Flotillin-mediated stabilization of unfolded proteins in bacterial membrane microdomains.

The function of many bacterial processes depends on the formation of functional membrane microdomains (FMMs), which resemble the lipid rafts of eukaryotic cells. However, the mechanism and the biological function of these membrane microdomains remain unclear. Here, we show that FMMs in the pathogen methicillin-resistant Staphylococcus aureus (MRSA) are dedicated to confining and stabilizing proteins unfolded due to cellular stress. The FMM scaffold protein flotillin forms a clamp-shaped oligomer that holds unfolded proteins, stabilizing them and favoring their correct folding. This process does not impose a direct energy cost on the cell and is crucial to survival of ATP-depleted bacteria, and thus to pathogenesis. Consequently, FMM disassembling causes the accumulation of unfolded proteins, which compromise MRSA viability during infection and cause penicillin re-sensitization due to PBP2a unfolding. Thus, our results indicate that FMMs mediate ATP-independent stabilization of unfolded proteins, which is essential for bacterial viability during infection.

Antimicrobial Peptides Increase Line Tension in Raft-Forming Lipid Membranes.

The formation of phase separated membrane domains is believed to be essential for the function of the cell. The precise composition and physical properties of lipid bilayer domains play crucial roles in regulating protein activity and governing cellular processes. Perturbation of the domain structure in human cells can be related to neurodegenerative diseases and cancer. Lipid rafts are also believed to be essential in bacteria, potentially serving as targets for antibiotics. An important question is how the membrane domain structure is affected by bioactive and therapeutic molecules, such as surface-active peptides, which target cellular membranes. Here we focus on antimicrobial peptides (AMPs), crucial components of the innate immune system, to gain insights into their interaction with model lipid membranes containing domains. Using small-angle neutron/X-ray scattering (SANS/SAXS), we show that the addition of several natural AMPs (indolicidin, LL-37, magainin II, and aurein 2.2) causes substantial growth and restructuring of the domains, which corresponds to increased line tension. Contrast variation SANS and SAXS results demonstrate that the peptide inserts evenly in both phases, and the increased line tension can be related to preferential and concentration dependent thinning of the unsaturated membrane phase. We speculate that the lateral restructuring caused by the AMPs may have important consequences in affecting physiological functions of real cells. This work thus shines important light onto the complex interactions and lateral (re)organization in lipid membranes, which is relevant for a molecular understanding of diseases and the action of antibiotics.

Membrane-bound Heat Shock Protein mHsp70 Is Required for Migration and Invasion of Brain Tumors.

Molecular chaperones, especially 70 kDa heat shock protein, in addition to their intracellular localization in cancer cells, can be exposed on the surface of the plasma membrane. We report that the membrane-associated chaperone mHsp70 of malignant brain tumors is required for high migratory and invasive activity of cancer cells. Live-cell inverted confocal microscopy of tumor samples from adult (n = 23) and pediatric (n = 9) neurooncologic patients showed pronounced protein expression on the membrane, especially in the perifocal zone. Mass spectrometry analysis of lipid rafts isolated from tumor cells confirmed the presence of the protein in the chaperone cluster (including representatives of other families, such as Hsp70, Hsc70, Hsp105, and Hsp90), which in turn, during interactome analysis, was associated with proteins involved in cell migration (e.g., Rac1, RhoC, and myosin-9). The use of small-molecule inhibitors of HSP70 (PES and JG98) led to a substantial decrease in the invasive potential of cells isolated from a tumor sample of patients, which indicates the role of the chaperone in invasion. Moreover, the use of HSP70 inhibitors in animal models of orthotopic brain tumors significantly delayed tumor progression, which was accompanied by an increase in overall survival. Data demonstrate that chaperone inhibitors, particularly JG98, disrupt the function of mHsp70, thereby providing an opportunity to better understand the diverse functions of this protein and offer aid in the development of novel cancer therapies. SIGNIFICANCE: Membrane-bound mHsp70 is required for brain tumor cell migration and invasion and therefore could be employed as a target for anticancer therapies.

Autophagy Promotes Enrichment of Raft Components within Extracellular Vesicles Secreted by Human 2FTGH Cells.

Autophagy plays a key role in removing protein aggregates and damaged organelles. In addition to its conventional degradative functions, autophagy machinery contributes to the release of cytosolic proteins through an unconventional secretion pathway. In this research, we analyzed autophagy-induced extracellular vesicles (EVs) in HT1080-derived human fibrosarcoma 2FTGH cells using transmission electron microscopy and atomic force microscopy (AFM). We preliminary observed that autophagy induces the formation of a subset of large heterogeneous intracellular vesicular structures. Moreover, AFM showed that autophagy triggering led to a more visible smooth cell surface with a reduced amount of plasma membrane protrusions. Next, we characterized EVs secreted by cells following autophagy induction, demonstrating that cells release both plasma membrane-derived microvesicles and exosomes. A self-forming iodixanol gradient was performed for cell subfractionation. Western blot analysis showed that endogenous LC3-II co-fractionated with CD63 and CD81. Then, we analyzed whether raft components are enriched within EV cargoes following autophagy triggering. We observed that the raft marker GD3 and ER marker ERLIN1 co-fractionated with LC3-II; dual staining by immunogold electron microscopy and coimmunoprecipitation revealed GD3-LC3-II association, indicating that autophagy promotes enrichment of raft components within EVs. Introducing a new brick in the crosstalk between autophagy and the endolysosomal system may have important implications for the knowledge of pathogenic mechanisms, suggesting alternative raft target therapies in diseases in which the generation of EV is active.

Confinement energy landscape classification reveals membrane receptor nano-organization mechanisms.

The cell membrane organization has an essential functional role through the control of membrane receptor confinement in micro- or nanodomains. Several mechanisms have been proposed to account for these properties, although some features have remained controversial, notably the nature, size, and stability of cholesterol- and sphingolipid-rich domains or lipid rafts. Here, we probed the effective energy landscape acting on single-nanoparticle-labeled membrane receptors confined in raft nanodomains- epidermal growth factor receptor (EGFR), Clostridium perfringens ε-toxin receptor (CPεTR), and Clostridium septicum α-toxin receptor (CSαTR)-and compared it with hop-diffusing transferrin receptors. By establishing a new analysis pipeline combining Bayesian inference, decision trees, and clustering approaches, we systematically classified single-protein trajectories according to the type of effective confining energy landscape. This revealed the existence of only two distinct organization modalities: confinement in a quadratic energy landscape for EGFR, CPεTR, and CSαTR (A), and free diffusion in confinement domains resulting from the steric hindrance due to F-actin barriers for transferrin receptor (B). The further characterization of effective confinement energy landscapes by Bayesian inference revealed the role of interactions with the domain environment in cholesterol- and sphingolipid-rich domains with (EGFR) or without (CPεTR and CSαTR) interactions with F-actin to regulate the confinement energy depth. These two distinct mechanisms result in the same organization type (A). We revealed that the apparent domain sizes for these receptor trajectories resulted from Brownian exploration of the energy landscape in a steady-state-like regime at a common effective temperature, independently of the underlying molecular mechanisms. These results highlight that confinement domains may be adequately described as interaction hotspots rather than rafts with abrupt domain boundaries. Altogether, these results support a new model for functional receptor confinement in membrane nanodomains and pave the way to the constitution of an atlas of membrane protein organization.

Intercellular adhesion molecule-1 suppresses TMZ chemosensitivity in acquired TMZ-resistant gliomas by increasing assembly of ABCB1 on the membrane.

AIMS: Despite aggressive treatment, the recurrence of glioma is an inevitable occurrence, leading to unsatisfactory clinical outcomes. A plausible explanation for this phenomenon is the phenotypic alterations that glioma cells undergo aggressive therapies, such as TMZ-therapy. However, the underlying mechanisms behind these changes are not well understood. METHODS: The TMZ chemotherapy resistance model was employed to assess the expression of intercellular adhesion molecule-1 (ICAM1) in both in vitro and in vivo settings. The potential role of ICAM1 in regulating TMZ chemotherapy resistance was investigated through knockout and overexpression techniques. Furthermore, the mechanism underlying ICAM1-mediated TMZ chemotherapy resistance was examined using diverse molecular biological methods, and the lipid raft protein was subsequently isolated to investigate the cellular subcomponents where ICAM1 operates. RESULTS: Acquired TMZ resistant (TMZ-R) glioma models heightened production of intercellular adhesion molecule-1 (ICAM1) in TMZ-R glioma cells. Additionally, we observed a significant suppression of TMZ-R glioma proliferation upon inhibition of ICAM1, which was attributed to the enhanced intracellular accumulation of TMZ. Our findings provide evidence supporting the role of ICAM1, a proinflammatory marker, in promoting the expression of ABCB1 on the cell membrane of TMZ-resistant cells. We have elucidated the mechanistic pathway by which ICAM1 modulates phosphorylated moesin, leading to an increase in ABCB1 expression on the membrane. Furthermore, our research has revealed that the regulation of moesin by ICAM1 was instrumental in facilitating the assembly of ABCB1 exclusively on the lipid raft of the membrane. CONCLUSIONS: Our findings suggest that ICAM1 is an important mediator in TMZ-resistant gliomas and targeting ICAM1 may provide a new strategy for enhancing the efficacy of TMZ therapy against glioma.

Expression of the membrane tetraspanin claudin 18 on cancer cells promotes T lymphocyte infiltration and antitumor immunity in pancreatic cancer.

Tumors weakly infiltrated by T lymphocytes poorly respond to immunotherapy. We aimed to unveil malignancy-associated programs regulating T cell entrance, arrest, and activation in the tumor environment. Differential expression of cell adhesion and tissue architecture programs, particularly the presence of the membrane tetraspanin claudin (CLDN)18 as a signature gene, demarcated immune-infiltrated from immune-depleted mouse pancreatic tumors. In human pancreatic ductal adenocarcinoma (PDAC) and non-small cell lung cancer, CLDN18 expression positively correlated with more differentiated histology and favorable prognosis. CLDN18 on the cell surface promoted accrual of cytotoxic T lymphocytes (CTLs), facilitating direct CTL contacts with tumor cells by driving the mobilization of the adhesion protein ALCAM to the lipid rafts of the tumor cell membrane through actin. This process favored the formation of robust immunological synapses (ISs) between CTLs and CLDN18-positive cancer cells, resulting in increased T cell activation. Our data reveal an immune role for CLDN18 in orchestrating T cell infiltration and shaping the tumor immune contexture.

Mechanism of Purinergic Regulation of Neurotransmission in Mouse Neuromuscular Junction: The Role of Redox Signaling and Lipid Rafts.

Acetylcholine is the main neurotransmitter at the vertebrate neuromuscular junctions (NMJs). ACh exocytosis is precisely modulated by co-transmitter ATP and its metabolites. It is assumed that ATP/ADP effects on ACh release rely on activation of presynaptic Gi protein-coupled P2Y13 receptors. However, downstream signaling mechanism of ATP/ADP-mediated modulation of neuromuscular transmission remains elusive. Using microelectrode recording and fluorescent indicators, the mechanism underlying purinergic regulation was studied in the mouse diaphragm NMJs. Pharmacological stimulation of purinoceptors with ADP decreased synaptic vesicle exocytosis evoked by both low and higher frequency stimulation. This inhibitory action was suppressed by antagonists of P2Y13 receptors (MRS 2211), Ca2+ mobilization (TMB8), protein kinase C (chelerythrine) and NADPH oxidase (VAS2870) as well as antioxidants. This suggests the participation of Ca2+ and reactive oxygen species (ROS) in the ADP-triggered signaling. Indeed, ADP caused an increase in cytosolic Ca2+ with subsequent elevation of ROS levels. The elevation of [Ca2+]in was blocked by MRS 2211 and TMB8, whereas upregulation of ROS was prevented by pertussis toxin (inhibitor of Gi protein) and VAS2870. Targeting the main components of lipid rafts, cholesterol and sphingomyelin, suppressed P2Y13 receptor-dependent attenuation of exocytosis and ADP-induced enhancement of ROS production. Inhibition of P2Y13 receptors decreased ROS production and increased the rate of exocytosis during intense activity. Thus, suppression of neuromuscular transmission by exogenous ADP or endogenous ATP can rely on P2Y13 receptor/Gi protein/Ca2+/protein kinase C/NADPH oxidase/ROS signaling, which is coordinated in a lipid raft-dependent manner.

Real-Time FRET-Based Measurements of Ca2+/PKA Dynamics in Plasma Membrane Microdomains of Primary Hippocampal Neurons.

Both Ca2+ and protein kinase A (PKA) are multifaceted and ubiquitous signaling molecules, essential for regulating the intricate network of signaling pathways. However, their dynamics within specialized membrane regions are still not well characterized. By using genetically encoded fluorescent indicators specifically targeted to distinct plasma membrane microdomains, we have established a protocol that permits observing Ca2+/PKA dynamics in discrete neuronal microdomains with high spatial and temporal resolution. The approach employs a fluorescence microscope with a sensitive camera and a dedicated CFP/YFP/mCherry filter set, enabling the simultaneous detection of donor-acceptor emission and red fluorescence signal. In this detailed step-by-step guide, we outline the experimental procedure, including isolation of rat primary neurons and their transfection with biosensors targeted to lipid rafts or non-raft regions of plasma membrane. We provide information on the necessary equipment and imaging setup required for recording, along with highlighting critical parameters and troubleshooting guidelines for real-time measurements. Finally, we provide examples of the observed Ca2+ and PKA changes in specific cellular compartments. The application of this technique may have significant implications for studying cross-talk between second messengers and their alterations in various pathological conditions. © 2024 Wiley Periodicals LLC.

Dynamic Simulations of Interaction of the PEG-DPPE Micelle-Encapsulated Short-Chain Ceramides with the Raft-Included Membrane.

The lipid raft subdomains in cancer cell membranes play a key role in signal transduction, biomolecule recruitment, and drug transmembrane transport. Augmented membrane rigidity due to the formation of a lipid raft is unfavorable for the entry of drugs, a limiting factor in clinical oncology. The short-chain ceramide (CER) has been reported to promote drug entry into membranes and disrupt lipid raft formation, but the underlying mechanism is not well understood. We recently explored the carrier-membrane fusion dynamics of PEG-DPPE micelles in delivering doxorubicin (DOX). Based on the phase-segregated membrane model composed of DPPC/DIPC/CHOL/GM1/PIP2, we aim to explore the dynamic mechanism of the PEG-DPPE micelle-encapsulating DOXs in association with the raft-included cell membrane modulated by C8 acyl tail CERs. The results show that the lipid raft remains integrated and DOX-resistant subjected to free DOXs and the micelle-encapsulating ones. Addition of CERs disorganizes the lipid raft by pushing CHOL aside from DPPC. It subsequently allows for a good permeability for PEG-DPPE micelle-encapsulated DOXs, which penetrate deeper as CER concentration increases. GM1 is significant in guiding drugs' redistributing between bilayer phases, and the anionic PIP2 further helps DOXs attain the inner bilayer surface. These results elaborate on the perturbing effect of CERs on lipid raft stability, which provides a new comprehensive approach for further design of drug delivery systems.

Selection of bifunctional RNAs with specificity for arginine and lipid membranes.

The molecular mechanisms of selective RNA loading into exosomes and other extracellular vesicles are not yet completely understood. In order to show that a pool of RNA sequences binds both the amino acid arginine and lipid membranes, we constructed a bifunctional RNA 10Arg aptamer specific for arginine and lipid vesicles. The preference of RNA 10Arg for lipid rafts was visualized and confirmed using FRET microscopy in neuroblastoma cells. The selection-amplification (SELEX) method using a doped (with the other three nucleotides) pool of RNA 10Arg sequences yielded several RNA 10Arg(D) sequences, and the affinities of these RNAs both to arginine and liposomes are improved in comparison to pre-doped RNA. Generation of these bispecific aptamers supports the hypothesis that an RNA molecule can bind both to RNA-binding proteins (RBPs) through arginine within the RBP-binding site and to membrane lipid rafts, thus facilitating RNA loading into exosomes and other extracellular vesicles.

Development of FRET Biosensor to Characterize CSK Subcellular Regulation.

C-terminal Src kinase (CSK) is the major inhibitory kinase for Src family kinases (SFKs) through the phosphorylation of their C-tail tyrosine sites, and it regulates various types of cellular activity in association with SFK function. As a cytoplasmic protein, CSK needs be recruited to the plasma membrane to regulate SFKs' activity. The regulatory mechanism behind CSK activity and its subcellular localization remains largely unclear. In this work, we developed a genetically encoded biosensor based on fluorescence resonance energy transfer (FRET) to visualize the CSK activity in live cells. The biosensor, with an optimized substrate peptide, confirmed the crucial Arg107 site in the CSK SH2 domain and displayed sensitivity and specificity to CSK activity, while showing minor responses to co-transfected Src and Fyn. FRET measurements showed that CSK had a relatively mild level of kinase activity in comparison to Src and Fyn in rat airway smooth muscle cells. The biosensor tagged with different submembrane-targeting signals detected CSK activity at both non-lipid raft and lipid raft microregions, while it showed a higher FRET level at non-lipid ones. Co-transfected receptor-type protein tyrosine phosphatase alpha (PTPα) had an inhibitory effect on the CSK FRET response. The biosensor did not detect obvious changes in CSK activity between metastatic cancer cells and normal ones. In conclusion, a novel FRET biosensor was generated to monitor CSK activity and demonstrated CSK activity existing in both non-lipid and lipid raft membrane microregions, being more present at non-lipid ones.

Domain-Targeted Membrane Partitioning of Specific Proteins with DNA Nanodevices.

The cell membrane exhibits a remarkable complexity of lipids and proteins that dynamically segregate into distinct domains to coordinate various cellular functions. The ability to manipulate the partitioning of specific membrane proteins without involving genetic modification is essential for decoding various cellular processes but highly challenging. In this work, by conjugating cholesterols or tocopherols at the three bottom vertices of the DNA tetrahedron, we develop two sets of nanodevices for the selective targeting of lipid-order (Lo) and lipid-disorder (Ld) domains on the live cell membrane. By incorporation of protein-recognition ligands, such as aptamers or antibodies, through toehold-mediated strand displacement, these DNA nanodevices enable dynamic translocation of target proteins between these two domains. We first used PTK7 as a protein model and demonstrated, for the first time, that the accumulation of PTK7 to the Lo domains could promote tumor cell migration, while sequestering it in the Ld domains would inhibit the movement of the cells. Next, based on their modular nature, these DNA nanodevices were extended to regulate the process of T cell activation through manipulating the translocation of CD45 between the Lo and the Ld domains. Thus, our work is expected to provide deep insight into the study of membrane structure and molecular interactions within diverse cell signaling processes.

Novel class of peptides disintegrating biological membranes to aid in the characterization of membrane proteins.

Styrene-maleic acid (SMA) and similar amphiphilic copolymers are known to cut biological membranes into lipid nanoparticles/nanodiscs containing membrane proteins apparently in their relatively native membrane lipid environment. Our previous work demonstrated that membrane raft microdomains resist such disintegration by SMA. The use of SMA in studying membrane proteins is limited by its heterogeneity and the inability to prepare defined derivatives. In the present paper, we demonstrate that some amphiphilic peptides structurally mimicking SMA also similarly disintegrate cell membranes. In contrast to the previously used copolymers, the simple peptides are structurally homogeneous. We found that their membrane-disintegrating activity increases with their length (reaching optimum at 24 amino acids) and requires a basic primary structure, that is, (XXD)n, where X represents a hydrophobic amino acid (optimally phenylalanine), D aspartic acid, and n is the number of repeats of these triplets. These peptides may provide opportunities for various well-defined potentially useful modifications in the study of membrane protein biochemistry. Our present results confirm a specific character of membrane raft microdomains.

Low Density Lipoprotein Cholesterol Decreases the Expression of Adenosine A2A Receptor and Lipid Rafts-Protein Flotillin-1: Insights on Cardiovascular Risk of Hypercholesterolemia.

High blood levels of low-density lipoprotein (LDL)-cholesterol (LDL-C) are associated with atherosclerosis, mainly by promoting foam cell accumulation in vessels. As cholesterol is an essential component of cell plasma membranes and a regulator of several signaling pathways, LDL-C excess may have wider cardiovascular toxicity. We examined, in untreated hypercholesterolemia (HC) patients, selected regardless of the cause of LDL-C accumulation, and in healthy participants (HP), the expression of the adenosine A2A receptor (A2AR), an anti-inflammatory and vasodilatory protein with cholesterol-dependent modulation, and Flotillin-1, protein marker of cholesterol-enriched plasma membrane domains. Blood cardiovascular risk and inflammatory biomarkers were measured. A2AR and Flotillin-1 expression in peripheral blood mononuclear cells (PBMC) was lower in patients compared to HP and negatively correlated to LDL-C blood levels. No other differences were observed between the two groups apart from transferrin and ferritin concentrations. A2AR and Flotillin-1 proteins levels were positively correlated in the whole study population. Incubation of HP PBMCs with LDL-C caused a similar reduction in A2AR and Flotillin-1 expression. We suggest that LDL-C affects A2AR expression by impacting cholesterol-enriched membrane microdomains. Our results provide new insights into the molecular mechanisms underlying cholesterol toxicity, and may have important clinical implication for assessment and treatment of cardiovascular risk in HC.

Lithocholic acid-based oligomers as drug delivery candidates targeting model of lipid raft.

This study presents a new approach to designing a lithocholic acid functionalized oligomer (OLithocholicAA-X) that can be used as a drug carrier with additional, beneficial activity. Namely, this novel oligomer can incorporate an anti-cancer drug due to the application of an effective backbone as its component (lithocholic acid) alone is known to have anticancer activity. The oligomer was synthesized and characterized in detail by nuclear magnetic resonance, attenuated total reflectance Fourier-transform infrared spectroscopy, ultraviolet-visible spectroscopy, thermal analysis, and mass spectrometry analysis. We selected lipid rafts as potential drug carrier-membrane binding sites. In this respect, we investigated the effects of OLithocholicAA-X on model lipid raft of normal and altered composition, containing an increased amount of cholesterol (Chol) or sphingomyelin (SM), using Langmuir monolayers and liposomes. The surface topography of the studied monolayers was additionally investigated by atomic force microscopy (AFM). The obtained results showed that the investigated oligomer has affinity for a system that mimics a normal lipid raft (SM:Chol 2:1). On the other hand, for systems with an excess of SM or Chol, thermodynamically unfavorable fluidization of the films occurs. Moreover, AFM topographies showed that the amount of SM determines the bioavailability of the oligomer, causing fragmentation of its lattice.

Overexpressing lipid raft protein STOML2 modulates the tumor microenvironment via NF-κB signaling in colorectal cancer.

Colorectal cancer (CRC) is characterized by a complex tumor inflammatory microenvironment, while angiogenesis and immunosuppression frequently occur concomitantly. However, the exact mechanism that controls angiogenesis and immunosuppression in CRC microenvironment remains unclear. Herein, we found that expression levels of lipid raft protein STOML2 were increased in CRC and were associated with advanced disease stage and poor survival outcomes. Intriguingly, we revealed that STOML2 is essential for CRC tumor inflammatory microenvironment, which induces angiogenesis and facilitates tumor immune escape simultaneously both in vitro and in vivo. Moreover, tumors with STOML2 overexpression showed effective response to anti-angiogenesis treatment and immunotherapy in vivo. Mechanistically, STOML2 regulates CRC proliferation, angiogenesis, and immune escape through activated NF-κB signaling pathway via binding to TRADD protein, resulting in upregulation of CCND1, VEGF, and PD-L1. Furthermore, treatment with NF-κB inhibitor dramatically reversed the ability of proliferation and angiogenesis. Clinically, we also observed a strong positive correlation between STOML2 expression and Ki67, CD31, VEGFC and PD-1 of CD8+T cell expression. Taken together, our results provided novel insights into the role of STOML2 in CRC inflammatory microenvironment, which may present a therapeutic opportunity for CRC.

Protein-Rich Rafts in Hybrid Polymer/Lipid Giant Unilamellar Vesicles.

Considerable attention has been dedicated to lipid rafts due to their importance in numerous cell functions such as membrane trafficking, polarization, and signaling. Next to studies in living cells, artificial micrometer-sized vesicles with a minimal set of components are established as a major tool to understand the phase separation dynamics and their intimate interplay with membrane proteins. In parallel, mixtures of phospholipids and certain amphiphilic polymers simultaneously offer an interface for proteins and mimic this segregation behavior, presenting a tangible synthetic alternative for fundamental studies and bottom-up design of cellular mimics. However, the simultaneous insertion of complex and sensitive membrane proteins is experimentally challenging and thus far has been largely limited to natural lipids. Here, we present the co-reconstitution of the proton pump bo3 oxidase and the proton consumer ATP synthase in hybrid polymer/lipid giant unilamellar vesicles (GUVs) via fusion/electroformation. Variations of the current method allow for tailored reconstitution protocols and control of the vesicle size. In particular, mixing of protein-free and protein-functionalized nanosized vesicles in the electroformation film results in larger GUVs, while separate reconstitution of the respiratory enzymes enables higher ATP synthesis rates. Furthermore, protein labeling provides a synthetic mechanism for phase separation and protein sequestration, mimicking lipid- and protein-mediated domain formation in nature. The latter means opens further possibilities for re-enacting phenomena like supercomplex assembly or symmetry breaking and enriches the toolbox of bottom-up synthetic biology.

Suppression of metastatic organ colonization and antiangiogenic activity of the orally bioavailable lipid raft-targeted alkylphospholipid edelfosine.

Metastasis is the leading cause of cancer mortality. Metastatic cancer is notoriously difficult to treat, and it accounts for the majority of cancer-related deaths. The ether lipid edelfosine is the prototype of a family of synthetic antitumor compounds collectively known as alkylphospholipid analogs, and its antitumor activity involves lipid raft reorganization. In this study, we examined the effect of edelfosine on metastatic colonization and angiogenesis. Using non-invasive bioluminescence imaging and histological examination, we found that oral administration of edelfosine in nude mice significantly inhibited the lung and brain colonization of luciferase-expressing 435-Lung-eGFP-CMV/Luc metastatic cells, resulting in prolonged survival. In metastatic 435-Lung and MDA-MB-231 breast cancer cells, we found that edelfosine also inhibited cell adhesion to collagen-I and laminin-I substrates, cell migration in chemotaxis and wound-healing assays, as well as cancer cell invasion. In 435-Lung and other MDA-MB-435-derived sublines with different organotropism, edelfosine induced G2/M cell cycle accumulation and apoptosis in a concentration- and time-dependent manner. Edelfosine also inhibited in vitro angiogenesis in human and mouse endothelial cell tube formation assays. The antimetastatic properties were specific to cancer cells, as edelfosine had no effects on viability in non-cancerous cells. Edelfosine accumulated in membrane rafts and endoplasmic reticulum of cancer cells, and membrane raft-located CD44 was downregulated upon drug treatment. Taken together, this study highlights the potential of edelfosine as an attractive drug to prevent metastatic growth and organ colonization in cancer therapy. The raft-targeted drug edelfosine displays a potent activity against metastatic organ colonization and angiogenesis, two major hallmarks of tumor malignancy.

In situ interaction between the hormone 17α-ethynylestradiol and the liquid-ordered phase composed of the lipid rafts sphingomyelin and cholesterol.

Hormone treatments are frequently associated with cardiovascular diseases and cancers in women. Additionally, the detrimental effects of their presence as contaminants in water remain a concern. The transport of hormones through cell membranes is essential for their biological action, but investigating cell permeability is challenging owing to the experimental difficulty in dealing with whole cells. In this paper, we study the interaction of the synthetic hormone 17α-ethynylestradiol (EE2) with membrane models containing the key raft components sphingomyelin (SM) and cholesterol (Chol). The models consisted of Langmuir monolayers and giant unilamellar vesicles (GUVs) that represent bilayers. EE2 induced expansion of SM monolayers upon interacting with the non-hydrated amide group of SM head, but it had practically no effect on SM GUVs because these group are not available for interaction in bilayers. In contrast, EE2 interacted with hydrated phosphate group (PO2-) and amide group of SM/Chol mixture monolayer, which could explain the loss in phase contrast of liquid-ordered GUVs suggesting pore formation. A comparison with reported EE2 effects on GUVs in the fluid phase, for which no loss in phase contrast was observed, indicates that the liquid-ordered phase consisting of lipid rafts is relevant to be associated with the changes on cell permeability caused by the hormones.