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Cell membranes are composed of a large variety of lipids and proteins. While the localization and function of membrane proteins have been extensively investigated, the distribution of membrane lipids, especially in the non-cytoplasmic leaflet of organelle membranes, remains largely unknown. Fluorescent biosensors have been widely used to study membrane lipid distribution; however, they have some limitations. By utilizing the quick-freezing and freeze-fracture replica labeling electron microscopy technique, we can uncover the precise distribution of membrane lipids within cells and assess the function of lipid-transporting proteins. In this review, I summarize recent progress in analyzing intracellular lipid distribution by utilizing this method. [ABSTRACT FROM AUTHOR]

Although the human immunodeficiency virus type 1 lipid envelope has been reported to be enriched with host cell sphingomyelin and cholesterol, the molecular mechanism of the enrichment is not well understood. Viral Gag protein plays a central role in virus budding. Here, we report the interaction between Gag and host cell lipids using different quantitative and super-resolution microscopy techniques in combination with specific probes that bind endogenous sphingomyelin and cholesterol. Our results indicate that Gag in the inner leaflet of the plasma membrane colocalizes with the outer leaflet sphingomyelin-rich domains and cholesterol-rich domains, enlarges sphingomyelin-rich domains, and strongly restricts the mobility of sphingomyelin-rich domains. Moreover, Gag multimerization induces sphingomyelin-rich and cholesterol-rich lipid domains to be in close proximity in a curvature-dependent manner. Our study suggests that Gag binds, coalesces, and reorganizes pre-existing lipid domains during assembly. Quantitative microscopies and specific lipid probes revealed that multimerizing HIV-1 Gag in the plasma membrane inner leaflet brings the outer leaflet sphingomyelin-rich and cholesterol-rich lipid domains closer in a curvature-dependent manner. [ABSTRACT FROM AUTHOR]

Sphingolipids play crucial roles in cellular membranes, myelin stability, and signalling responses to physiological cues and stress. Among them, sphingosine 1-phosphate (S1P) has been recognized as a relevant biomarker for neurodegenerative diseases, and its analogue FTY-720 has been approved by the FDA for the treatment of relapsing–remitting multiple sclerosis. Focusing on these targets, we here report three novel polymeric capture phases for the selective extraction of the natural biomarker and its analogue drug. To enhance analytical performance, we employed different synthetic approaches using a cationic monomer and a hydrophobic copolymer of styrene-DVB. Results have demonstrated high affinity of the sorbents towards S1P and fingolimod phosphate (FTY-720-P, FP). This evidence proved that lipids containing phosphate diester moiety in their structures did not constitute obstacles for the interaction of phosphate monoester lipids when loaded into an SPE cartridge. Our suggested approach offers a valuable tool for developing efficient analytical procedures. [ABSTRACT FROM AUTHOR]

The biosynthesis and secretion of Monascus pigments are closely related to the integrity of the cell membrane, which determines the composition of lipids and its content in cell membrane. The present study aimed to thoroughly describe the changes of lipid profiling in Monascus purpureus BWY-5, which was screened by carbon ion beam irradiation (12C6+) to almost single yield extracellular Monascus yellow pigments (extra-MYPs), by absolute quantitative lipidomics and tandem mass tags (TMT) based quantitative proteomic. 12C6+ irradiation caused non-lipid oxidation damage to Monascus cell membrane, leading to an imbalance in cell membrane lipid homeostasis. This imbalance was attributed to significant changes not only in the composition but also in the content of lipids in Monascus, especially the inhibition of glycerophospholipid biosynthesis. Integrity of plasma membrane was maintained by the increased production of ergosterol, monogalactosylmonoacylglycerol (MGMG) and sulfoquinovosylmonoacylglycerol (SQMG), while mitochondrial membrane homeostasis was maintained by the increase of cardiolipin production. The growth and extra-MYPs production of Monascus BWY-5 have been regulated by the promotion of sphingolipids (ceramide and sulfatide) biosynthesis. Simultaneous, energy homeostasis may be achieved by increase of TG synthesis and Ca2+/Mg2+-ATPase activity. These finding suggest ergosterol, cardiolipin, sphingolipids, MGMG and SQMG play a key facilitating role in cytomembrane lipid homeostasis maintaining for Monascus purpureus BWY-5, and then it is closely related to cell growth and extra-MYPs production. Key points: 1. Energy homeostasis in Monascus purpureus BWY-5 was achieved by increase of TG synthesis and Ca2+/Mg2+-ATPase activity. 2. Integrity of plasma membrane in Monascus purpureus BWY-5 was maintained by the increased production of ergosterol. 3. Mitochondrial membrane homeostasis in Monascus purpureus BWY-5 was maintaed by the increase of cardiolipin synthesis. [ABSTRACT FROM AUTHOR]

Platelet integrin αIIbβ3 is maintained in a bent inactive state (low affinity to physiologic ligand), but can rapidly switch to a ligand-competent (high-affinity) state in response to intracellular signals ("inside-out" activation). Once bound, ligands drive proadhesive "outside-in" signaling. Anti-αIIbβ3 drugs like eptifibatide can engage the inactive integrin directly, inhibiting thrombosis but inadvertently impairing αIIbβ3 hemostatic functions. Bidirectional αIIbβ3 signaling is mediated by reorganization of the associated αIIb and β3 transmembrane α-helices, but the underlying changes remain poorly defined absent the structure of the full-length receptor. We now report the cryo-EM structures of full-length αIIbβ3 in its apo and eptifibatide-bound states in native cell-membrane nanoparticles at near-atomic resolution. The apo form adopts the bent inactive state but with separated transmembrane α-helices, and a fully accessible ligand-binding site that challenges the model that this site is occluded by the plasma membrane. Bound eptifibatide triggers dramatic conformational changes that may account for impaired hemostasis. These results advance our understanding of integrin structure and function and may guide development of safer inhibitors. The structural basis of integrin signaling in health and disease is not fully understood. Here, the authors determine the cryoEM structure of full-length platelet integrin αIIbβ3 in its apo and eptifibatide-bound conformations in a native membrane environment. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Spinster (Spns) lipid transporters are critical for transporting sphingosine-1-phosphate (S1P) across cellular membranes. In humans, Spns2 functions as the main S1P transporter in endothelial cells, making it a potential drug target for modulating S1P signaling. Here, we employed an integrated approach in lipid membranes to identify unknown conformational states of a bacterial Spns from Hyphomonas neptunium (HnSpns) and to define its proton- and substrate-coupled conformational dynamics. Our systematic study reveals conserved residues critical for protonation steps and their regulation, and how sequential protonation of these proton switches coordinates the conformational transitions in the context of a noncanonical ligand-dependent alternating access. A conserved periplasmic salt bridge (Asp60TM2:Arg289TM7) keeps the transporter in a closed conformation, while proton-dependent conformational dynamics are significantly enhanced on the periplasmic side, providing a pathway for ligand exchange. The Spns lipid transporters use ion gradients to drive substrate transport, including bioactive sphingolipids. Here, Dastvan et al. investigated how binding of protons powers the conformational changes that enable broad transport by a bacterial Spns. [ABSTRACT FROM AUTHOR]

TMEM16 scramblases dissipate the plasma membrane lipid asymmetry to activate multiple eukaryotic cellular pathways. Scrambling was proposed to occur with lipid headgroups moving between leaflets through a membrane-spanning hydrophilic groove. Direct information on lipid-groove interactions is lacking. We report the 2.3 Å resolution cryogenic electron microscopy structure of the nanodisc-reconstituted Ca2+-bound afTMEM16 scramblase showing how rearrangement of individual lipids at the open pathway results in pronounced membrane thinning. Only the groove's intracellular vestibule contacts lipids, and mutagenesis suggests scrambling does not require specific protein-lipid interactions with the extracellular vestibule. We find scrambling can occur outside a closed groove in thinner membranes and is inhibited in thicker membranes, despite an open pathway. Our results show afTMEM16 thins the membrane to enable scrambling and that an open hydrophilic pathway is not a structural requirement to allow rapid transbilayer movement of lipids. This mechanism could be extended to other scramblases lacking a hydrophilic groove. Lipid scrambling is required for many cellular processes but the lipid-protein interactions that occur during transport are unknown. Using cryoEM and biochemical assays the authors show that membrane thinning is critical for lipid flipping. [ABSTRACT FROM AUTHOR]

Environmental stimuli are primarily perceived at the plasma membrane. Stimuli perception leads to membrane disintegration and generation of molecules which trigger lipid signaling. In plants, lipid signaling regulates important biological functions however, the molecular mechanism involved is unclear. Phospholipases C (PLCs) are important lipid-modifying enzymes in eukaryotes. In animals, PLCs by hydrolyzing phospholipids, such as phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2] generate diacylglycerol (DAG) and inositol- 1,4,5-trisphosphate (IP3). However, in plants their phosphorylated variants i.e., phosphatidic acid (PA) and inositol hexakisphosphate (IP6) are proposed to mediate lipid signaling. Specific substrate preferences divide PLCs into phosphatidylinositol–PLC (PI–PLC) and non-specific PLCs (NPC). PLC activity is regulated by various cellular factors including, calcium (Ca2+) concentration, phospholipid substrate, and post-translational modifications. Both PI–PLCs and NPCs are implicated in plants' response to stresses and development. Emerging evidences show that PLCs regulate structural and developmental features, like stomata movement, microtubule organization, membrane remodelling and root development under abiotic stresses. Thus, crucial insights are provided into PLC mediated regulatory mechanism of abiotic stress responses in plants. In this review, we describe the structure and regulation of plant PLCs. In addition, cellular and physiological roles of PLCs in abiotic stresses, phosphorus deficiency, aluminium toxicity, pollen tube growth, and root development are discussed. [ABSTRACT FROM AUTHOR]

Different types of cellular membranes have unique lipid compositions that are important for their functional identity. PI(4,5)P2 is enriched in the plasma membrane where it contributes to local activation of key cellular events, including actomyosin contraction and cytokinesis. However, how cells prevent PI(4,5)P2 from accumulating in intracellular membrane compartments, despite constant intermixing and exchange of lipid membranes, is poorly understood. Using the C. elegans early embryo as our model system, we show that the evolutionarily conserved lipid transfer proteins, PDZD-8 and TEX-2, act together with the PI(4,5)P2 phosphatases, OCRL-1 and UNC-26/synaptojanin, to prevent the build-up of PI(4,5)P2 on endosomal membranes. In the absence of these four proteins, large amounts of PI(4,5)P2 accumulate on endosomes, leading to embryonic lethality due to ectopic recruitment of proteins involved in actomyosin contractility. PDZD-8 localizes to the endoplasmic reticulum and regulates endosomal PI(4,5)P2 levels via its lipid harboring SMP domain. Accumulation of PI(4,5)P2 on endosomes is accompanied by impairment of their degradative capacity. Thus, cells use multiple redundant systems to maintain endosomal PI(4,5)P2 homeostasis. Cellular membranes have distinct lipid compositions despite intermixing, and it is unclear why plasma membrane lipids do not accumulate on endosomes. Here, the authors use the C. elegans embryo to identify lipid transfer proteins and phosphatases that are critical for endosomal lipid homeostasis. [ABSTRACT FROM AUTHOR]

The physical properties of lipids, such as viscosity, are homeostatically maintained in cells and are intimately involved in physiological roles. Measurement of the physical properties of plasma membranes has been achieved primarily through chemical or genetically encoded fluorescent probes. However, since most probes target plasma membranes, physical properties of lipids in intracellular organelles, including lipid droplets (LDs) are yet to be analyzed. Here, we present a novel Raman microscopy-based approach for quantifying the physical properties of intracellular lipids under deuterium-labeled fatty acid treatment conditions. Focusing on the fact that Raman spectra of carbon-deuterium vibration are altered depending on the surrounding lipid species, we quantitatively represented the physical properties of lipids as the gauche/trans conformational ratio of the introduced labeled fatty acids, which can be used as an indicator of viscosity. Intracellular Raman imaging revealed that the gauche/trans ratio of cytosolic regions was robustly preserved against perturbations attempting to alter the lipid composition. This was likely due to LDs functioning as a buffer against excess gauche/trans ratio, beyond its traditional role as an energy storage organelle. Our novel approach enables the observation of the physical properties of organelle lipids, which is difficult to perform with conventional probes, and is useful for quantitative assessment of the subcellular lipid environment. Uematsu and Shimizu use Raman spectroscopy to quantify changes in the physical properties of lipid droplets (LDs) and in non-LD cell regions associated with alterations in saturated and unsaturated fatty acid availability in culture medium. Their results suggest that Raman spectroscopy can yield a quantitative measurement of changes in lipid conformation and can quantify trans / gauche ratios in lipid chains. [ABSTRACT FROM AUTHOR]

Cells can expand their plasma membrane laterally by unfolding membrane undulations and by exocytosis. Here, we describe a third mechanism involving invaginations held shut by the membrane adapter, dynamin. Compartments open when Ca activates the lipid scramblase, TMEM16F, anionic phospholipids escape from the cytoplasmic monolayer in exchange for neutral lipids, and dynamins relax. Deletion of TMEM16F or dynamins blocks expansion, with loss of dynamin expression generating a maximally expanded basal plasma membrane state. Re-expression of dynamin2 or its GTPase-inactivated mutant, but not a lipid binding mutant, regenerates reserve compartments and rescues expansion. Dynamin2-GFP fusion proteins form punctae that rapidly dissipate from these compartments during TMEM16F activation. Newly exposed compartments extend deeply into the cytoplasm, lack numerous organellar markers, and remain closure-competent for many seconds. Without Ca, compartments open slowly when dynamins are sequestered by cytoplasmic dynamin antibodies or when scrambling is mimicked by neutralizing anionic phospholipids and supplementing neutral lipids. Activation of Ca-permeable mechanosensitive channels via cell swelling or channel agonists opens the compartments in parallel with phospholipid scrambling. Thus, dynamins and TMEM16F control large plasma membrane reserves that open in response to lateral membrane stress and Ca influx. Cells locally expand and retract their surface in response to environmental factors such as changes in membrane tension. Here the authors show the membrane adapter, dynamin2, locally constricts surface membrane to form an isolated but contiguous membrane reservoir that can open upon phospholipid scrambling via TMEM16F. [ABSTRACT FROM AUTHOR]

Phospholipid synthesis and fat storage as triglycerides are regulated by lipin phosphatidic acid phosphatases (PAPs), whose enzymatic PAP function requires association with cellular membranes. Using hydrogen deuterium exchange mass spectrometry, we find mouse lipin 1 binds membranes through an N-terminal amphipathic helix, the Ig-like domain and HAD phosphatase catalytic core, and a middle lipin (M-Lip) domain that is conserved in mammalian and mammalian-like lipins. Crystal structures of the M-Lip domain reveal a previously unrecognized protein fold that dimerizes. The isolated M-Lip domain binds membranes both in vitro and in cells through conserved basic and hydrophobic residues. Deletion of the M-Lip domain in lipin 1 reduces PAP activity, membrane association, and oligomerization, alters subcellular localization, diminishes acceleration of adipocyte differentiation, but does not affect transcriptional co-activation. This establishes the M-Lip domain as a dimeric protein fold that binds membranes and is critical for full functionality of mammalian lipins. Lipins need to bind cell membranes before they can function as phosphatidic acid phosphatases. Here, the authors elucidate the structural basis of lipin membrane-association and identify a lipin domain with a novel protein fold that is critical for membrane binding and full functionality of lipins. [ABSTRACT FROM AUTHOR]

Genome-wide association studies have identified SLC16A13 as a novel susceptibility gene for type 2 diabetes. The SLC16A13 gene encodes SLC16A13/MCT13, a member of the solute carrier 16 family of monocarboxylate transporters. Despite its potential importance to diabetes development, the physiological function of SLC16A13 is unknown. Here, we validate Slc16a13 as a lactate transporter expressed at the plasma membrane and report on the effect of Slc16a13 deletion in a mouse model. We show that Slc16a13 increases mitochondrial respiration in the liver, leading to reduced hepatic lipid accumulation and increased hepatic insulin sensitivity in high-fat diet fed Slc16a13 knockout mice. We propose a mechanism for improved hepatic insulin sensitivity in the context of Slc16a13 deficiency in which reduced intrahepatocellular lactate availability drives increased AMPK activation and increased mitochondrial respiration, while reducing hepatic lipid content. Slc16a13 deficiency thereby attenuates hepatic diacylglycerol-PKCε mediated insulin resistance in obese mice. Together, these data suggest that SLC16A13 is a potential target for the treatment of type 2 diabetes and non-alcoholic fatty liver disease. Schumann et al. demonstrate that the loss of a lactate transporter Slc16a13 increases mitochondrial respiration in the liver, which reduces hepatic lipid accumulation while increasing hepatic insulin sensitivity in mice fed a high-fat diet. This study suggests SLC16A13 as a potential target for the treatment of type 2 diabetes and non-alcoholic fatty liver disease. [ABSTRACT FROM AUTHOR]

Small GTPase proteins are ubiquitous and responsible for regulating several processes related to cell growth and differentiation. Mutations that stabilize their active state can lead to uncontrolled cell proliferation and cancer. Although these proteins are well characterized at the cellular scale, the molecular mechanisms governing their functions are still poorly understood. In addition, there is limited information about the regulatory function of the cell membrane which supports their activity. Thus, we have studied the dynamics and conformations of the farnesylated KRAS4b in various membrane model systems, ranging from binary fluid mixtures to heterogeneous raft mimics. Our approach combines long time-scale coarse-grained (CG) simulations and Markov state models to dissect the membrane-supported dynamics of KRAS4b. Our simulations reveal that protein dynamics is mainly modulated by the presence of anionic lipids and to some extent by the nucleotide state (activation) of the protein. In addition, our results suggest that both the farnesyl and the polybasic hypervariable region (HVR) are responsible for its preferential partitioning within the liquid-disordered (Ld) domains in membranes, potentially enhancing the formation of membrane-driven signaling platforms. [ABSTRACT FROM AUTHOR]

Changes in the membrane composition of sub-populations of cells can influence different properties with importance to tumour growth, metastasis and treatment efficacy. In this study, we use correlated fluorescence microscopy and ToF-SIMS with C60+ and (CO2)6k+ ion beams to identify and characterise sub-populations of cells based on successful transfection leading to over-expression of CCTδ, a component of the multi-subunit molecular chaperone named chaperonin-containing tailless complex polypeptide 1 (CCT). CCT has been linked to increased cell growth and proliferation and is known to affect cell morphology but corresponding changes in lipid composition of the membrane have not been measured until now. Multivariate analysis of the surface mass spectra from single cells, focused on the intact lipid ions, indicates an enrichment of phosphatidylethanolamine species in the transfected cells. While the lipid changes in this case are driven by the structural changes in the protein cytoskeleton, the consequence of phosphatidylethanolamine enrichment may have additional implications in cancer such as increased membrane fluidity, increased motility and an ability to adapt to a depletion of unsaturated lipids during cancer cell proliferation. This study demonstrates a successful fluorescence microscopy-guided cell by cell membrane lipid analysis with broad application to biological investigation. Graphical abstract [ABSTRACT FROM AUTHOR]

During pollen maturation, various organelles change their distribution and function during development as male gametophytes. We analyzed the behavior of lipid bodies and vacuoles involved in lipophagy in Arabidopsis pollen using serial section SEM and conventional TEM. At the bicellular pollen stage, lipid bodies in the vegetative cells lined up at the surface of the generative cell. Vacuoles then tightly attached, drew in, and degraded the lipid bodies and eventually occupied the space of the lipid bodies. Degradation of lipid began before transfer of the entire contents of the lipid body. At the tricellular stage, vacuoles instead of lipid bodies surrounded the sperm cells. The degradation of lipid bodies is morphologically considered microautophagy. The atg2-1 Arabidopsis mutant is deficient in one autophagy-related gene (ATG). In this mutant, the assembly of vacuoles around sperm cells was sparser than that in wild-type pollen. The deficiency of ATG2 likely prevents or slows lipid degradation, although it does not prevent contact between organelles. These results demonstrate the involvement of microlipophagy in the pollen development of Arabidopsis. [ABSTRACT FROM AUTHOR]

The membrane of cells and organelles are highly deformable fluid interfaces, and can take on a multitude of shapes. One distinctive and particularly interesting property of biological membranes is their ability to from long and uniform nanotubes. These nanoconduits are surprisingly omnipresent in all domains of life, from archaea, bacteria, to plants and mammals. Some of these tubes have been known for a century, while others were only recently discovered. Their designations are different in different branches of biology, e.g. they are called stromule in plants and tunneling nanotubes in mammals. The mechanical transformation of flat membranes to tubes involves typically a combination of membrane anchoring and external forces, leading to a pulling action that results in very rapid membrane nanotube formation – micrometer long tubes can form in a matter of seconds. Their radius is set by a mechanical balance of tension and bending forces. There also exists a large class of membrane nanotubes that form due to curvature inducing molecules. It seems plausible that nanotube formation and functionality in plants and animals may have been inherited from their bacterial ancestors during endosymbiotic evolution. Here we attempt to connect observations of nanotubes in different branches of biology, and outline their similarities and differences with the aim of providing a perspective on their joint functions and evolutionary origin. [ABSTRACT FROM AUTHOR]

Mannosylerythritol lipids (MEL) are glycolipids mainly produced by pseudo-yeasts. These molecules present remarkable biological activities widely explored in many fields, including medicine, pharmaceuticals, and cosmetics. This review presents the main biological activity of MEL on the HL60, K562, B16, PC12, and skin cells. There is strong evidence that MEL changes the levels of glycosphingolipids of HL-60 lineage, which induce differentiation into granulocytic cells. Regarding B16 cells, MEL can trigger both apoptosis (10 μM) and cell differentiation (5 μM), in which the MEL concentration is related to each metabolic pathway. MEL can also trigger differentiation in PC12 cells due to the increase in the GalCer content. In this specific case, the effects are transient, and the differentiated cells are unstable and tend to apoptosis. MEL-B can particularly maintain skin hydration and moisture due to their self-assembled structures that resemble the tissue cells. Moreover, MEL-B repair aquaporin expression in the HaCaT keratinocytes damaged with UVA irradiation, whereas MEL-C suppresses the expression of COX-2 protein in fibroblasts, indicating that these glycolipids activate the cellular antioxidant mechanism. Recent findings denoted the anti-melanogenic activity of MEL since they suppress tyrosinase enzyme at mRNA levels in B16 and NHMs cells. MEL act effectively on mammalian cells; however, there is no clear pattern of their metabolic effects. Also, gene expression levels seem to be related to two main factors: chemical structure and concentration. However, the specific signaling cascades that are induced by MEL remain inconclusive. Thus, further investigations are vital to understanding these mechanisms clearly. Key points: • The four MEL homologs promote different biological responses in mammalian cells. • MEL modifies the pattern of glycosphingolipids in the plasma membrane of tumor cells. • Activation/deactivation of phosphorylation of serine/threonine kinase proteins. [ABSTRACT FROM AUTHOR]

Despite over 50 years of research, a comprehensive understanding of how intramuscular triglyceride (IMTG) is stored in skeletal muscle and its contribution as a fuel during exercise is lacking. Immunohistochemical techniques provide information on IMTG content and lipid droplet (LD) morphology on a fibre type and subcellular-specific basis, and the lipid dye Oil Red O (ORO) is commonly used to achieve this. BODIPY 493/503 (BODIPY) is an alternative lipid dye with lower background staining and narrower emission spectra. Here we provide the first quantitative comparison of BODIPY and ORO for investigating exercise-induced changes in IMTG content and LD morphology on a fibre type and subcellular-specific basis. Estimates of IMTG content were greater when using BODIPY, which was predominantly due to BODIPY detecting a larger number of LDs, compared to ORO. The subcellular distribution of intramuscular lipid was also dependent on the lipid dye used; ORO detects a greater proportion of IMTG in the periphery (5 μm below cell membrane) of the fibre, whereas IMTG content was higher in the central region using BODIPY. In response to 60 min moderate-intensity cycling exercise, IMTG content was reduced in both the peripheral (− 24%) and central region (− 29%) of type I fibres (P < 0.05) using BODIPY, whereas using ORO, IMTG content was only reduced in the peripheral region of type I fibres (− 31%; P < 0.05). As well as highlighting some methodological considerations herein, our investigation demonstrates that important differences exist between BODIPY and ORO for detecting and quantifying IMTG on a fibre type and subcellular-specific basis. [ABSTRACT FROM AUTHOR]

Vacuolar and plasma membranes were isolated by a detergent-free method from beet roots (Beta vulgaris L.), and were fractionated in a sucrose density gradient of 15–60% by high-speed centrifugation at 200,000×g during 18 h. The membrane material distributed over the sucrose density gradient was analyzed for the presence of lipids characteristic of raft structures in different zones of the gradient. The quantitative and qualitative content of lipids and sterols, and the composition of fatty acids were analyzed. Some membrane structures differing in their biochemical characteristics were revealed to be located in different zones of the sucrose gradient. The results of the analysis allowed us to identify three zones in the sucrose gradient after the vacuolar membrane fractionation and two zones in the plasma membrane where membrane structures, which may be defined as rafts for their lipid composition, were presented. [ABSTRACT FROM AUTHOR]

Lipids form an integral, structural, and functional part of all life forms. They play a significant role in various cellular processes such as membrane fusion, fission, endocytosis, protein trafficking, and protein functions. Interestingly, recent studies have revealed their more impactful and critical involvement in infectious diseases, starting with the manipulation of the host membrane to facilitate pathogenic entry. Thereafter, pathogens recruit specific host lipids for the maintenance of favorable intracellular niche to augment their survival and proliferation. In this review, we showcase the lipid-mediated host pathogen interplay in context of life-threatening viral and bacterial diseases including the recent SARS-CoV-2 infection. We evaluate the emergent lipid-centric approaches adopted by these pathogens, while delineating the alterations in the composition and organization of the cell membrane within the host, as well as the pathogen. Lastly, crucial nexus points in their interaction landscape for therapeutic interventions are identified. Lipids act as critical determinants of bacterial and viral pathogenesis by altering the host cell membrane structure and functions. [ABSTRACT FROM AUTHOR]

Cancer cell possesses numerous adaptations to resist the immune system response and chemotherapy. One of the most significant properties of the neoplastic cells is the altered lipid metabolism, and consequently, the abnormal cell membrane composition. Like in the case of phosphatidylcholine, these changes result in the modulation of certain enzymes and accumulation of energetic material, which could be used for a higher proliferation rate. The changes are so prominent, that some lipids, such as phosphatidylserines, could even be considered as the cancer biomarkers. Additionally, some changes of biophysical properties of cell membranes lead to the higher resistance to chemotherapy, and finally to the disturbances in signalling pathways. Namely, the increased levels of certain lipids, like for instance phosphatidylserine, lead to the attenuation of the immune system response. Also, changes in lipid saturation prevent the cells from demanding conditions of the microenvironment. Particularly interesting is the significance of cell membrane cholesterol content in the modulation of metastasis. This review paper discusses the roles of each lipid type in cancer physiology. The review combined theoretical data with clinical studies to show novel therapeutic options concerning the modulation of cell membranes in oncology. [ABSTRACT FROM AUTHOR]

In this study, a lipid bilayer membrane model was used, in which the bilayer is tethered to a solid substrate with molecular tethers. The V-I characteristics of the lipid bilayers were found to be non-linear which suggests the presence of pores that are voltage-dependent. At high applied voltages, the conductance reached a limiting value, presumably indicating a limit on the maximum pore size. A decrease in the spacing between tethers (increasing tether density) caused a decrease in the membrane's conductance at high applied voltage, which is consistent with the maximum pore size being determined by the spacing between the tethers. The inclusion of 10 M% cholesterol within the membrane lipid caused a decrease in the membrane conductance. However, the inclusion of higher levels of cholesterol increased the membrane conductance. [ABSTRACT FROM AUTHOR]

The malaria parasite interfaces with its host erythrocyte (RBC) using a unique organelle, the parasitophorous vacuole (PV). The mechanism(s) are obscure by which its limiting membrane, the parasitophorous vacuolar membrane (PVM), collaborates with the parasite plasma membrane (PPM) to support the transport of proteins, lipids, nutrients, and metabolites between the cytoplasm of the parasite and the cytoplasm of the RBC. Here, we demonstrate that the PV has structure characterized by micrometer-sized regions of especially close apposition between the PVM and the PPM. To determine if these contact sites are involved in any sort of transport, we localize the PVM nutrient-permeable and protein export channel EXP2, as well as the PPM lipid transporter PfNCR1. We find that EXP2 is excluded from, but PfNCR1 is included within these regions of close apposition. We conclude that the host-parasite interface is structured to segregate those transporters of hydrophilic and hydrophobic substrates. While membrane contact sites between intracellular organelles are abundant, little is known about the contacts between membranes that delimit extracellular junctions within cells, such as intracellular parasites. Here authors demonstrate the segregation of a lipid transporter from a solute transporter in the malarial host-parasite interface. [ABSTRACT FROM AUTHOR]

The lipid bilayer is the basis of the structure and function of the cell membrane. The study of the molecular phenomena that affect biological membranes has a great impact on the understanding of cellular physiology. To understand these phenomena, it has become increasingly necessary to develop simple synthetic models that allow the most basic details of such processes to be reproduced. In this short communication, we took advantage of the properties of two well-established lipid model systems, GUVs and SLBs, with compositions mimicking the cell membrane present in mammals and bacteria, to study the thermotropic phase behavior of lipids as well as the effect of daptomycin, a cyclic lipopeptide used as an antibiotic. The study of mechanical and thermodynamical properties of these model systems could contribute to establish a theoretical framework to develop more efficient strategies for biological control. [ABSTRACT FROM AUTHOR]

In spite of tremendous progress in deciphering the molecular mechanisms involved in intracellular transport in cell culture and in the test tube, many aspects of this process in situ remain unclear. Here, we examined lipid transcytosis in enterocytes in adult rats. Apical clathrin-coated buds and the ER exit sites were not found. After starvation, the Golgi complex was in a non-transporting state and contained many vesicles, but no intercisternal connections and typical the cis-most and the trans-most cisternae. Following the addition of the lipids in the form of chyme, pre-chylomicrons (pre-ChMs) were initially found in the tubules of the smooth SER attached to the basolateral plasmalemma below the belt composed of adhesive junctions (AJ) and always connected with other cisternae. However, the ER exit sites were still absent. Pre-ChMs moved into the cis-most cisterna and were concentrated in cisternal distensions at the trans-side of the Golgi complex. This induced attachment of the cis-most and the trans-most cisternae to the Golgi complex. Post-Golgi carriers fused with the basolateral plasmalemma and delivered ChMs outside. Overloading of enterocytes with lipids resulted in an accumulation of lipid droplets, an increase of the diameter of ChMs, and shift of the Golgi complex to the transporting state with the formation of intercisternal connections, attachment of the cis-most and the trans-most cisternae and disappearance of vesicles. These data are discussed from the functional point of view. In spite of tremendous progress in deciphering the molecular mechanisms involved in intracellular transport in cell culture and in the test tube, many aspects of this process in situ remain unclear. Here, we examined lipid transcytosis in enterocytes in adult rats. Apical clathrin-coated buds and the ER exit sites were not found. After starvation, the Golgi complex was in a non-transporting state and contained many vesicles, but no intercisternal connections and typical the cis-most and the trans-most cisternae. Following the addition of the lipids in the form of chyme, pre-chylomicrons (pre-ChMs) were initially found in the tubules of the smooth SER attached to the basolateral plasmalemma below the belt composed of adhesive junctions (AJ) and always connected with other cisternae. However, the ER exit sites were still absent. Pre-ChMs moved into the cis-most cisterna and were concentrated in cisternal distensions at the trans-side of the Golgi complex. This induced attachment of the cis-most and the trans-most cisternae to the Golgi complex. Post-Golgi carriers fused with the basolateral plasmalemma and delivered ChMs outside. Overloading of enterocytes with lipids resulted in an accumulation of lipid droplets, an increase of the diameter of ChMs, and shift of the Golgi complex to the transporting state with the formation of intercisternal connections, attachment of the cis-most and the trans-most cisternae and disappearance of vesicles. These data are discussed from the functional point of view. [ABSTRACT FROM AUTHOR]

Membrane proteins are the major component of plasma membranes, and they play crucial roles in all organisms. To understand the influence of the presence of KcsA channel on cell membrane electroporation induced by picosecond pulse trains (psPT), in this paper, the electroporation of KcsA membrane protein system and bare lipid bilayer system (POPC) with the applied psPT are simulated using molecular dynamics (MD) method. First, we find that the average pore formation time of the KcsA system is longer than the bare system with the applied psPT. In the KcsA system, water protrusions appear more slowly. Then, the system size effects of psPT in the MD simulations are investigated. When the system size decreases, the average pore formation time of small KcsA membrane protein system is shorter than the bare system with the applied psPT. It is found that the psPT makes the protein fluctuation of small system increase greatly; meanwhile the instability of protein disturbs the water and then affects the water protrusion appearance time. Furthermore, it shows that the protein fluctuation of constant electric field is smaller than that of psPT and no field, and protein fluctuation increases with the psPT repetition frequency increasing. [ABSTRACT FROM AUTHOR]

Tuberculosis has become a cause of worldwide concern; emergence of resistance in various mycobacterial strains has led to an urgent demand for new therapeutic molecules. Antimicrobial peptides have emerged as potential candidates in antimicrobial drug development. We have tested a lead antibacterial peptide LP-23 and its cost-effective mimetic DP-23 for their antimicrobial effects in a non-pathogenic model of Mycobacterium. Their minimum inhibitory concentration (MIC) values against M. smegmatis were calculated by resazurin reduction assay. MIC of both peptidomimetics against M. smegmatis was found to be 6.25 μg/mL. In addition, hemolytic toxicity study of peptidomimetics suggested that the synthesized compounds were selective against bacteria. To better understand stability and mechanism of action of peptidomimetics, serum stability study and SEM analysis were carried out. Peptoid DP-23 was found to be more stable in serum then peptide LP-23, while SEM analysis indicated that these compounds target the cell membrane, impairing the membrane integrity of M. smegmatis. The activity and properties of LP-23 and DP-23 may make them new potential antibacterial agents against tuberculosis. [ABSTRACT FROM AUTHOR]

Cells maintain membrane fluidity by regulating lipid saturation, but the molecular mechanisms of this homeoviscous adaptation remain poorly understood. We have reconstituted the core machinery for regulating lipid saturation in baker's yeast to study its molecular mechanism. By combining molecular dynamics simulations with experiments, we uncover a remarkable sensitivity of the transcriptional regulator Mga2 to the abundance, position, and configuration of double bonds in lipid acyl chains, and provide insights into the molecular rules of membrane adaptation. Our data challenge the prevailing hypothesis that membrane fluidity serves as the measured variable for regulating lipid saturation. Rather, we show that Mga2 senses the molecular lipid-packing density in a defined region of the membrane. Our findings suggest that membrane property sensors have evolved remarkable sensitivities to highly specific aspects of membrane structure and dynamics, thus paving the way toward the development of genetically encoded reporters for such properties in the future. Cells maintain membrane fluidity by regulating lipid saturation, but the molecular mechanisms of this homeoviscous adaptation remain poorly understood. Here authors reconstituted the core machinery for regulating lipid saturation in baker's yeast to directly characterize its response to defined membrane environments and uncover its mode-of-action. [ABSTRACT FROM AUTHOR]

Plasma membranes are heterogeneous and laterally compartmentalized into distinct microdomains. These membrane microdomains consist of special lipids and proteins and are thought to act as signaling platforms. In plants, membrane microdomains have been detected by super-resolution microscopy, and there is evidence that they play roles in several biological processes. Here, we review current knowledge about the lipid and protein components of membrane microdomains. Furthermore, we summarize the dynamics of membrane microdomains in response to different stimuli. We also explore the biological functions associated with membrane microdomains as signal integration hubs. Finally, we outline challenges and questions for further studies. [ABSTRACT FROM AUTHOR]

The effect of different concentrations of NaCl on photosynthetic parameters and structural components of membranes of Hydrilla verticillata has been studied. The effect of NaCl in the concentration range of 5–10 g/L for 24 h leads to an increase in photosynthesis, pigment content, and lipid component rearrangement. A negative effect on membrane permeability and photosynthesis is observed under the action of NaCl at a concentration of 20 g/L. A decrease in the relative content of monogalactosyldiacylglycerol and an increase in digalactosyldiacylglycerol and sulfolipid are found in glycolipids. In the fraction of phospholipids, the relative content of phosphatidyl -choline, -ethanolamine, -inositol decreases and the contribution of phosphatidylglycerol increases with increasing NaCl concentration. Changes in the sterol component of membranes are associated with a decrease in the concentration of stigmasterol and increase in β-sitosterol. Adaptive rearrangements in the composition of pigments and membrane structure indicate a high functional activity of the photosynthetic apparatus of H. verticillata, which allows this species to achieve stability in saline ecotopes. [ABSTRACT FROM AUTHOR]

Local anesthetics (LAs) are known to act on membrane level; however, the molecular mechanism of their activity is still not fully understood. One hypothesis holds that these drugs can incorporate into lipid membrane of nerve cells and in this way change conformation of channel proteins responsible for transport of sodium ions. However, the action of anesthetics is not limited to nerve cells. These drugs also affect other types of cells and organelles, causing severe side effects. In this paper, we applied Langmuir monolayers-as model of cellular membranes-and investigated interactions between selected amide-type local anesthetics (lidocaine prilocaine, mepivacaine and ropivacaine, in the form of hydrochlorides) and lipid components of natural membranes: cholesterol, POPC and cardiolipin (CL) and their mixtures (POPC/cholesterol and POPC/CL/cholesterol), which can serve as simplified models of nerve cell membranes, erythrocytes, and mitochondria. The influence of the drug was monitored by registering the surface pressure (π) as a function of surface area per molecule (A) in a monolayer in the presence of the drug in the subphase. The structure of lipid monolayers on subphases containing and devoid of the studied drugs were visualized with Brewster angle microscopy (BAM). Langmuir monolayer studies complemented with surface visualization technique reveal the expansion and fluidization of lipid monolayers, with the most pronounced effect observed for cardiolipin. In mixed systems, the effect of LAs was found to depend on cholesterol proportion. The observed fluidization of membranes by local anesthetics may negatively affect cells functioning and therefore can explain side effects of these drugs both on the cardiovascular and nervous systems. [ABSTRACT FROM AUTHOR]

Literary data on very long-chain fatty acids (VLCFAs) that are present in polar lipids of the plant cell membranes are discussed. Large amounts of VLCFA are found in polar lipids of some cellular organelles as well as in nonextractable lipids from diverse plant objects, where the influence of surface lipids on the relative content of these FAs is excluded. In some plants, the VLCFA fraction in membrane lipids increases under several kinds of stress. Amounts and diversity of VLCFAs are lower in flowering plants as compared with the representatives of more ancient taxons—gymnosperms, ferns, and marine algae. Presence of VLCFAs in the composition of annular lipids of the cell membranes is assumed. Biosynthesis of VLCFAs, enzymes involved in the process, and encoding genes are discussed. [ABSTRACT FROM AUTHOR]

Plasma membrane forms the barrier between the cytoplasm and the environment. Cells constantly and selectively transport molecules across their plasma membrane without disrupting it. Any disruption in the plasma membrane compromises its selective permeability and is lethal, if not rapidly repaired. There is a growing understanding of the organelles, proteins, lipids, and small molecules that help cells signal and efficiently coordinate plasma membrane repair. This review aims to summarize how these subcellular responses are coordinated and how cellular signals generated due to plasma membrane injury interact with each other to spatially and temporally coordinate repair. With the involvement of calcium and redox signaling in single cell and tissue repair, we will discuss how these and other related signals extend from single cell repair to tissue level repair. These signals link repair processes that are activated immediately after plasma membrane injury with longer term processes regulating repair and regeneration of the damaged tissue. We propose that investigating cell and tissue repair as part of a continuum of wound repair mechanisms would be of value in treating degenerative diseases. [ABSTRACT FROM AUTHOR]

Extracellular vesicles such as exosomes, microvesicles, apoptotic bodies, and large oncosomes have been shown to participate in a wide variety of biological processes and are currently under intense investigation in many different fields of biomedicine. One of the key features of extracellular vesicles is that they have relatively large surface compared to their volume. Some extracellular vesicle surface molecules are shared with those of the plasma membrane of the releasing cell, while other molecules are characteristic for extracellular vesicular surfaces. Besides proteins, lipids, glycans, and nucleic acids are also players of extracellular vesicle surface interactions. Being secreted and present in high number in biological samples, collectively extracellular vesicles represent a uniquely large interactive surface area which can establish contacts both with cells and with molecules in the extracellular microenvironment. Here, we provide a brief overview of known components of the extracellular vesicle surface interactome and highlight some already established roles of the extracellular vesicle surface interactions in different biological processes in health and disease. [ABSTRACT FROM AUTHOR]

Membrane remodelling or the bending and rupture of the lipid bilayer occurs during diverse cellular processes such as cell division, synaptic transmission, vesicular transport, organelle biogenesis and sporulation. These activities are brought about by the localized change in membrane curvature, which in turn causes lipid-packing stress, of a planar lipid bilayer by proteins. For instance, vesicular transport processes are typically characterized by the cooperative recruitment of proteins that induce budding of a planar membrane and catalyse fission of the necks of membrane buds to release vesicles. The analysis of such membrane remodelling reactions has traditionally been restricted to electron microscopy-based approaches or force spectroscopic analysis of membrane tethers pulled from liposome-based model membrane systems. Our recent work has demonstrated the facile creation of tubular model membrane systems of supported membrane tubes (SMrTs), which mimic late-stage intermediates of typical vesicular transport reactions. This review addresses the nature of such an assay system and a fluorescence-intensity-based analysis of changes in tube dimensions that is indicative of the membrane remodelling capacity of proteins. [ABSTRACT FROM AUTHOR]

Since 2003, molecular dynamics simulations of lipid bilayers have provided valuable insights into the mechanisms underlying electropermeabilization (electroporation)-an electric field-induced increase in the permeability of biological membranes. The convention in these studies has been to apply the electric field normal to the plane of the membrane. In a typical electroporation application, however, where the electric field is reasonably uniform and unidirectional, the field is perpendicular to the membrane only at a few locations-for spherical cells only at the poles of the cells along the axis defined by the direction of the electric field. Everywhere else on the cell surface the field is applied at an angle that is oblique to the plane of the membrane. On a microscopic level, the invaginations and protrusions that characterize a living cell membrane also present many angles to the applied electric field. Here we report the results of molecular dynamics simulations of lipid electropore formation when the electric field is not normal to the membrane surface, which show that the tangential component of the field has a small but non-zero effect. [ABSTRACT FROM AUTHOR]

Molecular dynamics simulations of lipid bilayers in aqueous systems reveal how an applied electric field stabilizes the reorganization of the water-membrane interface into water-filled, membrane-spanning, conductive pores with a symmetric, toroidal geometry. The pore formation process and the resulting symmetric structures are consistent with other mathematical approaches such as continuum models formulated to describe the electroporation process. Some experimental data suggest, however, that the shape of lipid electropores in living cell membranes may be asymmetric. We describe here the axially asymmetric pores that form when mechanical constraints are applied to selected phospholipid atoms. Electropore formation proceeds even with severe constraints in place, but pore shape and pore formation time are affected. Since lateral and transverse movement of phospholipids may be restricted in cell membranes by covalent attachments to or non-covalent associations with other components of the membrane or to membrane-proximate intracellular or extracellular biomolecular assemblies, these lipid-constrained molecular models point the way to more realistic representations of cell membranes in electric fields. [ABSTRACT FROM AUTHOR]

Lipid biosensors are robust tools used in both in vitro and in vivo applications of lipid imaging and lipid detection. Lactadherin C2 (LactC2) was described in 2000 as being a potent and specific sensor for phosphatidylserine (PS) (Andersen et al. Biochemistry 39:6200-6206, 2000). PS is an anionic phospholipid enriched in the inner leaflet of the plasma membrane and has paramount roles in apoptosis, cells signaling, and autophagy. The myriad roles PS plays in membrane dynamics make monitoring PS levels and function an important endeavor. LactC2 has functioned as a tantamount PS biosensor namely in the field of cellular imaging. While PS specificity and high affinity of LactC2 for PS containing membranes has been well established, much less is known regarding LactC2 selectivity for subcellular pools of PS or PS within different membrane environments (e.g., in the presence of cholesterol). Thus, there has been a lack of studies that have compared LactC2 PS sensitivity based upon the acyl chain length and saturation or the presence of other host lipids such as cholesterol. Here, we use surface plasmon resonance as a label-free method to quantitatively assess the apparent binding affinity of LactC2 for membranes containing PS with different acyl chains, different fluidity, as well as representative lipid vesicle mimetics of cellular membranes. Results demonstrate that LactC2 is an unbiased sensor for PS, and can sensitively interact with membranes containing PS with different acyl chain saturation and interact with PS species in a cholesterol-independent manner. [ABSTRACT FROM AUTHOR]

Cross-presentation is a critical function of dendritic cells (DCs) required for induction of antitumor immune responses and success of cancer immunotherapy. It is established that tumor-associated DCs are defective in their ability to cross-present antigens. However, the mechanisms driving these defects are still unknown. We find that impaired crosspresentation in DCs is largely associated with defect in trafficking of peptide-MHC class I (pMHC) complexes to the cell surface. DCs in tumor-bearing hosts accumulate lipid bodies (LB) containing electrophilic oxidatively truncated (ox-tr) lipids. These ox-tr-LB, but not LB present in control DCs, covalently bind to chaperone heat shock protein 70. This interaction prevents the translocation of pMHC to cell surface by causing the accumulation of pMHC inside late endosomes/lysosomes. As a result, tumor-associated DCs are no longer able to stimulate adequate CD8 T cells responses. In conclusion, this study demonstrates a mechanism regulating cross-presentation in cancer and suggests potential therapeutic avenues. [ABSTRACT FROM AUTHOR]

Oxysterols are products of cholesterol oxidation. They can be formed endogenously (in both enzymatic and non-enzymatic reactions) as well as exogenously (delivered with food). Recent studies clearly demonstrate cytotoxic properties of these compounds, being mainly due to their incorporation into natural lipid bilayers. This process can influence mechanical and physicochemical properties of biomembrane-mainly by modifying the interactions between its components, which may result in the disruption of proper functioning of cell membrane and could lead to its degradation. Therefore, it can be assumed that oxysterols may affect the initiation of neurodegenerative diseases, including Alzheimer's disease. However, the mode of action of these molecules at the molecular level is not fully known. To get a better understanding of the role of oxysterols in neurodegeneration, it is of great importance to examine mutual interactions between oxysterols and neuronal membrane components. One of the most promising techniques that can be used to analyze such interactions is the Langmuir monolayer technique. In this work, we have prepared an artificial neuronal membrane modeled as multicomponent Langmuir monolayer built up with cholesterol, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and sphingomyelin (SM). To examine whether there are any changes in the membrane properties under oxidative stress, in this paper we have investigated the impact of the representative ring-oxidized oxysterol: 7-ketocholesterol (7-KC). Our results show that replacing cholesterol with 7-KC increases the interaction between molecules in the model membrane. [ABSTRACT FROM AUTHOR]

Nitrite is present as a noxious contaminant in drinking water and causes oxidative damage in various tissues of humans and animals. It is a well-known methemoglobin-forming agent that has been shown to damage blood cells. The protective effect of taurine, a semi-essential sulfur-containing amino acid, was studied on sodium nitrite (NaNO)-induced oxidative damage in human erythrocytes. Erythrocytes were incubated with NaNO, in the presence and absence of taurine, and changes in oxidative stress parameters determined. Pretreatment with taurine significantly ameliorated NaNO-induced oxidative damage to lipids, proteins, and plasma membrane. It also reduced the NaNO-induced increase in methemoglobin levels and ROS production. Taurine improved the antioxidant capacity of cells, restored the alterations in the activities of various metabolic enzymes, and prevented morphological changes in erythrocytes. Thus, taurine can be potentially used as a protective agent against the damaging effects of nitrite. [ABSTRACT FROM AUTHOR]

Integrins are heterodimeric (αβ) cell surface receptors that are potential therapeutic targets for a number of diseases. Despite the existence of structural data for all parts of integrins, the structure of the complete integrin receptor is still not available. We have used available structural data to construct a model of the complete integrin receptor in complex with talin F2-F3 domain. It has been shown that the interactions of integrins with their lipid environment are crucial for their function but details of the integrin/lipid interactions remain elusive. In this study an integrin/talin complex was inserted in biologically relevant bilayers that resemble the cell plasma membrane containing zwitterionic and charged phospholipids, cholesterol and sphingolipids to study the dynamics of the integrin receptor and its effect on bilayer structure and dynamics. The results of this study demonstrate the dynamic nature of the integrin receptor and suggest that the presence of the integrin receptor alters the lipid organization between the two leaflets of the bilayer. In particular, our results suggest elevated density of cholesterol and of phosphatidylserine lipids around the integrin/talin complex and a slowing down of lipids in an annulus of ~30 Å around the protein due to interactions between the lipids and the integrin/talin F2-F3 complex. This may in part regulate the interactions of integrins with other related proteins or integrin clustering thus facilitating signal transduction across cell membranes. [ABSTRACT FROM AUTHOR]

Lipids have numerous important functions in the human body, as they form the cells' plasma membranes and play a key role in many disease states, presumably also in osteoporosis. Here, the fatty acid composition of the outer plasma membranes of cells differentiated into the osteogenic and adipogenic direction is studied with surface-sensitive time-of-flight secondary ion mass spectrometry (ToF-SIMS). For data evaluation, principal component analysis (PCA) is applied. Human (bone-derived) mesenchymal stromal cells (hMSCs) from an osteoporotic donor and a control donor are compared to reveal differences in the fatty acid composition of the membranes. The chemical information is correlated to staining and real-time quantitative polymerase chain reaction (rt-qPCR) results to provide insight into the gene expression of several differentiation markers on the RNA level. Adipogenic differentiation of hMSCs from a non-osteoporotic donor correlates with increased relative intensities of all fatty acids under investigation. After osteogenic differentiation of non-osteoporotic cells, the relative mass signal intensities of unsaturated fatty acids such as oleic and linoleic acids are increased. However, the osteoporotic cells show increased levels of palmitic acid in the plasma membrane after exposure to osteogenic differentiation conditions, which correlates to an immature differentiation state relative to non-osteoporotic osteogenic cells. This immature differentiation state is confirmed by increased early osteogenic differentiation factor Runx2 on RNA level and by less calcium mineralization spots seen in von Kossa staining and ToF-SIMS images. [ABSTRACT FROM AUTHOR]

We present here a new method for calculating the radius of a transmembrane pore in a phospholipid bilayer. To compare size-related properties of pores in bilayers of various compositions, generated and maintained under different physical and chemical conditions, reference metrics are needed. Operational metrics can be associated with some observed behavior. For example, pore size can be defined by the largest object that will pass through the length of the pore. The novelty of the present approach resides in the characterization of electropore geometry via a statistical approach, based on essential dynamics rules. We define the pore size geometrically with an algorithm for determining the pore radius. In particular, we extract the radius from the tri-dimensional surface of a defined pore region. The method is applied to a pore formed in a phospholipid bilayer by application of an external electric field. Although the details described here are specific for lipid pores in molecular dynamics simulations, the method can be generalized for any kind of pores for which appropriate structural information is available. [ABSTRACT FROM AUTHOR]

The effects of local anesthetics (LAs), including aminoamides and aminoesters, on the characteristics of single gramicidin A (GA) channels in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers were studied. Aminoamides, namely lidocaine (LDC), prilocaine (PLC), mepivacaine (MPV), and bupivacaine (BPV), reduced the conductance of GA channels. Aminoesters influenced the current fluctuations induced by GA differently; procaine (PC) did not affect the fluctuations, whereas tetracaine (TTC) distinctly reduced the conductance of single GA channels. Using electrophysiological technique, we estimated the changes in the membrane boundary potential at the adsorption of LAs; LDC, PLC, MPV, BPV, and TTC substantially increased, while PC did not affect it. To elucidate which component of the membrane boundary potential, the surface or dipole potential, is responsible for the observed effects of LAs, we employed a fluorescence assay. We found that TTC led to a significant increase in the membrane dipole potential, whereas the adsorption of LDC, PLC, MPV, BPV, and PC did not produce any changes in the membrane dipole potential. We concluded that aminoamides affected the surface potential of lipid bilayers. Together, these data suggest that the effects of LAs on the conductance of single GA channels are caused by their influence on membrane electrostatic potentials; the regulation of GA pores by aminoamides is associated with the surface potential of membranes, whereas TTC modulation of channel properties is predominantly due to changes in dipole potential of lipid bilayers. These data might provide some significant implications for voltage-gated ion channels of cell membranes. [ABSTRACT FROM AUTHOR]

Copper is an essential metal for several physiological and metabolic processes, but a narrow range regulate its effect in phytoplankton cells. It can affect the production of biomolecules and be toxic at concentrations slightly above those required, e.g. decreasing photosynthesis and increasing respiration. The aims of this study were to analyse the changes in growth and chlorophyll a synthesis, and in biochemistry (total carbohydrates, proteins, lipids and fatty acids) of the freshwater microalga Selenastrum gracile after exposure to copper. Exponentially growing cells were exposed to 5 concentrations of free copper ions (Cu) ranging from 0.7 (control) to 13 × 10 M for up to 120 h. Free Cu ion concentrations were calculated through the chemical equilibrium model MINEQL. We observed that copper was responsible for a decrease in cell density and an increase in total protein and lipid production, but no effect on total carbohydrates was detected. The increase in phospholipids and sterols and a decrease in saturated fatty acids under copper exposure suggest a change in conformation of the cell membrane, by decreasing its fluidity. We suggest this serves the cell as a system to avoid the internalization of metal, thereby acting as a detoxifying mechanism. [ABSTRACT FROM AUTHOR]

The (re)organization of membrane components is of special importance to prepare mammalian sperm to fertilization. Establishing suitable methods to examine physico-chemical membrane parameters is of high interest. We characterized the behavior of fluorescent (NBD) analogs of sphingomyelin (SM), phosphatidylserine (PS), and cholesterol (Ch) in the acrosomal and postacrosomal macrodomain of boar sperm. Due to their specific transverse membrane distribution, a leaflet-specific investigation of membrane properties is possible. The behavior of lipid analogs in boar sperm was investigated by fluorescence lifetime imaging microscopy (FLIM), fluorescence recovery after photobleaching (FRAP), and fluorescence correlation spectroscopy (FCS). The results were compared with regard to the different temporal and spatial resolution of the methods. For the first time, fluorescence lifetimes of lipid analogs were determined in sperm cell membrane and found to be in a range characteristic for the liquid-disordered phase in artificial lipid membranes. FLIM analyses further indicate a more fluid microenvironment of NBD-Ch and NBD-PS in the postacrosomal compared to the acrosomal region. The concept of a more fluid cytoplasmic leaflet is supported by lower fluorescence lifetime and higher average D values (FCS) for NBD-PS in both head compartments. Whereas FLIM analyses did not indicate coexisting distinct liquid-ordered and -disordered domains in any of the head regions, comparisons between FRAP and FCS measurements suggest the incorporation of NBD-SM as well as NBD-PS in postacrosomal subpopulations with different diffusion velocity. The analog-specific results indicate that the lipid analogs used are suitable to report on the various physicochemical properties of different microenvironments. [ABSTRACT FROM AUTHOR]

The oleaginous yeast Rhodosporidium toruloides has great biotechnological potential. It accumulates a high amount of lipids which can be used for biofuels and also produces carotenoids which are valuable in the food and pharmaceutical industry. However, the location of these two hydrophobic products in the cell membrane prohibits its efficient harvesting and separation. Here, the transporter Pdr10 was engineered into R. toruloides and cultured in two-phase media containing oil. This enabled the production and in situ export of carotenoids into the oil and concurrent separation from intracellular lipids in the cells. When Pdr10 strain was cultured in the two-phase media, carotenoids and fatty acids yield increased from 1.9 to 2.9 μg/mg and 0.07 to 0.09 mg/mg, respectively. A total of 1.8 μg/mg carotenoids was exported by Pdr10 strain, as compared to 0.3 μg/mg in the wild type. In the Pdr10 strain, the composition of carotenoids and fatty acid it produced also changed. Torulene became the major carotene produced instead of torularhodin. Also, the unsaturated fatty acid C18:2 became the dominant fatty acid produced instead of the saturated C16:0, which was similar to the grape seed oil used in the two-phase media. This indicated that oil was being consumed by the cells, which was supported by the increased intracellular glycerol levels detected by gas chromatography-mass spectrometry (GC-MS). Our approach represents an easy and greener extraction method which could serve to increase the yield and facilitate separation of carotenoids and fatty acids. [ABSTRACT FROM AUTHOR]