond to nutrient stress by delivering cytosolic contents to vacuoles for degradation via the formation of a multi-layer vesicle named autophagosome. A set of autophagy-related (ATG) regulators are recruited to the phagophore assembly site for the initiation of phagophore, as well as its expansion and closure and subsequent delivery into the vacuole. However, it remains elusive that how the phagophore assembly is regulated under different stress conditions. Here, we described an unknown Arabidopsis (Arabidopsis thaliana) cytosolic ATG8-interaction protein family (ERC1/2), that binds ATG8 and NBR1 to promote autophagy. ERC1 proteins translocate to the phagophore membrane and develop into classical ring-like autophagosomes upon autophagic induction. However, ERC1 proteins form large droplets together with ATG8e proteins when in the absence of ATG8 lipidation activity. We described the property of these structures as phase-separated membraneless condensates by solving the in vivo organization with spatial and temporal resolution. Moreover, ERC1 condensates elicits a strong recruitment of the autophagic receptor NBR1. Loss of ERC1 suppressed NBR1 turnover and attenuated plant tolerance to heat stress condition. This work provides novel insights into the mechanical principle of phagophore initiation via an unreported ERC1-mediated biomolecular condensation for heat tolerance in Arabidopsis .

Structure and Characterization of Catanionic Complexes and Biocompatible Vesicles of N-Acyltaurine and Sarcosine Alkyl Ester: Encapsulation and Release Studies with 5-Fluorouracil.

Hydrated dispersions containing equimolar mixtures of cationic and anionic amphiphiles, referred to as catanionic systems, exhibit synergistic physicochemical properties, and mixing single-chain cationic and anionic lipids can lead to the spontaneous formation of vesicles as well as other phase structures. In the present work, we have characterized two catanionic systems prepared by mixing N-acyltaurines (NATs) and sarcosine alkyl esters (SAEs) bearing 11 and 12 C atoms in the acyl/alkyl chains. Turbidimetric and isothermal titration calorimetric studies revealed that both NATs form equimolar complexes with SAEs having matching acyl/alkyl chains. The three-dimensional structure of the sarcosine lauryl ester (lauryl sarcosinate, LS)-N-lauroyltaurine (NLT) equimolar complex has been determined by single-crystal X-ray diffraction. The LS-NLT equimolar complex is stabilized by electrostatic attraction and multiple hydrogen bonds, including classical, strong N-H···O hydrogen bonds as well as several C-H···O hydrogen bonds between the two amphiphiles. DSC studies showed that both equimolar complexes show single sharp phase transitions. Transmission electron microscopy and dynamic light scattering studies have demonstrated that the LS-NLT catanionic complex assemblies yield stable medium-sized vesicles (diameter 280-350 nm). These liposomes were disrupted at high pH, suggesting that the designed catanionic complexes can be used to develop base-labile drug delivery systems. In vitro studies with these catanionic liposomes showed efficient entrapment (73% loading) and release of the anticancer drug 5-fluorouracil in the physiologically relevant pH range of 6.0-8.0. The release rate was highest at pH 8.0, reaching about 78%, 90%, and 100% drug release at 2, 6, and 12 h, respectively. These observations indicate that LS-NLT catanionic vesicles will be useful for designing drug delivery systems, particularly for targeting organs such as the colon, which are inherently at basic pH.

Lipid vesicle formation by encapsulation of SMALPs in surfactant-stabilised droplets

Understanding the intricate functions of membrane proteins is pivotal in cell biology and drug discovery. The composition of the cell membrane is highly complex, with different types of membrane proteins and lipid species. Hence, studying cellular membranes in a complexity-reduced context is important to enhance our understanding of the roles of these different elements. However, reconstitution of membrane proteins in an environment that closely mimics the cell, like giant unilamellar vesicles (GUVs), remains challenging, often requiring detergents that compromise protein function. To address this challenge, we present a novel strategy to manufacture GUVs from styrene maleic acid lipid particles (SMALPs) that utilises surfactant-stabilised droplets as a template. As a first step towards the incorporation of membrane proteins, this work focusses on the conversion of pure lipid SMALPs in GUVs. To evaluate the method, we produced a new form of SMA linked to fluorescein, referred to as FSMA. We demonstrate the assembly of SMALPs at the surfactant-stabilised droplet interface, resulting in the formation of GUVs when released upon addition of a demulsifying agent. The released vesicles appear similar to electroformed vesicles imaged with confocal light microscopy, but a fluorescein leakage assay and cryo-TEM imaging reveal their porous nature, potentially as a result of residual interactions of SMA with the lipid bilayer. Our study represents a significant step towards opening new avenues for comprehensive protein research in a complexity-reduced, yet biologically relevant, setting.

The Rubicon-WIPI axis regulates exosome biogenesis during ageing.

Cells release intraluminal vesicles in multivesicular bodies as exosomes to communicate with other cells. Although recent studies suggest an intimate link between exosome biogenesis and autophagy, the detailed mechanism is not fully understood. Here we employed comprehensive RNA interference screening for autophagy-related factors and discovered that Rubicon, a negative regulator of autophagy, is essential for exosome release. Rubicon recruits WIPI2d to endosomes to promote exosome biogenesis. Interactome analysis of WIPI2d identified the ESCRT components that are required for intraluminal vesicle formation. Notably, we found that Rubicon is required for an age-dependent increase of exosome release in mice. In addition, small RNA sequencing of serum exosomes revealed that Rubicon determines the fate of exosomal microRNAs associated with cellular senescence and longevity pathways. Taken together, our current results suggest that the Rubicon-WIPI axis functions as a key regulator of exosome biogenesis and is responsible for age-dependent changes in exosome quantity and quality.

Hemifusomes and Interacting Proteolipid Nanodroplets: Formation of a Novel Cellular Organelle Complex.

Within cells, vesicle fusion, scission, and the formation of intraluminal vesicles are critical processes that facilitate traffic, degradation, and recycling of cellular components, and maintenance of cellular homeostasis. Despite significant advancements in elucidating the molecular mechanisms that drive these dynamic processes, the direct in situ visualization of membrane remodeling intermediates remains challenging. Here, through the application of cryo-electron tomography in mammalian cells, we have identified a previously undescribed vesicular organelle complex with unique membrane topology: heterotypic hemifused vesicles that share extended hemifusion diaphragms (HDs) with a 42 nm proteolipid nanodroplet (PND) at their rim. We have termed these organelle complexes "hemifusomes". The HDs of hemifusomes exhibit a range of sizes and curvatures, including the formation of lens-shaped compartments encapsulated within the membrane bilayer. The morphological diversity of the lens-shaped vesicle aligns with a step-wise process of their intraluminal budding, ultimately leading to their scission and the generation of intraluminal vesicles. We propose that hemifusomes function as versatile platforms for protein and lipid sorting and as central hubs for the biogenesis of intraluminal vesicles and the formation of multivesicular bodies.

Structural basis for lipid transfer by the ATG2A-ATG9A complex.

Autophagy is characterized by the formation of double-membrane vesicles called autophagosomes. Autophagy-related proteins (ATGs) 2A and 9A have an essential role in autophagy by mediating lipid transfer and re-equilibration between membranes for autophagosome formation. Here we report the cryo-electron microscopy structures of human ATG2A in complex with WD-repeat protein interacting with phosphoinositides 4 (WIPI4) at 3.2 Å and the ATG2A-WIPI4-ATG9A complex at 7 Å global resolution. On the basis of molecular dynamics simulations, we propose a mechanism of lipid extraction from the donor membranes. Our analysis revealed 3:1 stoichiometry of the ATG9A-ATG2A complex, directly aligning the ATG9A lateral pore with ATG2A lipid transfer cavity, and an interaction of the ATG9A trimer with both the N-terminal and the C-terminal tip of rod-shaped ATG2A. Cryo-electron tomography of ATG2A liposome-binding states showed that ATG2A tethers lipid vesicles at different orientations. In summary, this study provides a molecular basis for the growth of the phagophore membrane and lends structural insights into spatially coupled lipid transport and re-equilibration during autophagosome formation.

Mechanistic divergences of endocytic clathrin-coated vesicle formation in mammals, yeasts and plants.

Clathrin-coated vesicles (CCVs), generated by clathrin-mediated endocytosis (CME), are essential eukaryotic trafficking organelles that transport extracellular and plasma membrane-bound materials into the cell. In this Review, we explore mechanisms of CME in mammals, yeasts and plants, and highlight recent advances in the characterization of endocytosis in plants. Plants separated from mammals and yeast over 1.5 billion years ago, and plant cells have distinct biophysical parameters that can influence CME, such as extreme turgor pressure. Plants can therefore provide a wider perspective on fundamental processes in eukaryotic cells. We compare key mechanisms that drive CCV formation and explore what these mechanisms might reveal about the core principles of endocytosis across the tree of life. Fascinatingly, CME in plants appears to more closely resemble that in mammalian cells than that in yeasts, despite plants being evolutionarily further from mammals than yeast. Endocytic initiation appears to be highly conserved across these three systems, requiring similar protein domains and regulatory processes. Clathrin coat proteins and their honeycomb lattice structures are also highly conserved. However, major differences are found in membrane-bending mechanisms. Unlike in mammals or yeast, plant endocytosis occurs independently of actin, highlighting that mechanistic assumptions about CME across different systems should be made with caution.

Prominin 1 and Tweety Homology 1 both induce extracellular vesicle formation.

Prominin-1 (Prom1) is a five-transmembrane-pass integral membrane protein that associates with curved regions of the plasma membrane. Prom1 interacts with membrane cholesterol and actively remodels the plasma membrane. Membrane bending activity is particularly evident in photoreceptors, where Prom1 loss-of-function mutations cause failure of outer segment homeostasis, leading to cone-rod retinal dystrophy (CRRD). The Tweety Homology (Ttyh) protein family has been proposed to be homologous to Prominin, but it is not known whether Ttyh proteins have an analogous membrane-bending function. Here, we characterize the membrane-bending activity of human Prom1 and Ttyh1 in native bilayer membranes. We find that Prom1 and Ttyh1 both induce formation of extracellular vesicles (EVs) in cultured mammalian cells and that the EVs produced are physically similar. Ttyh1 is more abundant in EV membranes than Prom1 and produces EVs with membranes that are more tubulated than Prom1 EVs. We further show that Prom1 interacts more stably with membrane cholesterol than Ttyh1 and that this may contribute to membrane bending inhibition in Prom1 EVs. Intriguingly, a loss-of-function mutation in Prom1 associated with CRRD induces particularly stable cholesterol binding. These experiments provide mechanistic insight into Prominin function in CRRD and suggest that Prom and Ttyh belong to a single family of functionally related membrane-bending, EV-generating proteins.

Comprehensive analysis of the prognostic and immunological signature of TNFAIP8 family genes in human glioma

TNFAIP8 family molecules have been recognized for their involvement in the progression of tumors across a range of cancer types. Emerging experimental data suggests a role for certain TNFAIP8 family molecules in the development of glioma. Nonetheless, the comprehensive understanding of the genomic alterations, prognostic significance, and immunological profiles of TNFAIP8 family molecules in glioma remains incomplete. In the study, using the comprehensive bioinformatics tools, we explored the unique functions of 4 TNFAIP8 members including TNFAIP8, TNFAIP8L1, TNFAIP8L2 and TNFAIP8L3 in glioma. The expressions of TNFAIP8, TNFAIP8L1, TNFAIP8L2, and TNFAIP8L3 were notably upregulated in glioma tissues compared to normal tissues. Furthermore, survival analysis indicated that elevated expression levels of TNFAIP8, TNFAIP8L1 and TNFAIP8L2 were correlated with unfavorable outcomes in terms of overall survival (OS), disease-specific survival (DSS), and progression-free interval (PFI) among glioma patients. Genetic modifications, such as mutations and copy number alterations, within the TNFAIP8 family exhibited a significant association with extended OS, DSS and PFS in individuals diagnosed with glioma. The findings suggest a noteworthy correlation between TNFAIP8 family members and the age and 1p/19q codeletion status of glioma patients. We also found that there were significant relationships between TNFAIP8 family expression and tumor immunity in glioma. Furthermore, functional annotation of TNFAIP8 family members and their co-expressed genes in gliomas was carried out using GO and KEGG pathway analysis. The GO analysis revealed that the primary biological processes influenced by the TNFAIP8 family co-expressed genes included cell chemotaxis, temperature homeostasis, and endocytic vesicle formation. Additionally, the KEGG analysis demonstrated that TNFAIP8 family co-expressed genes are involved in regulating various pathways such as inflammatory mediator regulation of TRP channels, pathways in cancer, prolactin signaling pathway, and Fc gamma R-mediated phagocytosis. Overall, the findings suggest that TNFAIP8 family members may play a significant role in the development of glioma and have the potential to serve as prognostic indicators and therapeutic targets for individuals with glioma.

Influence of Solvophobicity of Biphenol-Derived Small Surface Ligands on the Formation of Size-Controllable Gold Nanoparticle Vesicles.

The self-assembly of gold nanoparticles (GNPs) into gold nanoparticle vesicles (GNVs) has been a topic of significant interest in recent years. However, the formation mechanism of GNVs is still not fully understood. In this article, we report that the new oligo(ethylene glycol)-terminated biphenol ligands (OBLs) show different solubility in tetrahydrofuran (THF) depending upon the number of terminal ethylene glycol units, resulting in a differential solvophobicity. The fluorine-free OBLs have the ability to self-assemble with GNPs into GNVs driven by the solvophobic feature of the ligands. The size of GNVs can be precisely controlled by tuning the interparticle attraction through changes in the unit number of terminal ethylene glycol or the water content in THF. Time-dependent studies revealed that the vesicle formation process consists of two stages: the rapid generation of vesicles, followed by their fusion to form thermodynamically stable GNVs with a saturated size. These two rapid processes are primarily influenced by the pronounced solvophobic attraction exerted by the surface ligands.

A Comprehensive Review on the Intricate Interplay between COVID-19 Immunization and the New Onset of Pemphigus Foliaceus.

Autoimmune bullous diseases (AIBDs) are characterized by the formation of vesicles, bullous lesions, and mucosal erosions. The autoantibodies target the cellular anchoring structures from the surface of epidermal keratinocyte named desmosomes, leading to a loss of cellular cohesion named acantholysis. AIBDs are classified into intraepidermal or subepidermal types based on clinical features, histological characteristics, and immunofluorescence patterns. Pemphigus foliaceus (PF) is an acquired, rare, autoimmune skin condition associated with autoantibodies that specifically target desmoglein-1, leading to a clinical presentation characterized by delicate cutaneous blisters, typically sparing the mucous membranes. Several factors, including genetic predisposition, environmental triggers, malignancies, medication use, and vaccination (for influenza, hepatitis B, rabies, tetanus, and more recently, severe acute respiratory syndrome Coronavirus 2 known as SARS-CoV-2), can potentially trigger the onset of pemphigus. With the advent of vaccines playing a pivotal role in combatting the 2019 coronavirus disease (COVID-19), extensive research has been conducted globally to ascertain their efficacy and potential cutaneous adverse effects. While reports of AIBDs post-COVID-19 vaccination exist in the medical literature, instances of PF following vaccination have been less commonly reported worldwide. The disease's pathophysiology is likely attributed to the resemblance between the ribonucleic acid (RNA) antigen present in these vaccines and cellular nuclear matter. The protein produced by the BNT-162b2 messenger ribonucleic acid (mRNA) vaccine includes immunogenic epitopes that could potentially trigger autoimmune phenomena in predisposed individuals through several mechanisms, including molecular mimicry, the activation of pattern recognition receptors, the polyclonal stimulation of B cells, type I interferon production, and autoinflammation. In this review, we present a comprehensive examination of the existing literature regarding the relationship between COVID-19 and PF, delving into their intricate interactions. This exploration improves the understanding of both pemphigus and mRNA vaccine mechanisms, highlighting the importance of close monitoring for PF post-immunization.

Pyocyanin stimulates membrane vesicle formation in Pseudomonas aeruginosa, but the synthesis is not required for enhanced vesiculation in biofilms

Bacterial membrane vesicles (MVs), particularly outer membrane vesicles (OMVs) in gram-negative bacteria, package their cargo for delivery to other bacteria or host cells. MV production is activated in the multicellular biofilm growth mode; however, the underlying mechanisms of enhanced MV production in biofilms are not fully understood. Pseudomonas aeruginosa is an opportunistic pathogen that causes chronic infections due to biofilm formation, making it difficult to treat. Pyocyanin is a phenazine synthesized by P. aeruginosa, which is a blue-pigment signaling molecule and generates H2O2. Herein, we investigated the interplay among pyocyanin, H2O2, and MVs in P. aeruginosa biofilms. Pyocyanin, H2O2, and MVs were sequentially produced in P. aeruginosa static liquid biofilms, and all phenotypes were repressed in a biofilm-deficient mutant ∆pelA∆pslA, which does not synthesize extracellular polysaccharides Pel and Psl. The addition of pyocyanin increased H2O2 generation and MV formation, suggesting that pyocyanin-mediated H2O2 generation facilitated MV formation in P. aeruginosa. Because catalase repressed MV formation in the biofilm, it suggested that oxidative stress may be a pathway for the activation of MV production in P. aeruginosa biofilms. On the other hand, MV formation in biofilms was not increased in the mutant of phzM, which encodes an enzyme for pyocyanin synthesis. Thus, while additional pyocyanin promotes MV formation, several factors other than pyocyanin, such as extracellular polysaccharides, play a major role in enhanced MV formation in P. aeruginosa biofilms. Our study provides new insights into vesiculation in P. aeruginosa biofilms.

Exploring the Properties of Curved Lipid Membranes: Comparative Analysis of Atomistic and Coarse-Grained Force Fields.

Curvature emerges as a fundamental membrane characteristic crucial for diverse biological processes, including vesicle formation, cell signaling, and membrane trafficking. Increasingly valuable insights into atomistic details governing curvature-dependent membrane properties are provided by computer simulations. Nevertheless, the underlying force field models are conventionally calibrated and tested in relation to experimentally derived parameters of planar bilayers, thereby leaving uncertainties concerning their consistency in reproducing curved lipid systems. In this study we compare the depiction of buckled phosphatidylcholine (POPC) and POPC-cholesterol membranes by four popular force field models. Aside from agreement with respect to general trends in curvature dependence of a number of parameters, we observe a few qualitative differences. Among the most prominent ones is the difference between atomistic and coarse grained force fields in their representation of relative compressibility of the polar headgroup region and hydrophobic lipid core. Through a number of downstream effects, this discrepancy can influence the way in which curvature modulates the behavior of membrane bound proteins depending on the adopted simulation model.

PITPβ promotes COPI vesicle fission through lipid transfer and membrane contact formation.

Intracellular transport among organellar compartments occurs in two general ways, by membrane-bound carriers or membrane contacts. Specific circumstances that involve the coordination of these two modes of transport remain to be defined. Studying Coat Protein I (COPI) transport, we find that phosphatidylcholine with short acyl chains (sPC) is delivered through membrane contact from the endoplasmic reticulum (ER) to sites of COPI vesicle formation at the Golgi to support the fission stage. Phosphatidylinositol transfer protein beta (PITPβ) plays a key role in this process, with the elucidation of this role advancing a new understanding of how PITPβ acts, providing a mechanistic understanding of a specific circumstance when vesicular transport requires membrane contact, and contributing to a basic understanding of how transport carriers in a model intracellular pathway are formed.