Membrane lipid saturation activates endoplasmic reticulum unfolded protein response transducers through their transmembrane domains

Endoplasmic reticulum (ER) stress sensors use a related luminal domain to monitor the unfolded protein load and convey the signal to downstream effectors, signaling an unfolded protein response (UPR) that maintains compartment-specific protein folding homeostasis. Surprisingly, perturbation of cellular lipid composition also activates the UPR, with important consequences in obesity and diabetes. However, it is unclear if direct sensing of the lipid perturbation contributes to UPR activation. We found that mutant mammalian ER stress sensors, IRE1α and PERK, lacking their luminal unfolded protein stress-sensing domain, nonetheless retained responsiveness to increased lipid saturation. Lipid saturation-mediated activation in cells required an ER-spanning transmembrane domain and was positively regulated in vitro by acyl-chain saturation in reconstituted liposomes. These observations suggest that direct sensing of the lipid composition of the ER membrane contributes to the UPR.     

INAUGURAL ARTICLE by a Recently Elected Academy Member:Stiffened lipid platforms at molecular force foci

How mechanical forces are sensed remains largely mysterious. The forces that gate prokaryotic and several eukaryotic channels were found to come from the lipid membrane. Our survey of animal cells found that membrane force foci all have cholesterol-gathering proteins and are reinforced with cholesterol. This result is evident in overt force sensors at the tips of stereocilia for vertebrate hearing and the touch receptor of Caenorhabditis elegans and mammalian neurons. For less specialized cells, cadherins sustain the force between neighboring cells and integrins between cells and matrix. These tension bearers also pass through and bind to a cholesterol-enriched platform before anchoring to cytoskeleton through other proteins. Cholesterol, in alliance with sphingomyelin and specialized proteins, enforces a more ordered structure in the bilayer. Such a stiffened platform can suppress mechanical noise, redirect, rescale, and confine force. We speculate that such platforms may be dynamic. The applied force may allow disordered-phase lipids to enter the platform-staging channel opening in the thinner mobile neighborhood. The platform may also contain specialized protein/lipid subdomains enclosing mechanosensitive channels to open with localized tension. Such a dynamic stage can mechanically operate structurally disparate channels or enzymes without having to tie them directly to cadherin, integrin, or other protein tethers.     

Lipid packing drives the segregation of transmembrane helices into disordered lipid domains in model membranes

Cell membranes are comprised of multicomponent lipid and protein mixtures that exhibit a complex partitioning behavior. Regions of structural and compositional heterogeneity play a major role in the sorting and self-assembly of proteins, and their clustering into higher-order oligomers. Here, we use computer simulations and optical microscopy to study the sorting of transmembrane helices into the liquid-disordered domains of phase-separated model membranes, irrespective of peptide-lipid hydrophobic mismatch. Free energy calculations show that the enthalpic contribution due to the packing of the lipids drives the lateral sorting of the helices. Hydrophobic mismatch regulates the clustering into either small dynamic or large static aggregates. These results reveal important molecular driving forces for the lateral organization and self-assembly of transmembrane helices in heterogeneous model membranes, with implications for the formation of functional protein complexes in real cells.     

Metabolic and immune-sensitive contacts between lipid droplets and endoplasmic reticulum reconstituted in vitro

Coordinated cell function requires a variety of subcellular organelles to exchange proteins and lipids across physical contacts that are also referred to as membrane contact sites. Such organelle-to-organelle contacts also evoke interest because they can appear in response to metabolic changes, immune activation, and possibly other stimuli. The microscopic size and complex, crowded geometry of these contacts, however, makes them difficult to visualize, manipulate, and understand inside cells. To address this shortcoming, we deposited endoplasmic reticulum (ER)-enriched microsomes purified from rat liver or from cultured cells on a coverslip in the form of a proteinaceous planar membrane. We visualized real-time lipid and protein exchange across contacts that form between this ER-mimicking membrane and lipid droplets (LDs) purified from the liver of rat. The high-throughput imaging possible in this geometry reveals that in vitro LD–ER contacts increase dramatically when the metabolic state is changed by feeding the animal and also when the immune system is activated. Contact formation in both cases requires Rab18 GTPase and phosphatidic acid, thus revealing common molecular targets operative in two very different biological pathways. An optical trap is used to demonstrate physical tethering of individual LDs to the ER-mimicking membrane and to estimate the strength of this tether. These methodologies can potentially be adapted to understand and target abnormal contact formation between different cellular organelles in the context of neurological and metabolic disorders or pathogen infection.

Lipid droplets (LDs) are triglyceride-rich cellular organelles that act as an energy depot, a reservoir for membrane biosynthesis, and a location where proteins are sequestered in response to specific stimuli (1–3). These functions require LDs to be dynamic, wherein the LDs exchange proteins and lipids with the endoplasmic reticulum (ER), mitochondria, peroxisomes, endosomes, and other organelles via physical contacts (4, 5). Abnormal LD–organelle interactions are implicated in neurological disease, metabolic disorder, fatty liver, and pathogen infection (5–7). Multispectral imaging showed that LD–ER contacts are the most promiscuous, with the ER contacting a majority of LDs (∼85%) irrespective of their location inside the cell (8). Accordingly, many researchers have used cell culture models to extract a wealth of information on the factors that sustain ER–LD contacts (1, 9–11). Most of these assays aim to understand the biogenesis of LDs, which is now well accepted to proceed through a triglyceride-rich, lens-like structure that expands in the ER lumen, eventually forming mature LDs on the cytosolic side of the ER lumen. Proteins such as seipin, sorting nexins (Snx14), and the NRZ-SNARE complex are required to sustain ER–LD contacts during these steps of LD-biogenesis (12–14).As an alternate line of investigation to these previous studies, we have attempted to address the physiologically relevant mechanisms that catabolize LDs inside mammalian liver to produce very-low-density lipoproteins (VLDL). We proposed that LD catabolism and ER–LD interactions are enhanced several-fold in the liver after feeding the animal (15, 16). Feeding stimulates insulin release, whereafter insulin activates the ARF1-GTPase and phospholipase-D to generate phosphatidic acid (PA) on the LD membrane. PA directly binds to kinesin-1 and recruits this motor to LDs. Kinesin induces rapid transport of LDs along microtubules to peripheral regions of the ER in hepatocytes, where ER–LD contacts likely form for catabolizing LDs and for delivering the resultant triglyceride to the ER lumen for assembling VLDL (15, 16). This entire pathway is toned down when insulin levels drop after fasting, causing ER–LD contacts to diminish and the liver to sequester LDs in a fasted state to protect other organs from lipotoxicity. Indeed, disrupting this homeostatic pathway caused massive accumulation of triglycerides in cardiac and skeletal tissue with potentially harmful consequences (15). In another exciting development, LDs were found to be a hub for immune activation in the liver, wherein the LDs could harbor and deliver antibacterial proteins for killing bacteria (3). It appeared to us that immune activation also promotes intimate ER–LD contacts in the liver (see supplementary figure 6 in ref. 3). We therefore wondered if LDs in liver cells first acquire antibacterial proteins from the ER via ER–LD contacts before these proteins can be used by the LDs to kill bacteria. Notably, this possibility was not explored by Bosch et al. (3). Two very different kinds of stimuli, namely feeding and immune activation, may therefore induce ER–LD contacts in the liver. Because the liver is a key player in lipid metabolism and systemic immune response, we saw an opportunity to understand how ER–LD contacts are modulated in the animal to serve physiologically important functions outside the well-understood and widely studied pathway of LD biogenesis.To address these questions, we developed an assay to deposit ER-enriched microsomes purified from rat liver, and also from cultured cells, on a coverslip in the form of an ER-mimicking planar membrane hereafter called the microsomal supported lipid bilayer (mSLB). We demonstrate that LDs purified from rat liver adhere to the mSLB via physical contacts, across which lipids and proteins are exchanged. Our results suggest formation of LD–mSLB fusion intermediates that mediate this exchange. In line with the above-discussed findings (3, 15), LD–mSLB contacts are dramatically enhanced after feeding or immune activation, thus bringing out their native-like nature and physiological relevance. Taking advantage of this in vitro assay, we show that the Rab18 GTPase and PA are common molecular players that engineer LD–mSLB contacts after feeding as well as after immune activation. This assay opens up possibilities to understand, in a controlled, in vitro environment, the molecular pathways that sustain LD–ER contacts in processes other than LD biogenesis. If adapted to other organelles, it may provide a tool to interrogate metabolic disorder, fatty liver, pathogen infection, and other human diseases arising out of abnormal organelle-to-organelle contact formation of diverse kinds.     

Altered lipid raft composition and defective cell death signal transduction in glycosylphosphatidylinositol anchor-deficient PIG-A mutant cells

Paroxysmal nocturnal haemoglobinuria (PNH) is a clonal disorder of haematopoietic stem cells caused by somatic PIGA mutations, resulting in a deficiency in glycosylphosphatidylinositol-anchored proteins (GPI-AP). Because GPI-AP associate with lipid rafts (LR), lack of GPI-AP on PNH cells may result in alterations in LR-dependent signalling. Conversely, PNH cells are a suitable model for investigating LR biology. LR from paired, wild-type GPI(+), and mutant GPI(−) cell lines (K562 and TF1) were isolated and analysed; GPI(−) LR contained important anti-apoptotic proteins, not found in LR from GPI(+) cells. When methyl-β-cyclodextrin (MβCD) was utilized to probe for functional differences between normal and GPI(−) LR, increased levels of phospho-p38 mitogen-activated protein kinase (MAPK), and phospho-p65 nuclear factor NF-κB were found in control and GPI(−) cells respectively. Subsequent experiments addressing the inhibition of phosphoinositide-3-kinase (PI3K) suggest that the PI3K/AKT pathway may be responsible for the resistance of K562 GPI(−)cells to negative effects of MβCD. In addition, transduction of tumour necrosis factor-α (TNF-α) signals in a LR-dependent fashion increased induction of p38 MAPK in GPI(+) and increased pro-survival NF-κB levels in K562 GPI(−) cells. Therefore, we suggest that the altered LR-dependent signalling in PNH-like cells may induce different responses to pro-inflammatory cytokines from those observed in cells with intact GPI-AP.     

The Elucidation of the Molecular Mechanism of the Extrusion Process

Extrusion is a popular method for producing homogenous population of unilamellar liposomes. The technique relies on forcing a lipid suspension through cylindrical pores in a polycarbonate membrane. The quantification of the extrusion and/or recalibration processes make possible the acquisition of experimental data, which can be correlated with the mechanical properties of the lipid bilayer. In this work, the force needed for the extrusion process was correlated with the mechanical properties of a lipid bilayer derived from other experiments. Measurements were performed using a home-made dedicated device capable of maintaining a stable volumetric flux of a liposome suspension through well-defined pores and to continuously measure the extrusion force. Based on the obtained results, the correlation between the lipid bilayer bending rigidity and extrusion force was derived. Specifically, it was found that the bending rigidity of liposomes formed from well-defined lipid mixtures agrees with data obtained by others using flicker-noise spectroscopy or micromanipulation. The other issue addressed in the presented studies was the identification of molecular mechanisms leading to the formation of unilamellar vesicles in the extrusion process. Finally, it was demonstrated that during the extrusion, lipids are not exchanged between vesicles, i.e., vesicles can divide but no membrane fusion or lipid exchange between bilayers was detected.     

Permeability of pure lipid bilayers to melatonin

Abstract: Melatonin, the chief hormone of the pineal gland, has been reported to interact with a variety of different cells. This ubiquitously acting hormone has been found to interact with protein receptors both at the cell membrane and in the nucleus. Moreover, melatonin was recently shown to be a very potent hydroxyl radical scavenger. The present work focuses on the interaction of melatonin with pure lipid bilayers. It is shown that melatonin can cross multilamellar lipid vesicles, which are used here as model systems for the lipid phase of biological membranes. Thus, the data prove that melatonin can easily pass through the cell membrane and bath every part of the cell, as previously suggested in the literature. Melatonin lipid association constant was calculated based on the change of the hormone fluorescence intensity due to its penetration into the hydrophobic lipid phase. Though melatonin was recently shown to be highly soluble in aqueous media, its lipid association constant is rather high, indicating that the biological action of the hormone is likely to be at the membrane level, either via its interaction with membrane receptors, and/or as a lipoperoxidation radical scavenger.     

Organization, dynamics, and segregation of Ras nanoclusters in membrane domains

Recent experiments have shown that membrane-bound Ras proteins form transient, nanoscale signaling platforms that play a crucial role in high-fidelity signal transmission. However, a detailed characterization of these dynamic proteolipid substructures by high-resolution experimental techniques remains elusive. Here we use extensive semiatomic simulations to reveal the molecular basis for the formation and domain-specific distribution of Ras nanoclusters. As model systems, we chose the triply lipidated membrane targeting motif of H-ras (tH) and a large bilayer made up of di16∶0-PC (DPPC), di18∶2-PC (DLiPC), and cholesterol. We found that 4–10 tH molecules assemble into clusters that undergo molecular exchange in the sub-μs to μs time scale, depending on the simulation temperature and hence the stability of lipid domains. Driven by the opposite preference of tH palmitoyls and farnesyl for ordered and disordered membrane domains, clustered tH molecules segregate to the boundary of lipid domains. Additionally, a systematic analysis of depalmitoylated and defarnesylated tH variants allowed us to decipher the role of individual lipid modifications in domain-specific nanocluster localization and thereby explain why homologous Ras isoforms form nonoverlapping nanoclusters. Moreover, the localization of tH nanoclusters at domain boundaries resulted in a significantly lower line tension and increased membrane curvature. Taken together, these results provide a unique mechanistic insight into how protein assembly promoted by lipid-modification modulates bilayer shape to generate functional signaling platforms.     

Protein area occupancy at the center of the red blood cell membrane

In the Fluid Mosaic Model for biological membrane structure, proposed by Singer and Nicolson in 1972, the lipid bilayer is represented as a neutral two-dimensional solvent in which the proteins of the membrane are dispersed and distributed randomly. The model portrays the membrane as dominated by a membrane lipid bilayer, directly exposed to the aqueous environment, and only occasionally interrupted by transmembrane proteins. This view is reproduced in virtually every textbook in biochemistry and cell biology, yet some critical features have yet to be closely examined, including the key parameter of the relative occupancy of protein and lipid at the center of a natural membrane. Here we show that the area occupied by protein and lipid at the center of the human red blood cell (RBC) plasma membrane is at least approximately 23% protein and less than approximately 77% lipid. This measurement is in close agreement with previous estimates for the RBC plasma membrane and the recently published measurements for the synaptic vesicle. Given that transmembrane proteins are surrounded by phospholipids that are perturbed by their presence, the occupancy by protein of more than approximately 20% of the RBC plasma membrane and the synaptic vesicle plasma membrane implies that natural membrane bilayers may be more rigid and less fluid than has been thought for the past several decades, and that studies of pure lipid bilayers do not fully reveal the properties of lipids in membranes. Thus, it appears to be the case that membranes may be more mosaic than fluid, with little unperturbed phospholipid bilayer.     

Comparative lipidomics and proteomics analysis of platelet lipid rafts using different detergents

Lipid rafts play a pivotal role in physiological functions of platelets. Their isolation using nonionic mild detergents is considered as the gold standard method, but there is no consensual detergent for lipid raft studies. We aimed to investigate which detergent is the most suitable for lipid raft isolation from platelet membrane, based on lipidomics and proteomics analysis.

Platelets were obtained from healthy donors. Twelve sucrose fractions were extracted by three different detergents, namely Brij 35, Lubrol WX, and Triton X100, at 0.05% and 1%. After lipidomics analysis and determination of fractions enriched in cholesterol (Ch) and sphingomyelin (SM), proteomics analysis was performed.

Lipid rafts were mainly observed in 1–4 fractions, and non-rafts were distributed on 5–12 fractions. Considering the concentration of Ch and SM, Lubrol WX 1% and Triton X100 1% were more suitable detergents as they were able to isolate lipid raft fractions that were more enriched than non-raft fractions. By proteomics analysis, overall, 822 proteins were identified in platelet membrane. Lipid raft fractions isolated with Lubrol WX 0.05% and Triton X100 1% contained mainly plasma membrane proteins. However, only Lubrol WX 0.05 and 1% and Triton X100 1% were able to extract non-denaturing proteins with more than 10 transmembrane domains.

Our results suggest that Triton X100 1% is the most suitable detergent for global lipid and protein studies on platelet plasma membrane. However, the detergent should be adapted if investigation of an association between specific proteins and lipid rafts is planned.     

Accumulation of styrene oligomers alters lipid membrane phase order and miscibility

Growth of plastic waste in the natural environment, and in particular in the oceans, has raised the accumulation of polystyrene and other polymeric species in eukyarotic cells to the level of a credible and systemic threat. Oligomers, the smallest products of polymer degradation or incomplete polymerization reactions, are the first species to leach out of macroscopic or nanoscopic plastic materials. However, the fundamental mechanisms of interaction between oligomers and polymers with the different cell components are yet to be elucidated. Simulations performed on lipid bilayers showed changes in membrane mechanical properties induced by polystyrene, but experimental results performed on cell membranes or on cell membrane models are still missing. We focus here on understanding how embedded styrene oligomers affect the phase behavior of model membranes using a combination of scattering, fluorescence, and calorimetric techniques. Our results show that styrene oligomers disrupt the phase behavior of lipid membranes, modifying the thermodynamics of the transition through a spatial modulation of lipid composition.

The increasing amount of plastic present in sea waters has become a major issue in recent years, with increasing concerns regarding the potential hazardous effects it may have on living systems (1, 2). Annual production of plastic has reached almost 300 million tons/y, of which 5 to 13 million tons are estimated to reach the oceans by different means (3). While initially the main concern for plastic contamination was the presence of microplastic, produced by polymeric degradation, recently the focus has shifted to nanoplastic (4, 5). Objects of this scale can easily enter the food chain via digestion and there is increasing evidence of plastic micro- and nanoobjects found in marine life forms (6–9). Moreover, nanometer-size polymer particles are also produced industrially for specific research and technological applications, such as imaging, sensing, and preparation of nanocomposites (10), providing a second route, besides degradation, for plastic-derived nanoparticles’ entry into sea waters. Despite a current lack of evidence on the presence of nanoobjects (4), studies have indeed shown that plastic nanoparticles can accumulate in the tissues of living organisms and disrupt their metabolism (11–13) and that size plays an important role in determining their accumulation (12, 13). However, a physicochemical characterization of the interaction between plastic nanoobjects and living organisms is still lacking, especially regarding the mechanisms of potential toxicity. As the first barrier encountered by any foreign object entering an organism, the cell membrane is the primary candidate of investigation in assessing possible toxicity of plastic nanofragments. In particular, the membrane lipid lateral organization plays a crucial role in many cellular signaling processes due to the presence in the membrane of small transient domains called “lipid rafts,” and even minute changes in membrane lipid organization can result in a potential alteration of these processes and pose a threat to cellular viability.Polystyrene is one of the most commonly used plastics in the world, contributing to a significant fraction of marine plastic wastes in the form of styrene oligomers (SO) (14), and it has been shown in several studies to directly affect lipid bilayer physical properties when accumulated within the membrane. Accumulation of styrene oligomers and polymers in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers indeed showed, in numerical simulations, to change the membrane mechanical properties and lateral lipid organization (15) and a stabilization of cholesterol-induced domains (16). However, experimental studies on the effects of polystyrene and oligomers in the cell membrane and model membranes are still lacking. It was previously reported that incorporation of styrene monomers into lipid membranes significantly changes the fluidity of the membrane (17), and a similar effect was indeed shown for charged polystyrene sulfonated chains in surfactant bilayers (18), but from an experimental point of view many aspects of the interaction are still elusive. In this work we investigate the effects of styrene oligomers (Mn = 500 Da) on the phase transition of lipid bilayers composed of unsaturated lipids or a mixture of unsaturated/saturated lipids, to obtain a more comprehensive picture of the role of membrane complexity on the effects of styrene oligomer accumulation. Indeed, small styrene chains display the largest mobility; they are the most likely to be transferred to the lipid membranes upon contact with a degrading piece of plastic. Also, when one considers the important role played by the gel–liquid transition point of the saturated lipid in the formation of domains in ternary lipid mixtures relevant for biomembranes, it becomes clear that the most fundamental question to be asked in this context is that of the influence of the oligomers on the gel–liquid transition of the saturated lipid. We investigated the changes in transition using differential scanning calorimetry (DSC), small-angle neutron scattering (SANS), and Laurdan fluorescence spectra to extract information on the structure and the thermodynamics. Moreover, we directly visualized the changes on the membranes at the micrometric scale using epifluorescence microscopy.     

Horizontal Bilayer for Electrical and Optical Recordings

Artificial bilayer containing reconstituted ion channels, transporters and pumps serve as a well-defined model system for electrophysiological investigations of membrane protein structure–function relationship. Appropriately constructed microchips containing horizontally oriented bilayers with easy solution access to both sides provide, in addition, the possibility to investigate these model bilayer membranes and the membrane proteins therein with high resolution fluorescence techniques up to the single-molecule level. Here, we describe a bilayer microchip system in which long-term stable horizontal free-standing and hydrogel-supported bilayers can be formed and demonstrate its prospects particularly for single-molecule fluorescence spectroscopy and high resolution fluorescence microscopy in probing the physicochemical properties like phase behavior of the bilayer-forming lipids, as well as in functional studies of membrane proteins.     

Critical fluctuations in domain-forming lipid mixtures

Critical fluctuations are investigated in lipid membranes near miscibility critical points in bilayers composed of dioleoylphosphatidylcholine, chain perdeuterated dipalmitoylphosphatidylcholine, and cholesterol. Phase boundaries are mapped over the temperature range from 10 degrees C to 60 degrees C by deuterium NMR. Tie-lines and three-phase triangles are evaluated across two-phase and three-phase regions, respectively. In addition, a line of miscibility critical points is identified. NMR resonances are broadened in the vicinity of critical points, and broadening is attributed to increased transverse relaxation rates arising from modulation of chain order with correlation times on a microsecond time scale. We conclude that spectral broadening arises from composition fluctuations in the membrane plane with dimensions of <50 nm and speculate that similar fluctuations are commonly found in cholesterol-containing membranes.     

Cis and trans interactions between atlastin molecules during membrane fusion

Atlastin (ATL), a membrane-anchored GTPase that mediates homotypic fusion of endoplasmic reticulum (ER) membranes, is required for formation of the tubular network of the peripheral ER. How exactly ATL mediates membrane fusion is only poorly understood. Here we show that fusion is preceded by the transient tethering of ATL-containing vesicles caused by the dimerization of ATL molecules in opposing membranes. Tethering requires GTP hydrolysis, not just GTP binding, because the two ATL molecules are pulled together most strongly in the transition state of GTP hydrolysis. Most tethering events are futile, so that multiple rounds of GTP hydrolysis are required for successful fusion. Supported lipid bilayer experiments show that ATL molecules sitting on the same (cis) membrane can also undergo nucleotide-dependent dimerization. These results suggest that GTP hydrolysis is required to dissociate cis dimers, generating a pool of ATL monomers that can dimerize with molecules on a different (trans) membrane. In addition, tethering and fusion require the cooperation of multiple ATL molecules in each membrane. We propose a comprehensive model for ATL-mediated fusion that takes into account futile tethering and competition between cis and trans interactions.The endoplasmic reticulum (ER) consists of tubules and sheets connected into a characteristic network by membrane fusion (1–3). In contrast to heterotypic fusion between viral and cellular membranes or between intracellular transport vesicles and target membranes, the fusing ER membranes are identical (i.e., homotypic fusion). The mechanism of heterotypic fusion has been studied extensively, leading to the concept that a conformational change of a viral fusion protein or the assembly of SNARE proteins pulls two opposing membranes together so that they can fuse (4–7). Recently, some insight has been obtained into homotypic ER fusion as well. This process is mediated by the atlastins (ATLs) in metazoans and by Sey1p/ROOT HAIR DEFECTIVE3 (RHD3)-related proteins in yeast and plants (8, 9).The ATLs and Sey1p/RHD3 are membrane-bound GTPases that belong to the dynamin family (reviewed in refs. 10, 11). They contain cytosolic N-terminal GTPase (G) and helical bundle domains, followed by two closely spaced transmembrane (TM) segments and a cytosolic C-terminal tail (12–14). A role for the GTPases in ER fusion is suggested by the observation that their depletion or mutational inactivation leads to long, nonbranched tubules or fragmented ER (8, 9). Nonbranched ER tubules are also observed on expression of dominant-negative ATL mutants (8, 12). In addition, the fusion of ER vesicles in Xenopus laevis egg extracts is prevented by ATL antibodies or a cytosolic fragment of ATL (8, 15), and the fusion of the ER in mating yeast cells is delayed on SEY1 deletion (16). Most convincingly, proteoliposomes containing purified ATL, Sey1p, or RHD3 undergo GTP-dependent fusion in vitro (9, 13, 16, 17). Defects in ATL-mediated ER fusion can cause hereditary spastic paraplegia, a neurodegenerative disease characterized by the shortening of the axons in corticospinal motor neurons, leading to progressive spasticity and weakness of the lower limbs (13, 18).How exactly the ATLs and Sey1p/RHD3 fuse membranes is not well understood. Crystal structures of ATL and biochemical experiments have not resulted in a coherent model. Initial structures of the N-terminal cytosolic domain of human atlastin-1 (ATL1) revealed two different conformations (13, 14). In both conformations, a dimer is formed between the two G domains, but in one conformation (crystal form 2; SI Appendix, Fig. S1), the subsequent three-helix bundle (3HB) domains point in opposite directions, whereas in the other (crystal form 1; SI Appendix, Fig. S1), they are parallel to one another. These structures were interpreted as “prefusion” and “postfusion” states, in which the two ATL molecules would either tether two opposing membranes or sit in the same membrane after fusion (13).Because the prefusion structure was crystallized in the presence of GDP and inorganic phosphate (P_i) and the postfusion structure was crystallized in the presence of GDP, it was proposed that GTP hydrolysis would induce this conformational change, resulting in the apposed membranes being pulled together for fusion (13). Both structures contained only bound GDP, however, raising doubts that the conformational change is induced by GTP hydrolysis. In addition, biochemical experiments suggested that ATL dimers form in the GTP-bound state and dissociate into monomers in GDP, given that dimerization was much more pronounced in the presence of nonhydrolyzable GTP analogs compared with GDP (13, 14); the high protein concentration during crystallization may have allowed dimerization in GDP.A third crystal structure (crystal form 3; SI Appendix, Fig. S1) led to further confusion. It was obtained in the presence of GMPPNP or GDP plus AlF₄^– (GDP/AlF₄^–) and represents a state before P_i release (19). This structure resembles the postfusion state rather than the expected prefusion state; the two 3HBs are again parallel, and in fact are even closer to one another. Finally, a structure of GDP-bound Drosophila ATL showed a prefusion-like conformation (20).The different structural and biochemical results have given rise to conflicting models for ATL-mediated membrane fusion. It has been proposed that GTP hydrolysis occurs after fusion, although this is difficult to reconcile with in vitro experiments showing that GTP hydrolysis is required for membrane fusion (21). Other models postulate that GTP binding is sufficient for the tethering of the two membranes through dimerization of ATL molecules sitting in opposing membranes (trans dimer) (9, 13, 22), or that GTP binding and hydrolysis occur in a monomer before trans dimerization (19). It also has been proposed that ATL molecules would interact only in cis, i.e., in the same membrane, rather than tethering opposing membranes before their fusion; the ATL molecules would induce high membrane curvature in both lipid bilayers, which would facilitate their fusion (23).Here we propose a model that provides a coherent picture of ATL function. We show that GTP hydrolysis is required for membrane tethering, although dimer formation per se can occur with nonhydrolyzable GTP analogs. GTP hydrolysis, and not just GTP binding, is required, because two ATL molecules are pulled together most strongly in the transition state during GTP hydrolysis, and because dimers forming on the same (cis) membrane need to be dissociated to allow ATL molecules to dimerize with molecules on a different (trans) membrane. Tethering and fusion require the cooperation of multiple ATL molecules in each membrane, and multiple cycles of GTP hydrolysis occur before a successful fusion event. These results have implications for ER fusion in vivo.     

Energetics of stalk intermediates in membrane fusion are controlled by lipid composition

We have used X-ray diffraction on the rhombohedral phospholipid phase to reconstruct stalk structures in different pure lipids and lipid mixtures with unprecedented resolution, enabling a quantitative analysis of geometry, as well as curvature and hydration energies. Electron density isosurfaces are used to study shape and curvature properties of the bent lipid monolayers. We observe that the stalk structure is highly universal in different lipid systems. The associated curvatures change in a subtle, but systematic fashion upon changes in lipid composition. In addition, we have studied the hydration interaction prior to the transition from the lamellar to the stalk phase. The results indicate that facilitating dehydration is the key to promote stalk formation, which becomes favorable at an approximately constant interbilayer separation of 9.0 ± 0.5 Å for the investigated lipid compositions.     

Protective effects of melatonin in reducing oxidative stress and in preserving the fluidity of biological membranes: a review

Free radicals generated within subcellular compartments damage macromolecules which lead to severe structural changes and functional alterations of cellular organelles. A manifestation of free radical injury to biological membranes is the process of lipid peroxidation, an autooxidative chain reaction in which polyunsaturated fatty acids in the membrane are the substrate. There is considerable evidence that damage to polyunsaturated fatty acids tends to reduce membrane fluidity. However, adequate levels of fluidity are essential for the proper functioning of biological membranes. Thus, there is considerable interest in antioxidant molecules which are able to stabilize membranes because of their protective effects against lipid peroxidation. Melatonin is an indoleamine that modulates a wide variety of endocrine, neural and immune functions. Over the last two decades, intensive research has proven this molecule, as well as its metabolites, to possess substantial antioxidant activity. In addition to their ability to scavenge several reactive oxygen and nitrogen species, melatonin increases the activity of the glutathione redox enzymes, that is, glutathione peroxidase and reductase, as well as other antioxidant enzymes. These beneficial effects of melatonin are more significant because of its small molecular size and its amphipathic behaviour, which facilitates ease of melatonin penetration into every subcellular compartment. In the present work, we review the current information related to the beneficial effects of melatonin in maintaining the fluidity of biological membranes against free radical attack, and further, we discuss its implications for ageing and disease.     

Interactive protein network of FXIII-A1 in lipid rafts of activated and non-activated platelets

Lipid-rafts are defined as membrane microdomains enriched in cholesterol and glycosphingolipids within platelet plasma membrane. Lipid raft-mediated clot retraction requires factor XIII and other interacting proteins. The aim of this study was to investigate the proteins that interact with factor XIII in raft and non-raft domains of activated and non-activated platelet plasma membrane. By lipidomics analysis, we identified cholesterol- and sphingomyelin-enriched areas as lipid rafts. Platelets were activated by thrombin. Proteomics analysis provided an overview of the pathways in which proteins of rafts and non-rafts participated in the interaction network of FXIII-A1, a catalytic subunit of FXIII. “Platelet activation” was the principal pathway among KEGG pathways for proteins of rafts, both before and after activation. Network analysis showed four types of interactions (activation, binding, reaction, and catalysis) in raft and non-raft domains in interactive network of FXIII-A1. FXIII-A1 interactions with other proteins in raft domains and their role in homeostasis highlight the specialization of the raft domain in clot retraction via the Factor XIII protein network.     

Fast molecular tracking maps nanoscale dynamics of plasma membrane lipids

We describe an optical method capable of tracking a single fluorescent molecule with a flexible choice of high spatial accuracy (∼10–20 nm standard deviation or ∼20–40 nm full-width-at-half-maximum) and temporal resolution (< 1 ms). The fluorescence signal during individual passages of fluorescent molecules through a spot of excitation light allows the sequential localization and thus spatio-temporal tracking of the molecule if its fluorescence is collected on at least three separate point detectors arranged in close proximity. We show two-dimensional trajectories of individual, small organic dye labeled lipids diffusing in the plasma membrane of living cells and directly observe transient events of trapping on < 20 nm spatial scales. The trapping is cholesterol-assisted and much more pronounced for a sphingo- than for a phosphoglycero-lipid, with average trapping times of ∼15 ms and < 4 ms, respectively. The results support previous STED nanoscopy measurements and suggest that, at least for nontreated cells, the transient interaction of a single lipid is confined to macromolecular dimensions. Our experimental approach demonstrates that fast molecular movements can be tracked with minimal invasion, which can reveal new important details of cellular nano-organization.     

Outer membrane permeability: Antimicrobials and diverse nutrients bypass porins in Pseudomonas aeruginosa

Gram-negative bacterial pathogens have an outer membrane that restricts entry of molecules into the cell. Water-filled protein channels in the outer membrane, so-called porins, facilitate nutrient uptake and are thought to enable antibiotic entry. Here, we determined the role of porins in a major pathogen, Pseudomonas aeruginosa, by constructing a strain lacking all 40 identifiable porins and 15 strains carrying only a single unique type of porin and characterizing these strains with NMR metabolomics and antimicrobial susceptibility assays. In contrast to common assumptions, all porins were dispensable for Pseudomonas growth in rich medium and consumption of diverse hydrophilic nutrients. However, preferred nutrients with two or more carboxylate groups such as succinate and citrate permeated poorly in the absence of porins. Porins provided efficient translocation pathways for these nutrients with broad and overlapping substrate selectivity while efficiently excluding all tested antibiotics except carbapenems, which partially entered through OprD. Porin-independent permeation of antibiotics through the outer-membrane lipid bilayer was hampered by carboxylate groups, consistent with our nutrient data. Together, these results challenge common assumptions about the role of porins by demonstrating porin-independent permeation of the outer-membrane lipid bilayer as a major pathway for nutrient and drug entry into the bacterial cell.

Antimicrobial resistance is a major worldwide threat to human health. The World Health Organization has classified Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii as the most concerning pathogens (“critical priority”) (1). All three pathogens are Gram-negative bacteria with the characteristic inner and outer membranes. The outer membrane is a stringent permeability barrier that restricts the entry of most molecules and therefore presents a major challenge for the development of urgently needed novel antibiotics (2–5).The outer membrane consists of an asymmetric lipid bilayer with lipopolysaccharide (LPS) in the outer leaflet and phospholipids in the inner leaflet and various outer-membrane proteins that are embedded in, or attached to, the lipid bilayer. LPS contains negatively charged phosphate and carboxylate groups that are cross-linked by divalent Mg²⁺ and Ca²⁺ cations, resulting in stable clusters of LPS molecules that reduce the permeation of small molecules by 10- to 100-fold compared to phospholipid bilayers (6). Some outer membrane proteins form water-filled channels (so-called porins) that facilitate translocation of molecules through the outer membrane (4, 5). Enterobacteriaceae have general “unspecific” porins that permit the entry of molecules with a size of up to 600 Da. By contrast, P. aeruginosa and A. baumannii have a large set of “specific” porins that permit the entry of only few molecules with sizes below 200 Da. In addition, all three pathogens have porins with mainly structural roles in stabilizing the link between outer membrane and the underlying peptidoglycan layer (OmpA and OprF). It has been proposed that a small fraction of these structural porin molecules form large unspecific pores that permit entry of larger molecules at low rates (7), but this model remains controversial.Antimicrobials and nutrients can penetrate the outer membrane by two different pathways, through the lipid bilayer or through porins. Hydrophobic molecules might predominantly use the lipid pathway, while hydrophilic molecules might prefer porins. However, the quantitative relevance of each pathway for outer-membrane permeability remains unknown (3, 8, 9). Even slow permeation pathways that mediate concentration-equilibration times in the order of minutes (instead of seconds) can yield relevant intracellular drug concentrations in bacteria with generation times of more than 20 min, unless drug-efflux pumps and/or hydrolases diminish drug levels (2).Translocation pathways and their selectivity for specific physicochemical properties of molecules are crucial for the rational improvement of drug entry into Gram-negative bacteria. The important contribution of large cation-selective porins such as OmpF and OmpC for outer-membrane translocation into Enterobacteriaceae enabled the establishment of rules for medicinal chemistry to improve whole-cell activities of antimicrobials against these bacteria (10–12). These porins have been extensively studied, and in particular OmpF has a major impact on susceptibility to various β-lactam antibiotics (13). However, an Escherichia coli ΔompC ΔompF double mutant retains substantial susceptibility to diverse other antibiotics (9), suggesting alternative translocation pathways.For P. aeruginosa, physicochemical parameters favoring translocation have been more difficult to identify (10, 14, 15). Both P. aeruginosa and A. baumannii have lower outer-membrane permeability than Enterobacteriaceae for hydrophilic molecules because they lack unspecific porins (16), making antimicrobial development particularly difficult for these critical pathogens. Specific porins might facilitate antibiotic entry into P. aeruginosa (17), but clear evidence for standard assay conditions is only available for penetration of carbapenems through OprD (18). Functional studies of individual porins in P. aeruginosa are hampered by the large diversity of specific porins that are thought to each enable uptake of a few nutrients (19). Phenotypes of inactivating one particular porin might be masked by the numerous remaining other porins. To circumvent these issues, individual porins have been purified and reconstituted in artificial membranes, or expressed in E. coli, to determine their substrate specificity. However, the results might not reflect porin functions in their native context because their channel properties differ depending on the lipid environment (20, 21).In this study, we overcame these difficulties using extensive mutagenesis. In contrast to previous assumptions, we show that wild-type P. aeruginosa PA14 and a PA14 Δ40 mutant that lacks all identifiable 40 porin genes have indistinguishable susceptibility to diverse antibiotics. Moreover, the Δ40 strain grew normally on rich media, and nutrient consumption assays revealed substantial porin-independent uptake of diverse hydrophilic nutrients. Bringing back individual porins accelerated uptake of some neutral/zwitterionic molecules and was essential for efficient consumption of negatively charged carboxylate-containing compounds. Instead of narrow substrate specificity, porins actually had broad overlapping substrate selectivity. These results demonstrate an unexpected but efficient porin-independent translocation pathway through the outer-membrane lipid bilayer for diverse hydrophilic compounds and all antipseudomonal antibiotics. A detailed understanding of this pathway will facilitate the development of novel antibiotics.