Membrane Restructuring Research Articles

Probing the interactions of the HIV-1 matrix protein-derived polybasic region with lipid bilayers: insights from AFM imaging and force spectroscopy.

The human immunodeficiency virus type 1 (HIV-1) matrix protein contains a highly basic region, MA-HBR, crucial for various stages of viral replication. To elucidate the interactions between the polybasic peptide MA-HBR and lipid bilayers, we employed liquid-based atomic force microscopy (AFM) imaging and force spectroscopy on lipid bilayers of differing compositions. In 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayers, AFM imaging revealed the formation of annulus-shaped protrusions upon exposure to the polybasic peptide, accompanied by distinctive mechanical responses characterized by enhanced bilayer puncture forces. Importantly, our AFM-based force spectroscopy measurements unveiled that MA-HBR induces interleaflet decoupling within the cohesive bilayer organization. This is evidenced by a force discontinuity observed within the bilayer's elastic deformation regime. In POPC/cholesterol bilayers, MA-HBR caused similar yet smaller annular protrusions, demonstrating an intriguing interplay with cholesterol-rich membranes. In contrast, in bilayers containing anionic 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) lipids, MA-HBR induced unique annular protrusions, granular nanoparticles, and nanotubules, showcasing its distinctive effects in anionic lipid-enriched environments. Notably, our force spectroscopy data revealed that anionic POPS lipids weakened interleaflet adhesion within the bilayer, resulting in interleaflet decoupling, which potentially contributes to the specific bilayer perturbations induced by MA-HBR. Collectively, our findings highlight the remarkable variations in how the polybasic peptide, MA-HBR, interacts with lipid bilayers of differing compositions, shedding light on its role in host membrane restructuring during HIV-1 infection.

Passive transport of Ca2+ ions through lipid bilayers imaged by widefield second harmonic microscopy

In biology, release of Ca2+ ions in the cytosol is essential to trigger or control many cell functions. Calcium signaling acutely depends on lipid membrane permeability to Ca2+. For proper understanding of membrane permeability to Ca2+, both membrane hydration and the structure of the hydrophobic core must be taken into account. Here, we vary the hydrophobic core of bilayer membranes and observe different types of behavior in high-throughput wide-field second harmonic imaging. Ca2+ translocation is observed through mono-unsaturated (DOPC:DOPA) membranes, reduced upon the addition of cholesterol, and completely inhibited for branched (DPhPC:DPhPA) and poly-unsaturated (SLPC:SLPA) lipid membranes. We propose, using molecular dynamics simulations, that ion transport occurs through ion-induced transient pores, which requires nonequilibrium membrane restructuring. This results in different rates at different locations and suggests that the hydrophobic structure of lipids plays a much more sophisticated regulating role than previously thought.

Insights into plastic biodegradation: community composition and functional capabilities of the superworm (Zophobas morio) microbiome in styrofoam feeding trials

Plastics are inexpensive and widely used organic polymers, but their high durability hinders biodegradation. Polystyrene, including extruded polystyrene (also known as styrofoam), is among the most commonly produced plastics worldwide and is recalcitrant to microbial degradation. In this study, we assessed changes in the gut microbiome of superworms (Zophobas morio) reared on bran, polystyrene or under starvation conditions over a 3 weeks period. Superworms on all diets were able to complete their life cycle to pupae and imago, although superworms reared on polystyrene had minimal weight gains, resulting in lower pupation rates compared to bran reared worms. The change in microbial gut communities from baseline differed considerably between diet groups, with polystyrene and starvation groups characterized by a loss of microbial diversity and the presence of opportunistic pathogens. Inferred microbial functions enriched in the polystyrene group included transposon movements, membrane restructuring and adaptations to oxidative stress. We detected several encoded enzymes with reported polystyrene and styrene degradation abilities, supporting previous reports of polystyrene-degrading bacteria in the superworm gut. By recovering metagenome-assembled genomes (MAGs) we linked phylogeny and functions and identified genera including Pseudomonas , Rhodococcus and Corynebacterium that possess genes associated with polystyrene degradation. In conclusion, our results provide the first metagenomic insights into the metabolic pathways used by the gut microbiome of superworms to degrade polystyrene. Our results also confirm that superworms can survive on polystyrene feed, but this diet has considerable negative impacts on host gut microbiome diversity and health.

Interactions of HIV gp41's membrane-proximal external region and transmembrane domain with phospholipid membranes from 31P NMR.

HIV-1 entry into cells requires coordinated changes of the conformation and dynamics of both the fusion protein, gp41, and the lipids in the cell membrane and virus envelope. Commonly proposed features of membrane deformation during fusion include high membrane curvature, lipid disorder, and membrane surface dehydration. The virus envelope and target cell membrane contain a diverse set of phospholipids and cholesterol. To dissect how different lipids interact with gp41 to contribute to membrane fusion, here we use 31P solid-state NMR spectroscopy to investigate the curvature, dynamics, and hydration of POPE, POPC and POPS membranes, with and without cholesterol, in the presence of a peptide comprising the membrane proximal external region (MPER) and transmembrane domain (TMD) of gp41. Static 31P NMR spectra indicate that the MPER-TMD induces strong negative Gaussian curvature (NGC) to the POPE membrane but little curvature to POPC and POPC:POPS membranes. The NGC manifests as an isotropic peak in the static NMR spectra, whose intensity increases with the peptide concentration. Cholesterol inhibits the NGC formation and stabilizes the lamellar phase. Relative intensities of magic-angle spinning 31P cross-polarization and direct-polarization spectra indicate that all three phospholipids become more mobile upon peptide binding. Finally, 2D 1H-31P correlation spectra show that the MPER-TMD enhances water 1H polarization transfer to the lipids, indicating that the membrane surfaces become more hydrated. These results suggest that POPE is an essential component of the high-curvature fusion site, and lipid dynamic disorder is a general feature of membrane restructuring during fusion.

Actin Cytoskeletal Dynamics in Single-Cell Wound Repair.

The plasma membrane protects the eukaryotic cell from its surroundings and is essential for cell viability; thus, it is crucial that membrane disruptions are repaired quickly to prevent immediate dyshomeostasis and cell death. Accordingly, cells have developed efficient repair mechanisms to rapidly reseal ruptures and reestablish membrane integrity. The cortical actin cytoskeleton plays an instrumental role in both plasma membrane resealing and restructuring in response to damage. Actin directly aids membrane repair or indirectly assists auxiliary repair mechanisms. Studies investigating single-cell wound repair have often focused on the recruitment and activation of specialized repair machinery, despite the undeniable need for rapid and dynamic cortical actin modulation; thus, the role of the cortical actin cytoskeleton during wound repair has received limited attention. This review aims to provide a comprehensive overview of membrane repair mechanisms directly or indirectly involving cortical actin cytoskeletal remodeling.

Serum metabolites associated with brain amyloid beta deposition, cognition and dementia progression.

Metabolomics in the Alzheimer’s Disease Neuroimaging Initiative cohort provides a powerful tool for mapping biochemical changes in Alzheimer’s disease, and a unique opportunity to learn about the association between circulating blood metabolites and brain amyloid-β deposition in Alzheimer’s disease. We examined 140 serum metabolites and their associations with brain amyloid-β deposition, cognition and conversion from mild cognitive impairment to Alzheimer’s disease in the Alzheimer’s Disease Neuroimaging Initiative. Processed [18F] Florbetapir PET images were used to perform a voxel-wise statistical analysis of the effect of metabolite levels on amyloid-β accumulation across the whole brain. We performed a multivariable regression analysis using age, sex, body mass index, apolipoprotein E ε4 status and study phase as covariates. We identified nine metabolites as significantly associated with amyloid-β deposition after multiple comparison correction. Higher levels of one acylcarnitine (C3; propionylcarnitine) and one biogenic amine (kynurenine) were associated with decreased amyloid-β accumulation and higher memory scores. However, higher levels of seven phosphatidylcholines (lysoPC a C18:2, PC aa C42:0, PC ae C42:3, PC ae C44:3, PC ae C44:4, PC ae C44:5 and PC ae C44:6) were associated with increased brain amyloid-β deposition. In addition, higher levels of PC ae C44:4 were significantly associated with lower memory and executive function scores and conversion from mild cognitive impairment to Alzheimer’s disease dementia. Our findings suggest that dysregulation of peripheral phosphatidylcholine metabolism is associated with earlier pathological changes noted in Alzheimer’s disease as measured by brain amyloid-β deposition as well as later clinical features including changes in memory and executive functioning. Perturbations in phosphatidylcholine metabolism may point to issues with membrane restructuring leading to the accumulation of amyloid-β in the brain. Additional studies are needed to explore whether these metabolites play a causal role in the pathogenesis of Alzheimer’s disease or if they are biomarkers for systemic changes during preclinical phases of the disease.

Conformational plasticity underlies membrane fusion induced by an HIV sequence juxtaposed to the lipid envelope

Envelope glycoproteins from genetically-divergent virus families comprise fusion peptides (FPs) that have been posited to insert and perturb the membranes of target cells upon activation of the virus-cell fusion reaction. Conserved sequences rich in aromatic residues juxtaposed to the external leaflet of the virion-wrapping membranes are also frequently found in viral fusion glycoproteins. These membrane-proximal external regions (MPERs) have been implicated in the promotion of the viral membrane restructuring event required for fusion to proceed, hence, proposed to comprise supplementary FPs. However, it remains unknown whether the structure–function relationships governing canonical FPs also operate in the mirroring MPER sequences. Here, we combine infrared spectroscopy-based approaches with cryo-electron microscopy to analyze the alternating conformations adopted, and perturbations generated in membranes by CpreTM, a peptide derived from the MPER of the HIV-1 Env glycoprotein. Altogether, our structural and morphological data support a cholesterol-dependent conformational plasticity for this HIV-1 sequence, which could assist cell-virus fusion by destabilizing the viral membrane at the initial stages of the process.

RICE SEEDLING ESTABLISHMENT AS INFLUENCED BY CULTIVARS AND SEED PRIMING WITH POTASSIUM NITRATE

Poor crop establishment is one of the major constraints to obtain the higher potential of rice, particularly in areas prone to environmental stresses. Therefore, present study was conducted to evaluate the effects of potassium nitrate on germination dynamics, seedling growth and associated physiological and biochemical events of two rice cultivars. For this purpose, various seed priming treatments were tested in lab and greenhouse. The percent concentrations of potassium nitrate were 0.25, 0.50, 0.75, 1.0 and 1.25 in both lab evaluation and greenhouse experiment. Non-primed seeds were maintained as a control for comparison. The results depicted that soaking rice seeds at higher concentrations of KNO3 could delay emergence time and final emergence (%) in both cultivars under lab and greenhouse conditions. Seed priming with 0.75% KNO3 significantly increased the stand establishment and seedling vigor attributes of both cultivars compared with other concentrations and naked rice seeds both in lab evaluation and greenhouse screening. Similarly, highest values for photosynthesis rate, evaporation rate and CO2 index were observed in experimental units receiving primed seeds with 0.75% KNO3 under greenhouse screening. Interestingly, no variance was observed among both rice cultivars. Overall, higher seed emergence, seedling vigor and associated biochemical attributes due to seed priming with 0.75% KNO3 was associated with decrease in alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDH) activities in lab and greenhouse screening.

Photobiological and lipidic responses reveal the drought tolerance of Aster tripolium cultivated under severe and moderate drought: Perspectives for arid agriculture in the mediterranean

In the past Aster tripolium has already proved to be a good candidate for saline agriculture in soils with low water availability. Thus, the aim of the present work was to disentangle the photobiological and biochemical mechanisms underlying the response of A. tripolium to PEG-induced drought stress, by exposing plants to PEG-induced moderate and severe drought conditions. Plant primary productivity was maintained under moderate drought conditions, due to the presence of alternative electron donors fueling the PSII. Additionally, the high anthocyanin production under drought conditions, act as photoprotective shields against photoinhibition. Moreover, the increased quinone turnover rate simultaneously with a net rate of RC closure and density increase, acted as a counteractive measure, allowing high energy fluxes into the photosystems under drought conditions. PSI showed an activity reduction, indicating that under drought conditions the ETC activity acts as an energetic escape route. Furthermore, membrane remodeling could also be observed under drought. The total fatty acid and omega-3 linolenic acid (18:3) contents were maintained, under osmotic stress. Membrane restructuring with lower amounts of polyunsaturated fatty acids (18:3) is considered an adaptation to osmotically stressful environments. Increased 18:1 and 16:1t fatty acids production improve the LHCs and chloroplast membrane stabilization, allowing the LHC to maintain its efficient functioning. The results here presented are very similar to the ones observed in the past regarding A. tripolium feedback to salinity stress, indicating that the mechanisms to overcome osmotic stress, either due to increased salinity or reduced water availability, are the same.

Unexpected Cholesterol-Induced Destabilization of Lipid Membranes near Transmembrane Carbon Nanotubes

Cholesterol is a crucial component of mammalian cell membranes that takes part in many vital processes. It is generally accepted that cholesterol stabilizes the membrane and induces transitions into ordered states. In contrast to expectations, we demonstrate that cholesterol can destabilize the membrane by creating a nanodomain around a perpendicularly embedded ultrashort carbon nanotube (CNT), and we show that cholesterol triggers the translocation of an ultrashort CNT through the cell membrane. Using atomistic simulations, we report the existence of a nanoscale domain around an ultrashort carbon nanotube within a crossover distance of 0.9nm from the surface of the nanotube, where the properties of the bilayer are different from the bulk: the domain is characterized by increased fluctuations, increased thickness, and increased order of the lipids with respect to the bulk. Cholesterol decreases the thickness and order of lipids and increases the fluctuations with respect to a pure lipid bilayer. Experimentally, we confirm that cholesterol nanodomains provoke spontaneous translocation of nanotubes through a lipid bilayer even for low membrane tensions. A specially designed microfluidic device allows us to trace the kinetic pathway of the translocation process and establish the threshold cholesterol concentration of 20% for translocation. The reported nanoscale cholesterol-induced membrane restructuring near the ultrashort CNT in lipid membranes enables precise control and specific targeting of a membrane using cholesterol. As an example, it may allow for specific targeting between cholesterol-rich mammalian cells and cholesterol-poor bacterial cells.

Hypoxia-induced remodelling of goldfish membranes

Hypoxia-tolerant animals use metabolic suppression as an essential strategy to survive low oxygen. Ectotherms can alter membrane lipid composition in response to changes in environmental temperature, but it is currently unknown whether chronic hypoxia can also elicit membrane restructuring. The goal of this study was to investigate a possible physiological link between membrane remodelling and metabolic suppression in goldfish exposed to prolonged hypoxia (4 weeks at 10% air saturation). We have tested the hypothesis that chronic hypoxia would modulate membrane lipid composition in ways that are consistent with known mechanisms of ion pump inhibition. Because homeoviscous membrane restructuring could interfere with the response to hypoxia, measurements were made at 2 temperatures. Results show that hypoxic goldfish suppress metabolic rate by 74% (at 13 °C) and 63% (at 20 °C). This study is the first to reveal that cold-acclimated animals undergo extensive, tissue-specific restructuring of membrane lipids as they reach minimal metabolic rates. However, hypoxia does not affect membrane composition in fish acclimated to 20 °C. The strong membrane response of cold-acclimated fish involves increases in cholesterol abundance (in white muscle and gills) and in fatty acid saturation, mainly caused by a reduction in %22:6 (docosahexaenoic acid in gills and liver). Major ion pumps like Na+/K+-ATPase are known to be inhibited by cholesterol and activated by 22:6. Because ion pumping by membrane-bound ATPases accounts for a large fraction of basal cellular energy use, we propose that the membrane responses reported here could be a novel mechanism to promote metabolic suppression in cold-acclimated animals.

Influenza A matrix protein M1 induces lipid membrane deformation via protein multimerization.

The matrix protein M1 of the Influenza A virus (IAV) is supposed to mediate viral assembly and budding at the plasma membrane (PM) of infected cells. In order for a new viral particle to form, the PM lipid bilayer has to bend into a vesicle toward the extracellular side. Studies in cellular models have proposed that different viral proteins might be responsible for inducing membrane curvature in this context (including M1), but a clear consensus has not been reached. In the present study, we use a combination of fluorescence microscopy, cryogenic transmission electron microscopy (cryo-TEM), cryo-electron tomography (cryo-ET) and scanning fluorescence correlation spectroscopy (sFCS) to investigate M1-induced membrane deformation in biophysical models of the PM. Our results indicate that M1 is indeed able to cause membrane curvature in lipid bilayers containing negatively charged lipids, in the absence of other viral components. Furthermore, we prove that protein binding is not sufficient to induce membrane restructuring. Rather, it appears that stable M1–M1 interactions and multimer formation are required in order to alter the bilayer three-dimensional structure, through the formation of a protein scaffold. Finally, our results suggest that, in a physiological context, M1-induced membrane deformation might be modulated by the initial bilayer curvature and the lateral organization of membrane components (i.e. the presence of lipid domains).

Identification of key genes and its chromosome regions linked to drought responses in leaves across different crops through meta-analysis of RNA-Seq data

BackgroundOur study is the first to provide RNA-Seq data analysis related to transcriptomic responses towards drought across different crops. The aim was to identify and map which genes play a key role in drought response on leaves across different crops. Forty-two RNA-seq samples were analyzed from 9 published studies in 7 plant species (Arabidopsis thaliana, Solanum lycopersicum, Zea mays, Vitis vinifera, Malus X domestica, Solanum tuberosum, Triticum aestivum).ResultsTwenty-seven (16 up-regulated and 11 down-regulated) drought-regulated genes were commonly present in at least 7 of 9 studies, while 351 (147 up-regulated and 204 down-regulated) were commonly drought-regulated in 6 of 9 studies. Across all kind of leaves, the drought repressed gene-ontologies were related to the cell wall and membrane re-structuring such as wax biosynthesis, cell wall organization, fatty acid biosynthesis. On the other hand, drought-up-regulated biological processes were related to responses to osmotic stress, abscisic acid, water deprivation, abscisic-activated signalling pathway, salt stress, hydrogen peroxide treatment. A common metabolic feature linked to drought response in leaves is the repression of terpenoid pathways. There was an induction of AL1 (alfin-like), UGKYAH (trihelix), WRKY20, homeobox genes and members of the SET domain family in 6 of 9 studies. Several genes involved in detoxifying and antioxidant reactions, signalling pathways and cell protection were commonly modulated by drought across the 7 species. The chromosome (Chr) mapping of these key abiotic stress genes highlighted that Chr 4 in Arabidopsis thaliana, Chr 1 in Zea mays, Chr 2 and Chr 5 in Triticum aestivum contained a higher presence of drought-related genes compared to the other remaining chromosomes. In seedling studies, it is worth notice the up-regulation of ERF4 and ESE3 (ethylene), HVA22 (abscisic acid), TIR1 (auxin) and some transcription factors (MYB3, MYB94, MYB1, WRKY53 and WRKY20). In mature leaves, ERF1 and Alfin-like 1 were induced by drought while other transcription factors (YABBY5, ARR2, TRFL2) and genes involved phospholipid biosynthesis were repressed.ConclusionsThe identified and mapped genes might be potential targets of molecular breeding activities to develop cultivars with enhanced drought resistance and tolerance across different crops.

Lipopolysaccharide-Dependent Membrane Permeation and Lipid Clustering Caused by Cyclic Lipopeptide Colistin.

Polyanionic lipopolysaccharides (LPS) play an important role in regulating the permeability of the outer membrane (OM) of Gram-negative bacteria. Impairment of the LPS-enriched OM is essential in initiating the bactericidal activity of polymyxins. We are interested in how colistin (polymyxin E) affects the membrane permeability of LPS/phospholipid bilayers. Our vesicle leakage experiment showed that colistin binding enhanced bilayer permeability; the maximum increase in the bilayer permeability was positively correlated with the LPS fraction. Addition of magnesium ions abolished the effect of LPS in enhancing bilayer permeabilization. To describe the vesicle leakage behavior from a structural perspective, we performed liquid atomic force microscopy (AFM) measurements on planar lipid bilayers. We found that colistin caused the formation of nano- and macroclusters that protruded from the bilayer by ∼2 nm. Moreover, cluster development was promoted by increasing the fraction of LPS or colistin concentration but inhibited by magnesium ions. To explain our experimental data, we proposed a lipid clustering model where colistin binds to LPS to form large-scale complexes segregated from zwitterionic phospholipids. The discontinuity (and thickness mismatch) at the edge of LPS–colistin clusters will create a passage that allows solutes to permeate through. The proposed model is consistent with all data obtained from our leakage and AFM experiments. Our results of LPS-dependent membrane restructuring provided useful insights into the mechanism that could be used by polymyxins in impairing the permeability barrier of the OM of Gram-negative bacteria.

Cholesterol and phosphatidylethanolamine lipids exert opposite effects on membrane modulations caused by the M2 amphipathic helix

Membrane curvature remodeling induced by amphipathic helices (AHs) is essential in many biological processes. Here we studied a model amphipathic peptide, M2AH, derived from influenza A M2. We are interested in how M2AH may promote membrane curvature by altering membrane physical properties. We used atomic force microscopy (AFM) to examine changes in membrane topographic and mechanical properties. We used electron paramagnetic resonance (EPR) spectroscopy to explore changes in lipid chain mobility and chain orientational order. We found that M2AH perturbed lipid bilayers by generating nanoscale pits. The structural data are consistent with lateral expansion of lipid chain packing, resulting in a mechanically weaker bilayer. Our EPR spectroscopy showed that M2AH reduced lipid chain mobility and had a minimal effect on lipid chain orientational order. The EPR data are consistent with the surface-bound state of M2AH that acts as a chain mobility inhibitor. By comparing results from different lipid bilayers, we found that cholesterol enhanced the activity of M2AH in inducing bilayer pits and altering lipid chain mobility. The results were explained by considering specific M2AH-cholesterol recognition and/or cholesterol-induced expansion of interlipid distance. Both AFM and EPR experiments revealed a modest effect of anionic lipids. This highlights that membrane interaction of M2AH is mainly driven by hydrophobic forces. Lastly, we found that phosphatidylethanolamine (PE) lipids inhibited the activity of M2AH. We explained our data by considering interlipid hydrogen-bonding that can stabilize bilayer organization. Our results of lipid-dependent membrane modulations are likely relevant to M2AH-induced membrane restructuring.

1385 Dual mechanism of Type VII collagen transfer by bone marrow mesenchymal stem cell extracellular vesicles to recessive dystrophic epidermolysis bullosa fibroblasts

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe blistering disease resulting from a lack of type VII collagen production. Recent clinical trials have shown efficacy of bone marrow-derived stem cells (BM-MSCs) in the treatment of epidermolysis bullosa, including improved basement membrane restructuring and cutaneous wound healing. The mechanism as to how type VII collagen is transferred from donor stem cell to recipient RDEB cells has not been defined. Here, we report that BM-MSC-derived extracellular vesicles serve at least two roles: 1) to help transport the insoluble type VII collagen alpha chains within the extracellular space; and 2) to feed RDEB fibroblasts with messenger RNA that codes for type VII collagen, resulting in COL7A1 translation and synthesis of normal type VII collagen alpha chain proteins, eventually secreted by RDEB fibroblasts. Utilizing a chemoselective ligation detection method, we found RDEB cells that were treated simultaneously with BM-MSC EVs and an L-methionine analog, L-homopropargylglycine (HPG), synthesized collagen VII alpha chain protein that contained the alkyne group of HPG to react (i.e. undergo the Click-iT® reaction) with azide-modified Alexa 594, enabling detection and suggesting de novo synthesis of type VII collagen by RDEB fibroblasts. These dual mechanisms could result in a net positive “transfer” of type VII collagen to the extracellular environment of skin in RDEB patients, likely improving both basement membrane integrity and wound healing. Therapies that utilize EVs to enhance delivery of type VII collagen, both in terms of nucleic acids and protein, have potential to improve the functions of skin in RDEB patients.

Dual mechanism of type VII collagen transfer by bone marrow mesenchymal stem cell extracellular vesicles to recessive dystrophic epidermolysis bullosa fibroblasts

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe blistering disease resulting from a lack of type VII collagen production. Recent clinical trials have shown efficacy of bone marrow-derived mesenchymal stem cells (BM-MSCs) in the treatment of epidermolysis bullosa, including improved basement membrane restructuring and cutaneous wound healing. The mechanism as to how type VII collagen is transferred from donor stem cell to recipient RDEB cells has not been defined. Here, we submit the model that BM-MSC-derived extracellular vesicles serve at least two roles: 1) to help transport type VII collagen within the extracellular space; and 2) to feed RDEB fibroblasts with messenger RNA that codes for type VII collagen, resulting in COL7A1 translation and synthesis of type VII collagen alpha chain proteins by RDEB fibroblasts. Utilizing a chemoselective ligation detection method, we found RDEB cells that were treated simultaneously with BM-MSC EVs and an l-methionine analog, l-homopropargylglycine (HPG), synthesized collagen VII alpha chain protein that contained the alkyne group of HPG to react (i.e. undergo the Click-iT® reaction) with azide-modified Alexa 594, suggesting de novo synthesis of type VII collagen by RDEB fibroblasts. Thus, our results support a model in which BM-MSC EVs help increase type VII collagen levels available to recipient cells by 1) donating BM-MSC type VII collagen protein and 2) inducing RDEB fibroblasts to make their own type VII collagen protein. These findings allow us to hypothesize that the secretome of BM-MSCs could have therapeutic value in the treatment of RDEB-related skin disorders.

Membrane Restructuring Events during the Enzymatic Generation of Ceramides with Very Long-Chain Polyunsaturated Fatty Acids.

In rat sperm heads, sphingomyelin (SM) species that contain very long-chain polyunsaturated fatty acid (V-SM) become ceramides (V-Cer) after inducing in vitro the acrosomal reaction. The reason for such a specific location of this conversion, catalyzed by a sphingomyelinase (SMase), has received little investigation so far. Here, the effects of SMase were compared in unilamellar vesicles (large unilamellar vesicles (LUVs), giant unilamellar vesicles (GUVs)) containing phosphatidylcholine, and either V-SM or a palmitate-rich SM (P-SM). In uniformly sized LUVs at 37 °C, more V-Cer was generated and more rapidly than P-Cer. Nephelometry and dynamic light scattering showed that LUVs tended to form large lipid particles more intensely, and Förster resonance energy transfer (FRET) increases suggested that lateral lipid mixing was more marked when V-Cer rather than P-Cer was produced. As reported by 6-dodecanoyl-2-dimethyl-aminopnaphthalene (Laurdan) and 1,6-diphenyl-1,3,5,-hexatriene (DPH), the production of V-Cer resulted in higher and faster restriction in lipid mobility than that of P-Cer, implying a stronger increase in membrane dehydration and microviscosity. Moreover, DPH anisotropy suggested a higher solubility of V-Cer than that of P-Cer in the liquid-disordered phase. At room temperature, liquid-condensed lateral domains appeared in P-SM- but not in V-SM-containing GUVs. The former maintained their size while losing their contents gradually during SMase action, whereas the latter became permeable earlier and reduced their size in few minutes until suddenly collapsing. The fast and potent generation of V-Cer may contribute to the membrane restructuring events that occur on the acrosome-reacted sperm head.

Expansion of Sphingosine Kinase and Sphingosine-1-Phosphate Receptor Function in Normal and Cancer Cells: From Membrane Restructuring to Mediation of Estrogen Signaling and Stem Cell Programming.

Sphingolipids, sphingolipid metabolizing enzymes, and their receptors network are being recognized as part of the signaling mechanisms, which govern breast cancer cell growth, migration, and survival during chemotherapy treatment. Approximately 70% of breast cancers are estrogen receptor (ER) positive and, thus, rely on estrogen signaling. Estrogen activates an intracellular network composed of many cytoplasmic and nuclear mediators. Some estrogen effects can be mediated by sphingolipids. Estrogen activates sphingosine kinase 1 (SphK1) and amplifies the intracellular concentration of sphingosine-1-phosphate (S1P) in breast cancer cells during stimulation of proliferation and survival. Specifically, Estrogen activates S1P receptors (S1PR) and induces growth factor receptor transactivation. SphK, S1P, and S1PR expression are causally associated with endocrine resistance and progression to advanced tumor stages in ER-positive breast cancers in vivo. Recently, the network of SphK/S1PR was shown to promote the development of ER-negative cancers and breast cancer stem cells, as well as stimulating angiogenesis. Novel findings confirm and broaden our knowledge about the cross-talk between sphingolipids and estrogen network in normal and malignant cells. Current S1PRs therapeutic inhibition was indicated as a promising chemotherapy approach in non-responsive and advanced malignancies. Considering that sphingolipid signaling has a prominent role in terminally differentiated cells, the impact should be considered when designing specific SphK/S1PR inhibitors. This study analyzes the dynamic of the transformation of sphingolipid axis during a transition from normal to pathological condition on the level of the whole organism. The sphingolipid-based mediation and facilitation of global effects of estrogen were critically accented as a bridging mechanism that should be explored in cancer prevention.

Temperature induced lipid membrane restructuring and changes in nanomechanics

The naturally occurring milk sphingomyelin is of particular interest owing to its complex composition and involvement in the formation of the milk fat globule membrane (MFGM). Knowledge of membrane organization and nanomechanical stability has proved to be crucial in understanding their properties and functions. In this work, two model membrane systems composed of 1, 2 dioleoyl-sn-glycero-3-phosphocholine (DOPC), egg sphingomyelin (egg-SM) and cholesterol, and DOPC, milk sphingomyelin (milk-SM) and cholesterol were exposed to both RT and 10°C. The morphological and nanomechanical changes were investigated using atomic force microscopy (AFM) imaging and force mapping below RT using a designed liquid cell with temperature-control. In both systems, the size and shape of SM/Chol-enriched liquid ordered domains (Lo) and DOPC-enriched liquid disordered phase (Ld) were monitored at controlled temperatures. AFM based force-mapping showed that rupture forces were consistently higher for Lo domains than Ld phases and were decreased for Ld with decreasing temperature while an increase in breakthrough force was observed in Lo domains. More interestingly, dynamic changes and defect formations in the hydrated lipid bilayers were mostly detected at low temperature, suggesting a rearrangement of lipid molecules to relieve additional tension introduced upon cooling. Noteworthy, in these model membrane systems, tension-driven defects generally heal on reheating the sample. The results of this work bring new insights to low temperature induced membrane structural reorganization and mechanical stability changes which will bring us one step closer to understand more complex systems such as the MFGM.