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Scientific Reports Jan 2024Biochemical approaches revealed that tetraspanins are multi-regulatory proteins forming a web, where they act in tetraspanin-enriched-microdomains (TEMs). A microscopic...
Biochemical approaches revealed that tetraspanins are multi-regulatory proteins forming a web, where they act in tetraspanin-enriched-microdomains (TEMs). A microscopic criterion differentiating between web and TEMs is lacking. Using super-resolution microcopy, we identify co-assemblies between the tetraspanins CD9 and CD81 and CD151 and CD81. CD9 assemblies contain as well the CD9/CD81-interaction partner EWI-2. Moreover, CD9 clusters are proximal to clusters of the CD81-interaction partner CD44 and CD81-/EWI-2-interacting ezrin-radixin-moesin proteins. Assemblies scatter unorganized across the cell membrane; yet, upon EWI-2 elevation, they agglomerate into densely packed arranged-crowds in a process independent from actin dynamics. In conclusion, microscopic clusters are equivalent to biochemical tetraspanin-assemblies, defining in their entirety the tetraspanin web. Cluster-agglomeration enriches tetraspanins, which makes agglomerations to a microscopic complement of TEMs. The microscopic classification of tetraspanin assemblies advances our understanding of this enigmatic protein family, whose members play roles in a plethora of cellular functions, diseases, and pathogen infections.
Topics: Tetraspanins; Cell Membrane; Actins; Transcription Factors
PubMed: 38267610
DOI: 10.1038/s41598-024-52615-1 -
Biomedicine & Pharmacotherapy =... Feb 2024Metastasis 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...
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 G/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.
Topics: Animals; Mice; Humans; Mice, Nude; Antineoplastic Agents; Neoplasms; Phospholipid Ethers; Apoptosis; Membrane Microdomains
PubMed: 38266621
DOI: 10.1016/j.biopha.2024.116149 -
Frontiers in Microbiology 2023The causative agent of the COVID-19 pandemic, SARS-CoV-2, is a virus that targets mainly the upper respiratory tract. However, it can affect other systems such as the...
The causative agent of the COVID-19 pandemic, SARS-CoV-2, is a virus that targets mainly the upper respiratory tract. However, it can affect other systems such as the gastrointestinal (GI) tract. Therapeutic strategies for this virus are still inconclusive and understanding its entry mechanism is important for finding effective treatments. Cholesterol is an important constituent in the structure of cellular membranes that plays a crucial role in a variety of cellular events. In addition, it is important for the infectivity and pathogenicity of several viruses. ACE2, the main receptor of SARS-CoV-2, is associated with lipid rafts which are microdomains composed of cholesterol and sphingolipids. In this study, we investigate the role of statins, lipid-lowering drugs, on the trafficking of ACE2 and the impact of cholesterol modulation on the interaction of this receptor with S1 in Caco-2 cells. The data show that fluvastatin and simvastatin reduce the expression of ACE2 to variable extents, impair its association with lipid rafts and sorting to the brush border membrane resulting in substantial reduction of its interaction with the S1 subunit of the spike protein. By virtue of the substantial effects of statins demonstrated in our study, these molecules, particularly fluvastatin, represent a promising therapeutic intervention that can be used off-label to treat SARS-CoV-2.
PubMed: 38260879
DOI: 10.3389/fmicb.2023.1335458 -
International Journal of Molecular... Jan 2024Caveolae constitute membrane microdomains where receptors and ion channels functionally interact. Caveolin-3 (cav-3) is the key structural component of muscular...
Caveolae constitute membrane microdomains where receptors and ion channels functionally interact. Caveolin-3 (cav-3) is the key structural component of muscular caveolae. Mutations in lead to caveolinopathies, which result in both muscular dystrophies and cardiac diseases. In cardiomyocytes, cav-1 participates with cav-3 to form caveolae; skeletal myotubes and adult skeletal fibers do not express cav-1. In the heart, the absence of cardiac alterations in the majority of cases may depend on a conserved organization of caveolae thanks to the expression of cav-1. We decided to focus on three specific cav-3 mutations (Δ62-64YTT; T78K and W101C) found in heterozygosis in patients suffering from skeletal muscle disorders. We overexpressed both the WT and mutated cav-3 together with ion channels interacting with and modulated by cav-3. Patch-clamp analysis conducted in caveolin-free cells (MEF-KO), revealed that the T78K mutant is dominant negative, causing its intracellular retention together with cav-3 WT, and inducing a significant reduction in current densities of all three ion channels tested. The other cav-3 mutations did not cause significant alterations. Mathematical modelling of the effects of cav-3 T78K would impair repolarization to levels incompatible with life. For this reason, we decided to compare the effects of this mutation in other cell lines that endogenously express cav-1 (MEF-STO and CHO cells) and to modulate cav-1 expression with an shRNA approach. In these systems, the membrane localization of cav-3 T78K was rescued in the presence of cav-1, and the current densities of hHCN4, hKv1.5 and hKir2.1 were also rescued. These results constitute the first evidence of a compensatory role of cav-1 in the heart, justifying the reduced susceptibility of this organ to caveolinopathies.
Topics: Adult; Animals; Cricetinae; Humans; Caveolin 1; Caveolin 3; Cricetulus; Mutation; CHO Cells; Ion Channels
PubMed: 38256054
DOI: 10.3390/ijms25020980 -
Journal of Environmental Management Feb 2024Tropical Atlantic blooms of pelagic Sargassum species are associated with severe inundation events along the coasts of Caribbean and West African nations that cause...
Tropical Atlantic blooms of pelagic Sargassum species are associated with severe inundation events along the coasts of Caribbean and West African nations that cause extensive ecological and socioeconomic harm. The use of in-water harvesting as a management strategy avoids the plethora of challenges associated with shoreline inundations. Moreover, with a growing interest in the valorisation of this raw material, in-water harvesting provides the best opportunity to collect substantial amounts of 'fresh' sargassum that can be used in a variety of applications. However, in-water harvesting of sargassum will remove organisms associated with the floating habitat, resulting in loss of biodiversity, thus creating a potential management dilemma. To address this management concern, we assessed the clinging fauna associated with sargassum rafts at various distances from shore. From a total of 119 dipnet samples of sargassum, we recorded 18 taxa, across 6 phyla (Arthropoda, Mollusca, Chordata, Platyhelminthes, Nemathelminthes, Annelida) with the phylum Arthropoda being the most speciose (n = 10). Our multivariate and model selection analyses support that distance from shore and season are the most important drivers of variability in community composition and that season is the most important driver of individual abundance and number of taxa across samples. Overall, rafts within 0-3000 m of the shoreline of Barbados harbored low biodiversity and were dominated by small invertebrates (mean size: 5.5 mm) of no commercial value. Results suggest that biodiversity trade-offs associated with in-water sargassum harvesting in coastal areas are likely to be negligible.
Topics: Animals; Sargassum; Water; Caribbean Region; Ecosystem; Arthropods; Membrane Microdomains
PubMed: 38242025
DOI: 10.1016/j.jenvman.2024.120077 -
Frontiers in Physiology 2023The plus-end directed actin-dependent motor protein, myosin Va, is of particular relevance for outward vesicular protein trafficking and for restraining specific cargo... (Review)
Review
The plus-end directed actin-dependent motor protein, myosin Va, is of particular relevance for outward vesicular protein trafficking and for restraining specific cargo vesicles within the actin cortex. The latter is a preferred site of cAMP production, and the specificity of cAMP signaling is largely mediated through the formation of microdomains that spatially couple localized metabotropic receptor activity and cAMP production to selected effectors and downstream targets. This review summarizes the core literature on the role of myosin Va for the creation of such a cAMP microdomain at the mammalian nerve-muscle synapse that serves the activity-dependent recycling of nicotinic acetylcholine receptors (nAChRs)-a principal ligand-gated ion channel which is imperative for voluntary muscle contraction. It is discussed that i) the nerve-muscle synapse is a site with a unique actin-dependent microstructure, ii) myosin Va and protein kinase A regulatory subunit Iα as well as nAChR and its constitutive binding partner, rapsyn, colocalize in endocytic/recycling vesicles near the postsynaptic membrane, and iii) impairment of myosin Va or displacement of protein kinase A regulatory subunit Iα leads to the loss of nAChR stability. Regulation of this signaling process and underlying basic pieces of machinery were covered in previous articles, to which the present review refers.
PubMed: 38239886
DOI: 10.3389/fphys.2023.1342994 -
Microbial Cell (Graz, Austria) 2024FurE is a H symporter specific for the cellular uptake of uric acid, allantoin, uracil, and toxic nucleobase analogues in the fungus nidulans. Being member of the NCS1...
FurE is a H symporter specific for the cellular uptake of uric acid, allantoin, uracil, and toxic nucleobase analogues in the fungus nidulans. Being member of the NCS1 protein family, FurE is structurally related to the APC-superfamily of transporters. APC-type transporters are characterised by a 5+5 inverted repeat fold made of ten transmembrane segments (TMS1-10) and function through the rocking-bundle mechanism. Most APC-type transporters possess two extra C-terminal TMS segments (TMS11-12), the function of which remains elusive. Here we present a systematic mutational analysis of TMS11-12 of FurE and show that two specific aromatic residues in TMS12, Trp473 and Tyr484, are essential for ER-exit and trafficking to the plasma membrane (PM). Molecular modeling shows that Trp473 and Tyr484 might be essential through dynamic interactions with residues in TMS2 (Leu91), TMS3 (Phe111), TMS10 (Val404, Asp406) and other aromatic residues in TMS12. Genetic analysis confirms the essential role of Phe111, Asp406 and TMS12 aromatic residues in FurE ER-exit. We further show that co-expression of FurE-Y484F or FurE-W473A with wild-type FurE leads to a dominant negative phenotype, compatible with the concept that FurE molecules oligomerize or partition in specific microdomains to achieve concentrative ER-exit and traffic to the PM. Importantly, truncated FurE versions lacking TMS11-12 are unable to reproduce a negative effect on the trafficking of co-expressed wild-type FurE. Overall, we show that TMS11-12 acts as an intramolecular chaperone for proper FurE folding, which seems to provide a structural code for FurE partitioning in ER-exit sites.
PubMed: 38225947
DOI: 10.15698/mic2024.01.811 -
Cellular and Molecular Life Sciences :... Jan 2024Colorectal cancer (CRC) is characterized by a complex tumor inflammatory microenvironment, while angiogenesis and immunosuppression frequently occur concomitantly....
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 CD8T 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.
Topics: Humans; Cell Line, Tumor; Colorectal Neoplasms; NF-kappa B; Signal Transduction; Tumor Microenvironment; Up-Regulation; Membrane Microdomains; Membrane Proteins
PubMed: 38214751
DOI: 10.1007/s00018-023-05105-y -
Biomacromolecules Feb 2024Considerable attention has been dedicated to lipid rafts due to their importance in numerous cell functions such as membrane trafficking, polarization, and signaling....
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 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.
Topics: Unilamellar Liposomes; Polymers; Phospholipids; Membrane Proteins; Membrane Microdomains; Adenosine Triphosphate
PubMed: 38190609
DOI: 10.1021/acs.biomac.3c00972 -
Journal of the American Chemical Society Jan 2024The peroxidation of membrane lipids by free radicals contributes to aging, numerous diseases, and ferroptosis, an iron-dependent form of cell death. Peroxidation changes...
The peroxidation of membrane lipids by free radicals contributes to aging, numerous diseases, and ferroptosis, an iron-dependent form of cell death. Peroxidation changes the structure and physicochemical properties of lipids, leading to bilayer thinning, altered fluidity, and increased permeability of membranes in model systems. Whether and how lipid peroxidation impacts the lateral organization of proteins and lipids in biological membranes, however, remains poorly understood. Here, we employ cell-derived giant plasma membrane vesicles (GPMVs) as a model to investigate the impact of lipid peroxidation on ordered membrane domains, often termed membrane rafts. We show that lipid peroxidation induced by the Fenton reaction dramatically enhances the phase separation propensity of GPMVs into coexisting liquid-ordered (Lo) and liquid-disordered (Ld) domains and increases the relative abundance of the disordered phase. Peroxidation also leads to preferential accumulation of peroxidized lipids and 4-hydroxynonenal (4-HNE) adducts in the disordered phase, decreased lipid packing in both Lo and Ld domains, and translocation of multiple classes of raft proteins out of ordered domains. These findings indicate that the peroxidation of plasma membrane lipids disturbs many aspects of membrane rafts, including their stability, abundance, packing, and protein and lipid composition. We propose that these disruptions contribute to the pathological consequences of lipid peroxidation during aging and disease and thus serve as potential targets for therapeutic intervention.
Topics: Lipid Peroxidation; Phase Separation; Cell Membrane; Membrane Lipids; Proteins; Membrane Microdomains; Lipid Bilayers
PubMed: 38171000
DOI: 10.1021/jacs.3c10132