Complex Membrane Structures Research Articles

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Phospholipid synthesis fueled by lipid droplets drives the structural development of poliovirus replication organelles

Rapid development of complex membranous replication structures is a hallmark of picornavirus infections. However, neither the mechanisms underlying such dramatic reorganization of the cellular membrane architecture, nor the specific role of these membranes in the viral life cycle are sufficiently understood. Here we demonstrate that the cellular enzyme CCTα, responsible for the rate-limiting step in phosphatidylcholine synthesis, translocates from the nuclei to the cytoplasm upon infection and associates with the replication membranes, resulting in the rerouting of lipid synthesis from predominantly neutral lipids to phospholipids. The bulk supply of long chain fatty acids necessary to support the activated phospholipid synthesis in infected cells is provided by the hydrolysis of neutral lipids stored in lipid droplets. Such activation of phospholipid synthesis drives the massive membrane remodeling in infected cells. We also show that complex membranous scaffold of replication organelles is not essential for viral RNA replication but is required for protection of virus propagation from the cellular anti-viral response, especially during multi-cycle replication conditions. Inhibition of infection-specific phospholipid synthesis provides a new paradigm for controlling infection not by suppressing viral replication but by making it more visible to the immune system.

Iron-Sulfur Cluster Biosynthesis in Algae with Complex Plastids.

Plastids surrounded by four membranes harbor a special compartment between the outer and inner plastid membrane pair, the so-called periplastidal compartment (PPC). This cellular structure is usually presumed to be the reduced cytoplasm of a eukaryotic phototrophic endosymbiont, which was integrated into a host cell and streamlined into a plastid with a complex membrane structure. Up to date, no mitochondrion or mitochondrion-related organelle has been identified in the PPC of any representative. However, two prominent groups, the cryptophytes and the chlorarachniophytes, still harbor a reduced cell nucleus of symbiont origin, the nucleomorph, in their PPCs. Generally, many cytoplasmic and nucleus-located eukaryotic proteins need an iron–sulfur cofactor for their functionality. Beside some exceptions, their synthesis is depending on a so-called iron–sulfur complex (ISC) assembly machinery located in the mitochondrion. This machinery provides the cytoplasm with a still unknown sulfur component, which is then converted into iron–sulfur clusters via a cytosolic iron–sulfur protein assembly (CIA) machinery. Here, we investigated if a CIA machinery is present in mitochondrion-lacking PPCs. By using bioinformatic screens and in vivo-localizations of candidate proteins, we show that the presence of a PPC-specific CIA machinery correlates with the presence of a nucleomorph. Phylogenetic analyses of PPC- and host specific CIA components additionally indicate a complex evolution of the CIA machineries in organisms having plastids surrounded by four membranes.

How to minimize dye-induced perturbations while studying biomembrane structure and dynamics: PEG linkers as a rational alternative

Organic dye-tagged lipid analogs are essential for many fluorescence-based investigations of complex membrane structures, especially when using advanced microscopy approaches. However, lipid analogs may interfere with membrane structure and dynamics, and it is not obvious that the properties of lipid analogs would match those of non-labeled host lipids. In this work, we bridged atomistic simulations with super-resolution imaging experiments and biomimetic membranes to assess the performance of commonly used sphingomyelin-based lipid analogs. The objective was to compare, on equal footing, the relative strengths and weaknesses of acyl chain labeling, headgroup labeling, and labeling based on poly-ethyl-glycol (PEG) linkers in determining biomembrane properties. We observed that the most appropriate strategy to minimize dye-induced membrane perturbations and to allow consideration of Brownian-like diffusion in liquid-ordered membrane environments is to decouple the dye from a membrane by a PEG linker attached to a lipid headgroup. Yet, while the use of PEG linkers may sound a rational and even an obvious approach to explore membrane dynamics, the results also suggest that the dyes exploiting PEG linkers interfere with molecular interactions and their dynamics. Overall, the results highlight the great care needed when using fluorescent lipid analogs, in particular accurate controls.

Immunohistochemical Analysis of Myelin Structures.

Immunochemistry (immunocytochemistry for cells and immunohistochemistry for tissues) is a method used to label specific antigens, based on highly specific antibody-epitope interactions. The resulting labeling can be visualized and imaged through microscopy adapted to the type of detection system used (fluorophore, peroxidase, etc.). In the nervous system, myelin is a complex membrane structure, generated by myelinating glial cells, which ensheath axons and facilitate electrical conduction. Myelin alteration has been shown to occur in various neurological diseases, in which it is associated with functional deficits. Here, we focus on myelin detection by immunofluorescence using immunochemistry protocols based on antibodies directed against major myelin proteins.

NHE- and diffusion-dependent proton fluxes across the tubular system membranes of fast-twitch muscle fibers of the rat.

The complex membrane structure of the tubular system (t-system) in skeletal muscle fibers is open to the extracellular environment, which prevents measurements of H+ movement across its interface with the cytoplasm by conventional methods. Consequently, little is known about the t-system's role in the regulation of cytoplasmic pH, which is different from extracellular pH. Here we describe a novel approach to measure H+-flux measurements across the t-system of fast-twitch fibers under different conditions. The approach involves loading the t-system of intact rat fast-twitch fibers with a strong pH buffer (20 mM HEPES) and pH-sensitive fluorescent probe (10 mM HPTS) before the t-system is sealed off. The pH changes in the t-system are then tracked by confocal microscopy after rapid changes in cytoplasmic ionic conditions. T-system sealing is achieved by removing the sarcolemma by microdissection (mechanical skinning), which causes the tubules to pinch off and seal tight. After this procedure, the t-system repolarizes to physiological levels and can be electrically stimulated when placed in K+-based solutions of cytosolic-like ionic composition. Using this approach, we show that the t-system of fast-twitch skeletal fibers displays amiloride-sensitive Na+/H+ exchange (NHE), which decreases markedly at alkaline cytosolic pH and has properties similar to that in mammalian cardiac myocytes. We observed mean values for NHE density and proton permeability coefficient of 339 pmol/m2 of t-system membrane and 158 µm/s, respectively. We conclude that the cytosolic pH in intact resting muscle can be quantitatively explained with respect to extracellular pH by assuming that these values apply to the t-system membrane and the sarcolemma.

Alkyl substituent effect on photosensitized inactivation of Escherichia coli by pyridinium-bonded P-porphyrins

Activity of singlet oxygen sensitizers for photoinactivation of bacteria and photodynamic therapy of tumor cells has been evaluated using nonpathogenic model cells, such as Escherichia coli, Saccharomyces cerevisiae, and HeLa cells. Among them, E. coli, gram-negative bacterium, has complex membrane structures in the cell wall, resulting in an impermeable barrier to antimicrobial agents. Therefore, few singlet oxygen sensitizers have photoinactivation activities toward E. coli at low concentrations. Recently polycationic porphyrins have received much attention as a new type of singlet oxygen sensitizers because they have strong binding affinities for DNA and proteins. Here, we prepared 13 types of di- and tricationic P- and Sb-porphyrin sensitizers substituted with the N-alkylpyridinium (APy) group at the axial ligand or the meso position to examine their bactericidal activity toward E. coli under visible-light irradiation. Photobactericidal activities were evaluated using half-life (T1/2 in min) of E. coli and minimum effective concentrations of the porphyrin sensitizers. Di-cationic P-porphyrins containing the Apy group at meso position exhibited bactericidal activity under dark conditions. Tricationic porphyrins showed a higher bactericidal activity than monocationic porphyrins. It was found that the bactericidal activity depended on the alkyl chain length of APy. Tricationic porphyrin with N-heptylpyridinium in two axial ligands was the most reactive for the photoinactivation of E. coli.

Mitochondria-Associated Membranes and ER Stress.

The endoplasmic reticulum (ER) is a crucial organelle for coordinating cellular Ca2+ signaling and protein synthesis and folding. Moreover, the dynamic and complex membranous structures constituting the ER allow the formation of contact sites with other organelles and structures, including among others the mitochondria and the plasma membrane (PM). The contact sites that the ER form with mitochondria is a hot topic in research, and the nature of the so-called mitochondria-associated membranes (MAMs) is continuously evolving. The MAMs consist of a proteinaceous tether that physically connects the ER with mitochondria. The MAMs harness the main functions of both organelles to form a specialized subcompartment at the interface of the ER and mitochondria. Under homeostatic conditions, MAMs are crucial for the efficient transfer of Ca2+ from the ER to mitochondria, and for proper mitochondria bioenergetics and lipid synthesis. MAMs are also believed to be the master regulators of mitochondrial shape and motility, and to form a crucial site for autophagosome assembly. Not surprisingly, MAMs have been shown to be a hot spot for the transfer of stress signals from the ER to mitochondria, most notably under the conditions of loss of ER proteostasis, by engaging the unfolded protein response (UPR). In this chapter after an introduction on ER biology and ER stress, we will review the emerging and key signaling roles of the MAMs, which have a root in cellular processes and signaling cascades coordinated by the ER.

A dynamic view of the endoplasmic reticulum

Cellular Structure The endoplasmic reticulum (ER) is a complex membranous structure that extends from the nuclear envelope to the cell periphery. It has important roles in many cellular processes, and numerous proteins are involved in maintaining its structure. Nixon-Abell et al. used

The Impact of Formula fortified with Milk Fat Globule Membrane on the Neonatal Intestine

BackgroundBreastmilk provides the ideal nutrient source during development. However, in some cases it is not available in adequate amounts, if at all (especially following premature delivery), to the developing infant resulting in a need for formula feeding. As achieving appropriate growth during this early time is essential for proper development and protection from noxious stimuli, there is a need for the composition of formula to mimic that of breastmilk as closely as possible. Fat in breastmilk, a major source of energy for the newborn, contains a unique and very complex triple membrane structure referred to as the Milk Fat Globule Membrane (MFGM) resulting in strikingly large fat molecules (average size of 5μm in human milk). Surprisingly, the fat component in formula is derived from vegetable sources rather than MFGM, which differ greatly in size and composition. Interestingly, although studies in several adult models of infection and inflammation have attributed protective effects to MFGM supplementation, its role in the newborn intestine and its development has not thoroughly been explored.ObjectiveTo examine the effects of Milk Fat Globule Membrane (MFGM) on postnatal development of the small intestine and colon.MethodsRat pups were artificially reared using the pup‐in‐a‐cup model to examine the effects of MFGM (1.2 or 6 mg/mL) supplementation on early intestinal development. Rat pups underwent gastronomy at postnatal day (PD) 5 and were supplemented with formula containing soy (control formula) or MFGM+soy until PD15, at which point they were euthanized along with mother reared littermates, resulting in three groups. Small (jeujunal, ileal) and large intestinal (distal colon) samples were collected for histological assessment and immunohistochemistry (IHC).ResultsAlthough no overt differences were noted in pup weight or overall length of the small or large intestine at PD15 between the three groups, histological assessment revealed significant changes in the epithelial architecture along the length of the intestine (jejunum, ileum and colon). MFGM supplementation resulted in significant dose dependent changes at the intestinal surface, resulting in crypt depths similar to those recorded in mother reared (MR) pups at PD15. IHC analysis of intestinal sections revealed similar positive staining between MFGM and MR pups for major cell types within the epithelium (goblet cells and enterocytes), rates of cell proliferation, and tight junction proteins.ConclusionMFGM normalizes many aspects of intestinal development similar to that observed in mother reared littermates. Its supplementation in formula may be beneficial in neonates who do not have access to breast milk, particularly pre‐term infants to accelerate intestinal development.Support or Funding InformationGanive Bhinder is funded by a Canadian Institutes of Health Research Phd Studentship and University of British Columbia 4 year fellowship.Milk Fat Globule Membrane for this study was provided by Mead Johnson Nutrition.

The Hepatitis C Virus-Induced Membranous Web and Associated Nuclear Transport Machinery Limit Access of Pattern Recognition Receptors to Viral Replication Sites.

Hepatitis C virus (HCV) is a positive-strand RNA virus of the Flaviviridae family and a major cause of liver disease worldwide. HCV replicates in the cytoplasm, and the synthesis of viral proteins induces extensive rearrangements of host cell membranes producing structures, collectively termed the membranous web (MW). The MW contains the sites of viral replication and assembly, and we have identified distinct membrane fractions derived from HCV-infected cells that contain replication and assembly complexes enriched for viral RNA and infectious virus, respectively. The complex membrane structure of the MW is thought to protect the viral genome limiting its interactions with cytoplasmic pattern recognition receptors (PRRs) and thereby preventing activation of cellular innate immune responses. Here we show that PRRs, including RIG-I and MDA5, and ribosomes are excluded from viral replication and assembly centers within the MW. Furthermore, we present evidence that components of the nuclear transport machinery regulate access of proteins to MW compartments. We show that the restricted assess of RIG-I to the MW can be overcome by the addition of a nuclear localization signal sequence, and that expression of a NLS-RIG-I construct leads to increased immune activation and the inhibition of viral replication.

Capacitive Membrane Activity in Isolated Cardiac Myocytes

Cardiac myocytes are cells with complex membrane structure of unknown dynamics. A good physical measure of the state of the sarcolemma is membrane capacitance. High-resolution capacitance measurements were instrumental in disclosing secretory and endocytotic mechanisms in simpler cells. We used a new method of membrane capacitance recording based on deconvolution of current responses to square wave voltage stimuli (MAT-MECAS: https://sourceforge.net/projects/mat-mecas/) to study membrane dynamics of isolated cardiac myocytes; specifically, the spontaneous and stimulated membrane capacitance changes. We observed spontaneous discrete capacitance events of 1 − 20 fF that may correspond to fusion and fission of single vesicles of 180 − 800 nm in diameter. In addition, large spontaneous stepwise increases of membrane capacitance of 80 − 250 fF were also observed. These large capacitance increases were occasionally preceded by a transient opening of a fusion pore with a conductance of several hundreds of pS that lasted for a few hundreds of milliseconds. Such large stepwise changes of membrane capacitance might be related to the opening of the mouth of transversal tubules. Discrete capacitance increases were also observed after stimulation of myocytes by a train of calcium currents. In these experiments, the discrete capacitance increases of 50 − 160 fF occurred 3 to 20 seconds after stimulation. This might reflect calcium induced fusion of intracellular membrane bodies with the surface membrane. In conditions of permanently raised intracellular concentration of calcium ions, membrane capacitance showed increased activity of step-like capacitance events. These observations support the role of intracellular calcium in regulation of membrane dynamics in cardiac myocytes. Altogether, these findings provide the first evidence of vesicle fusion events related to physiologically relevant membrane activity in cardiac myocytes.Supported by: APVV-0721-10, VEGA 2/0147/14.

Steel Arch Stabilized by a Textile Membrane

A slender steel arch supporting textile membranes in a complex membrane structure with respect to in-plane and out-of plane stability is investigated. In the last decades the textile membranes are widely used to cover both common and exclusive structures due to progress of new membrane materials with eminent properties. In comparison with use of a traditional roof decking these structures are much more economical and attractive. However, the complex analysis and computational design methods are demanding and still not codified. The real physical model of a membrane structure supported by two inner steel arches and covering a concert stage was constructed and tested in laboratory of CTU in Prague. The membrane used was the Précontraint 702S Ferrari1 prestressed textile membrane with PVC coating. The arches were hot-formed in a workshop from Grade S355J0 steel. The paper presents experimental results for: i) isolated inner arch, ii) complex structure of the membrane with supporting arches, both under symmetrical and asymmetrical loading. Furthermore the numerical nonlinear FEM model created in SOFiSTiK software package and its validation for the above structural configurations and loading is presented. Subsequent parametrical studies cover various levels of the membrane prestressing to show its significance for nonlinear behaviour and stability of the inner arch. Finally some recommendations concerning numerical modelling are presented.

Layer-by-Layer Assembly of Supported Lipid Bilayer Poly-L-Lysine Multilayers.

Multilayer lipid membranes perform many important functions in biology, such as electrical isolation (myelination of axons), increased surface area for biocatalytic purposes (thylakoid grana and mitochondrial cristae), and sequential processing (golgi cisternae). Here we develop a simple layer-by-layer methodology to form lipid multilayers via vesicle rupture onto existing supported lipid bilayers (SLBs) using poly l-lysine (PLL) as an electrostatic polymer linker. The assembly process was monitored at the macroscale by quartz crystal microbalance with dissipation (QCM-D) and the nanoscale by atomic force microscopy (AFM) for up to six lipid bilayers. By varying buffer pH and PLL chain length, we show that longer chains (≥300 kDa) at pH 9.0 form thicker polymer supported multilayers, while at low pH and shorter length PLL, we create close packed layers (average lipid bilayers separations of 2.8 and 0.8 nm, respectively). Fluorescence recovery after photobleaching (FRAP) and AFM were used to show that the diffusion of lipid and three different membrane proteins in the multilayered membranes has little dependence on lipid stack number or separation between membranes. These approaches provide a straightforward route to creating the complex membrane structures that are found throughout nature, allowing possible applications in areas such as energy production and biosensing while developing our understanding of the biological processes at play.

Efficient and CO2-tolerant oxygen transport membranes prepared from high-valence B-site substituted cobalt-free SrFeO3−δ

The simultaneous high oxygen permeability and high chemical stability of perovskites for use as oxygen transport membranes are of critical importance for applications in oxyfuel processes and as membrane reactors for coupling reactions. Here cobalt-free and CO2-tolerant SrFe0.8M0.2O3−δ (M=Zr, Mo, and W) were exploited as materials for oxygen transport membranes, which exhibited stable cubic phase structures in both air and CO2-containing atmospheres. At 850°C and under the air/helium gradient across the membranes, oxygen permeation fluxes of 0.387, 0.216 and 0.201mLcm−2min−1 [STP] were reached for SrFe0.8Zr0.2O3−δ, SrFe0.8Mo0.2O3−δ and SrFe0.8W0.2O3−δ (membrane thickness: 1mm), respectively. More importantly, relatively stable oxygen permeation fluxes of 0.262, 0.145 and 0.164mLcm−2min−1 were still reached for above three membranes correspondingly and maintained for almost 600min when the sweep gas was switched to 10% CO2-containing helium when compared to the un-doped SrFeO3−δ membrane. Our findings suggest that the stable phase structure and improved CO2 resistance can be effectively achieved by facile doping of high-valence and redox-inactive transition metal ions into the SrFeO3−δ parent oxide. This process provides an efficient way for the development of CO2-tolerant oxygen transport membranes without applying other complex membrane structures (e.g., dual-phase membranes) or noble metals.

Modelling of deformable structures in the general framework of the discrete element method

The discrete element method (DEM) is particularly suited for the numerical simulation of granular soils interacting with various types of deformable structures and inclusions. Numerous studies have been dedicated to the accurate modelling of particle shape, yet there is a lack of a general framework for modelling deformable structures of arbitrary shapes such as textiles, grids, membranes, tubes and containers. This paper presents a novel generalised approach to this problem in three dimensions. Minkowski sums of polytopes and spheres are used to describe the topology via three simple primitives: spheres, cylinders and thick facets. The cylinders and facets are deformable and can be connected to form grids and other membrane-like structures. A conventional elastic–plastic contact model is adapted to reflect all possible interactions. The implementation is verified by considering spheres moving along a complex membrane structure and a buckling tube. In addition, simulated pull-out tests on a grid and a membrane and bouncing tests of a hollow deformable sphere are reported. The versatility and capabilities of the approach and the potential applications to soil–inclusion problems are demonstrated.

Lack of GCN5 remarkably enhances the resistance against prolonged endoplasmic reticulum stress-induced apoptosis through up-regulation of Bcl-2 gene expression

The endoplasmic reticulum (ER), a complex membrane structure, has important roles in all eukaryotic cells. Catastrophe of its functions would lead to ER stress that causes various diseases such as cancer, neurodegenerative diseases, diabetes and so on. Prolonged ER stress could trigger apoptosis via activation of various signal transduction pathways. To investigate physiological roles of histone acetyltransferase GCN5 in regulation of ER stress, we analyzed responses of homozygous GCN5-deficient DT40 mutants, ΔGCN5, against ER stress. GCN5-deficiency in DT40 caused drastic resistance against apoptosis induced by pharmacological ER stress agents (thapsigargin and tunicamycin). Pharmaceutical analysis using specific Bcl-2 inhibitors showed that the drastic resistance against prolonged ER stress-induced apoptosis is, in part, due to up-regulation of Bcl-2 gene expression in ΔGCN5. These data revealed that GCN5 is involved in regulation of prolonged ER stress-induced apoptosis through controlling Bcl-2 gene expression.

Proteomic analysis of proteins surrounding occludin and claudin-4 reveals their proximity to signaling and trafficking networks.

Tight junctions are complex membrane structures that regulate paracellular movement of material across epithelia and play a role in cell polarity, signaling and cytoskeletal organization. In order to expand knowledge of the tight junction proteome, we used biotin ligase (BioID) fused to occludin and claudin-4 to biotinylate their proximal proteins in cultured MDCK II epithelial cells. We then purified the biotinylated proteins on streptavidin resin and identified them by mass spectrometry. Proteins were ranked by relative abundance of recovery by mass spectrometry, placed in functional categories, and compared not only among the N- and C- termini of occludin and the N-terminus of claudin-4, but also with our published inventory of proteins proximal to the adherens junction protein E-cadherin and the tight junction protein ZO-1. When proteomic results were analyzed, the relative distribution among functional categories was similar between occludin and claudin-4 proximal proteins. Apart from already known tight junction- proteins, occludin and claudin-4 proximal proteins were enriched in signaling and trafficking proteins, especially endocytic trafficking proteins. However there were significant differences in the specific proteins comprising the functional categories near each of the tagging proteins, revealing spatial compartmentalization within the junction complex. Taken together, these results expand the inventory of known and unknown proteins at the tight junction to inform future studies of the organization and physiology of this complex structure.

Quantitative Proteomic Analysis of Host-virus Interactions Reveals a Role for Golgi Brefeldin A Resistance Factor 1 (GBF1) in Dengue Infection

Dengue virus is considered to be the most important mosquito-borne virus worldwide and poses formidable economic and health care burdens on many tropical and subtropical countries. Dengue infection induces drastic rearrangement of host endoplasmic reticulum membranes into complex membranous structures housing replication complexes; the contribution(s) of host proteins and pathways to this process is poorly understood but is likely to be mediated by protein-protein interactions. We have developed an approach for obtaining high confidence protein-protein interaction data by employing affinity tags and quantitative proteomics, in the context of viral infection, followed by robust statistical analysis. Using this approach, we identified high confidence interactors of NS5, the viral polymerase, and NS3, the helicase/protease. Quantitative proteomics allowed us to exclude a large number of presumably nonspecific interactors from our data sets and imparted a high level of confidence to our resulting data sets. We identified 53 host proteins reproducibly associated with NS5 and 41 with NS3, with 13 of these candidates present in both data sets. The host factors identified have diverse functions, including retrograde Golgi-to-endoplasmic reticulum transport, biosynthesis of long-chain fatty-acyl-coenzyme As, and in the unfolded protein response. We selected GBF1, a guanine nucleotide exchange factor responsible for ARF activation, from the NS5 data set for follow up and functional validation. We show that GBF1 plays a critical role early in dengue infection that is independent of its role in the maintenance of Golgi structure. Importantly, the approach described here can be applied to virtually any organism/system as a tool for better understanding its molecular interactions.

Phospholipid transport via mitochondria.

In eukaryotic cells, complex membrane structures called organelles are highly developed to exert specialized functions. Mitochondria are one of such organelles consisting of the outer and inner membranes (OM and IM) with characteristic protein and phospholipid compositions. Maintaining proper phospholipid compositions of the membranes is crucial for mitochondrial integrity, thereby contributing to normal cell activities. As cellular locations for phospholipid synthesis are restricted to specific compartments such as the endoplasmic reticulum (ER) membrane and the mitochondrial inner membrane, newly synthesized phospholipids have to be transported and distributed properly from the ER or mitochondria to other cellular membranes. Although understanding of molecular mechanisms of phospholipid transport are much behind those of protein transport, recent studies using yeast as a model system began to provide intriguing insights into phospholipid exchange between the ER and mitochondria as well as between the mitochondrial OM and IM. In this review, we summarize the latest findings of phospholipid transport via mitochondria and discuss the implicated molecular mechanisms.