Understanding the mechanisms of antibiotic resistance is critical for the development of new therapeutics. Traditional methods for testing bacteria are often limited in their efficiency and reusability. Single bacterial cells can be studied at high throughput using double emulsions, although the lack of control over the oil shell permeability and limited access to the droplet interior present serious drawbacks. Here, a straightforward strategy for studying bacteria-encapsulating double emulsion-templated giant unilamellar vesicles (GUVs) is introduced. This microfluidic approach serves to simultaneously load bacteria inside synthetic GUVs and to permeabilize their membrane with the pore-forming peptide melittin. This enables antibiotic delivery or the influx of fresh medium into the GUV lumen for highly parallel cultivation and antimicrobial efficacy testing. Polymer-based GUVs proved to be efficient culture and analysis microvessels, as microfluidics allow easy selection and encapsulation of bacteria and rapid modification of culture conditions for antibiotic development. Further, a method for in situ profiling of biofilms within GUVs for high-throughput screening is demonstrated. Conceivably, synthetic GUVs equipped with biopores can serve as a foundation for the high-throughput screening of bacterial colony interactions during biofilm formation and for investigating the effect of antibiotics on biofilms.

Pesticide misuse has well-documented detrimental effects on ecosystems, with Nile tilapia (Oreochromis niloticus) being particularly vulnerable. The current study focuses on the impact of widely used sugarcane crop pesticides, Imazapic (IMZ) and Methyl Parathion (MP), on tilapia gill tissues and their lipid membranes. This investigation was motivated by the specific role of the lipid membrane in transport regulation. Bioinspired cell membrane models, including Langmuir monolayers and liposomes (LUVs and GUVs), were utilized to explore the interaction of IMZ and MP. The results revealed electrostatic interactions between IMZ and MP and the polar head groups of lipids, inducing morphological alterations in the lipid bilayer. Tilapia gill tissue exposed to the pesticides exhibited hypertrophic increases in primary and secondary lamellae, total lamellar fusion, vasodilation, and lifting of the secondary lamellar epithelium. These alterations can lead to compromised oxygen absorption by fish and subsequent mortality. This study not only highlights the harmful effects of the pesticides IMZ and MP, but also emphasizes the crucial role of water quality in ecosystem well-being, even at minimal pesticide concentrations. Understanding these impacts can better inform management practices to safeguard aquatic organisms and preserve ecosystem health in pesticide-affected environments.

A new decyl chain [-(CH2)9CH3] riboflavin conjugate has been synthesized and investigated. A nucleophilic substitution (SN2) reaction was used for coupling the alkyl chain to riboflavin (Rf), a model natural photosensitizer. As expected, the alkylated compound (decyl-Rf) is significantly more lipophilic than its precursor and efficiently intercalates within phospholipid bilayers, increasing its fluorescence quantum yield. The oxidative damage to lipid membranes photoinduced by decyl-Rf was investigated in large and giant unilamellar vesicles (LUVs and GUVs, respectively) composed of different phospholipids. Using a fluorogenic probe, fast radical formation and singlet oxygen generation was demonstrated upon UVA irradiation in vesicles containing decyl-Rf. Photosensitized formation of conjugated dienes and hydroperoxides, and membrane leakage in LUVs rich in poly-unsaturated fatty acids were also investigated. The overall assessment of the results shows that decyl-Rf is a significantly more efficient photosensitizer of lipids than its unsubstituted precursor and that the association to lipid membranes is key to trigger phospholipid oxidation. Alkylation of hydrophilic photosensitizers as a simple and general synthetic tool to obtain efficient photosensitizers of biomembranes, with potential applications, is discussed.

Giant vesicles are model membrane systems that can be characterized with microscopy. Whereas most giant synthetic vesicles are created with a single phospholipid species, vesicles with mixed membrane compositions, including single-tailed and double-tailed lipids, serve as more accurate models of biological membranes and also have applications in the origins of life and drug delivery fields. Here we describe several approaches that can be used to create giant vesicles with mixed lipid compositions.

Micromechanics techniques are playing an increasing role in characterization of biomembranes. The mechanical properties of membranes play an important role for a whole range of cellular processes. Lipid-protein biomembranes display lateral heterogeneity, domain formation, and morphological changes at mesoscopic and nanoscopic length scales. An attempt is made to introduce how membrane\'s material properties can be measured. Both fluctuation analysis and micro-pipette aspiration experiments have been used to quantify the micromechanics of membranes. The relationship between the structure and function of biomembranes is a critical concern in modern biology. This overview calls for a deeper understanding of how the cell complexity might be related to the mechanical properties of the lipid-protein membrane. Mechanical properties can influence cellular response to processes like adhesion, transport, differentiation, proliferation and migration.

Artificial vesicles are important tools in membrane research because they enable studying membrane properties in controlled conditions. Giant unilamellar vesicles (GUVs) are specially interesting due to their similarity in size to eukaryotic cells. We focus on optimization of GUV production from phosphatidylcholine/sphingomyelin/cholesterol mixtures using the electroformation method. This mixture has been extensively researched lately due to its relevance for the formation of lipid rafts. We measured the effect of voltage, frequency, lipid film thickness, and cholesterol (Chol) concentration on electroformation successfulness using spin-coating for reproducible lipid film deposition. Special attention is given to the effect of Chol concentrations above the phospholipid bilayer saturation threshold. Such high concentrations are of interest to groups studying the role of Chol in the fiber cell plasma membranes of the eye lens or development of atherosclerosis. Utilizing atomic force and fluorescence microscopy, we found the optimal lipid film thickness to be around 30 nm, and the best frequency-voltage combinations in the range of 2-6 V and 10-100 Hz. Increasing the Chol content, we observed a decrease in GUV yield and size. However, the effect was much less pronounced when the optimal lipid film thickness was used. The results underline the need for simultaneous optimization of both electrical parameters and thickness in order to produce high-quality GUVs for experimental research.

Many signal transductions resulting from ligand-receptor interactions occur at the cell surface. These signaling pathways play essential roles in cell polarization, membrane morphogenesis, and the modulation of membrane tension at the cell surface. However, due to the large number of membrane-binding proteins, including actin-membrane linkers, and transmembrane proteins present at the cell surface, the molecular mechanisms underlying the regulation at the cell surface are yet unclear. Here, we describe the molecular functions of one of the key players at the cell surface, ezrin/radixin/moesin (ERM) proteins from a biophysical point of view. We focus our discussion on biophysical properties of ERM proteins revealed by using biophysical tools in live cells and in vitro reconstitution systems. We first describe the structural properties of ERM proteins and then discuss the interactions of ERM proteins with PI(4,5)P2 and the actin cytoskeleton. These properties of ERM proteins revealed by using biophysical approaches have led to a better understanding of their physiological functions in cells and tissues.
UNASSIGNED: The online version contains supplementary material available at 10.1007/s12551-021-00928-0.

Fatty acids readily assemble into bilayer membranes at a pH near their apparent pKa. Fatty acid vesicles are not only useful for research in the fields of origins of life, soft matter science, biophysics, and drug delivery, but are also cost-effective and easy to manipulate, making them ideal for teaching students about self-assembly and lipid bilayers. Here, we describe simple ways to make giant, unilamellar fatty acid vesicles suitable for microscopy and encapsulation studies.

Since its inception more than thirty years ago, electroformation has become the most commonly used method for growing giant unilamellar vesicles (GUVs). Although the method seems quite straightforward at first, researchers must consider the interplay of a large number of parameters, different lipid compositions, and internal solutions in order to avoid artifactual results or reproducibility problems. These issues motivated us to write a short review of the most recent methodological developments and possible pitfalls. Additionally, since traditional manual analysis can lead to biased results, we have included a discussion on methods for automatic analysis of GUVs. Finally, we discuss possible improvements in the preparation of GUVs containing high cholesterol contents in order to avoid the formation of artifactual cholesterol crystals. We intend this review to be a reference for those trying to decide what parameters to use as well as an overview providing insight into problems not yet addressed or solved.

Membrane fission triggered by the endosomal sorting complex required for transport (ESCRT) is an important process observed in several pathogenic and non-pathogenic cellular events. From a synthetic-biology viewpoint, ESCRT proteins represent an interesting machinery for the construction of cell mimetic sub-compartments produced by fission. Since their discovery, the studies on ESCRT-III-mediated action, have mainly focused on protein dynamics, ignoring the role of lipid organization and membrane phase state. Recently, it has been suggested that membrane buds formed by the action of ESCRT-III are generated from transient microdomains in endosomal membranes. However, the interplay between membrane domain formation and ESCRT remodeling pathways has not been investigated. Here, giant unilamellar vesicles made of ternary lipid mixtures, either homogeneous in phase or exhibiting liquid-ordered/liquid-disordered phase coexistence, were employed as a model membrane system. These vesicles were incubated with purified recombinant ESCRT-III proteins from the parasite Entamoeba histolytica. In homogeneous membranes, we observe that EhVps32 can trigger domain formation while EhVps20 preferentially co-localizes in the liquid disordered phase. The addition of EhVps24 appears to induce the formation of intraluminal vesicles produced from the liquid-ordered phase. In phase separated membranes, the intraluminal vesicles are also generated from the liquid-ordered phase and presumably emerge from the phase boundary region. Our findings reinforce the hypothesis that ESCRT-mediated remodeling depends on the membrane phase state. Furthermore, the obtained results point to a potential synthetic biology approach for establishing eukaryotic mimics of artificial cells with microcompartments of specific membrane composition, which can also differ from that of the mother vesicle.