Amphiphilic components of the murchison carbonaceous chondrite: Surface properties and membrane formation

We have investigated physicochemical properties of amphiphilic compounds in carbonaceous meteorites. The primary aim was to determine whether such materials represent plausible sources of lipid-like compounds that could have been involved as membrane components in primitive cells. Samples of the Murchison CM2 chondrite were extracted with chloroform-methanol, and the chloroform-soluble material was separated by two-dimensional thin layer chromatography. Fluorescnece, iodine stains and charring were used to identify major components on the plates. These were than scraped and eluted as specific fractions which were investigated by fluorescence and absorption spectra, surface chemical methods, gas chromatography-mass spectrometry, and electron microscopy. Fraction 5 was strongly fluorescent, and contained pyrene and fluoranthene, the major polycyclic aromatic hydrocarbons of the Murchison chondrite. This fraction was also present in extracts from the Murray and Mighei CM2 chondrites. Fraction 3 was surface active, forming apparent monomolecular films at air-water interfaces. Surface force measurements suggested that fraction 3 contained acidic groups. Fraction 1 was also surface active, and certain components could self-assemble into membranous vesicles which encapsulated polar solutes. The observations reported here demonstrate that organic compounds plausibly available on the primitive Earth through meteoritic infall are surface active, and have the ability to self-assemble into membranes.     

Stability of Model Membranes in Extreme Environments

The first forms of cellular life required a source of amphiphilic compounds capable of assembling into stable boundary structures. Membranes composed of fatty acids have been proposed as model systems of primitive membranes, but their bilayer structure is stable only within a narrow pH range and low ionic strength. They are particularly sensitive to aggregating effects of divalent cations (Mg+2, Ca+2, Fe+2) that would be present in Archaean sea water. Here we report that mixtures of alkyl amines and fatty acids form vesicles at strongly basic and acidic pH ranges which are resistant to the effects of divalent cations up to 0.1 M. Vesicles formed by mixtures of decylamine and decanoic acid (1:1 mole ratio) are relatively permeable to pyranine, a fluorescent anionic dye, but permeability could be reduced by adding 2 mol% of a polycyclic aromatic hydrocarbon such as pyrene. Permeability to the dye was also reduced by increasing the chain length of the amphiphiles. For instance, 1:1 mole ratio mixtures of dodecylamine and dodecanoic acid were able to retain pyranine dye during and following gel filtration. We conclude that primitive cell membranes were likely to be composed of mixtures of amphiphilic and hydrophobic molecules that manifested increased stability over pure fatty acid membranes.     

Hijacking components of the cellular secretory pathway for replication of poliovirus RNA

Infection of cells with poliovirus induces a massive intracellular membrane reorganization to form vesicle-like structures where viral RNA replication occurs. The mechanism of membrane remodeling remains unknown, although some observations have implicated components of the cellular secretory and/or autophagy pathways. Recently, we showed that some members of the Arf family of small GTPases, which control secretory trafficking, became membrane-bound after the synthesis of poliovirus proteins in vitro and associated with newly formed membranous RNA replication complexes in infected cells. The recruitment of Arfs to specific target membranes is mediated by a group of guanine nucleotide exchange factors (GEFs) that recycle Arf from its inactive, GDP-bound state to an active GTP-bound form. Here we show that two different viral proteins independently recruit different Arf GEFs (GBF1 and BIG1/2) to the new structures that support virus replication. Intracellular Arf-GTP levels increase approximately 4-fold during poliovirus infection. The requirement for these GEFs explains the sensitivity of virus growth to brefeldin A, which can be rescued by the overexpression of GBF1. The recruitment of Arf to membranes via specific GEFs by poliovirus proteins provides an important clue toward identifying cellular pathways utilized by the virus to form its membranous replication complex.     

Complex dynamic development of poliovirus membranous replication complexes

Replication of all positive-strand RNA viruses is intimately associated with membranes. Here we utilize electron tomography and other methods to investigate the remodeling of membranes in poliovirus-infected cells. We found that the viral replication structures previously described as "vesicles" are in fact convoluted, branching chambers with complex and dynamic morphology. They are likely to originate from cis-Golgi membranes and are represented during the early stages of infection by single-walled connecting and branching tubular compartments. These early viral organelles gradually transform into double-membrane structures by extension of membranous walls and/or collapsing of the luminal cavity of the single-membrane structures. As the double-membrane regions develop, they enclose cytoplasmic material. At this stage, a continuous membranous structure may have double- and single-walled membrane morphology at adjacent cross-sections. In the late stages of the replication cycle, the structures are represented mostly by double-membrane vesicles. Viral replication proteins, double-stranded RNA species, and actively replicating RNA are associated with both double- and single-membrane structures. However, the exponential phase of viral RNA synthesis occurs when single-membrane formations are predominant in the cell. It has been shown previously that replication complexes of some other positive-strand RNA viruses form on membrane invaginations, which result from negative membrane curvature. Our data show that the remodeling of cellular membranes in poliovirus-infected cells produces structures with positive curvature of membranes. Thus, it is likely that there is a fundamental divergence in the requirements for the supporting cellular membrane-shaping machinery among different groups of positive-strand RNA viruses.     

Origin of amphiphilic molecules and their role in primary structure formation

Attempts to solve two fundamental questions are described: the first concerns which mechanisms were responsible for the self-assembly of membrane structures on the prebiotic Earth, and the second concerns the routes by which considerable amounts of membrane amphiphiles formed from simpler hydrocarbons. The physicochemical properties of several amphiphilic compounds extracted from the Murchison carbonaceous chondrite were studied, using infra-red and fluorescent spectroscopy, measurements of surface activity, chromato-mass spectrometry, and polarization and electron microscopy. The results supported previous observations that amphiphilic and aromatic hydrocarbons were present in significant quantities, and the first demonstration of surface activity among a number of acidic derivatives of hydrocarbons is reported. In addition, one fraction of the surface-active compounds can form bilayer structures, showing that membranes could have self-assembled on the prebiotic Earth. Photochemical oxidation of hydrocarbons is shown to be a likely source of the amphiphilic molecules required for the self-assembly of primary membrane structures.     

Mutations that disrupt the morphogenesis and localization of a sperm-specific organelle in Caenorhabditis elegans

Nematode sperm contain unusual organelles, membranous organelles, which undergo dramatic morphological changes during spermatogenesis. Early in spermatogenesis, the membranous organelle functions to transport sperm specific components to the spermatids; later, during the formation of the crawling spermatozoa, it adds new components to the cell surface as it fuses with the plasma membrane. Genetic analysis of spermatogenesis in the nematode Caenorhabditis elegans has revealed mutations that specifically disrupt the proper cellular localization and morphogenesis of this organelle. In animals homozygous for the either the known deficiency hcDf1 or the probable deficiency h12, the membranes of the membranous organelles are aberrantly covered with ribosomes. A mutation in the spermatogenesis-defective spe-10 gene causes severe defects in the morphogenesis of a fibrous body-membranous organelle complex. In both cases, these mutations also disrupt the proper localization of both nuclei and membranous organelles in haploid spermatids and spermatozoa.     

银耳（Tremella fuciformis）原基分化前期双核菌丝细胞和分生孢子的边缘体与质膜体

本文报道银耳（Ｔｒｅｍｅｌｌａｆｕｃｉｆｏｒｍｉｓ）原基分化前期．在双核菌丝的幼细胞、成熟细胞和分生孢子中,与质膜相关联的两类膜结构──边缘体和质膜体的形成与功能。根据相似结构的存在．支持小泡或多泡体排出质膜之外附在细胞壁上成为边缘体和参于细胞壁合成的假定。银耳原基分化前期．双核菌丝迅速分裂的幼细胞．其质膜内陷产生泡状质膜体,内含数个小泡,或产生膜状质膜体;在成熟细胞中．质膜内陷通常形成回旋的膜结构──膜状质膜体．内含１—２个电子致密小泡．当这两类质膜体脱离质膜进入细胞质后,有的膜层和小泡局部被消化．因此,推断质膜体具有内吞和输送养料的作用。另外．在桶孔隔膜闭塞一侧电子致密度高的细胞质中．还观察到一种罕见的只有单个膜层的质膜体．其内充满３个电子致密小泡．估计它的形成与功能同膜状质膜体相似。作者认为．桶孔闭塞和质膜体的出现是与银耳原基细胞分化有关联的两个重要特征。最后,在成熟细胞中,尚可以观察到质膜体的膜层能够散开形成内质网．因此．内质网也可以来源于质膜体。     

Hydrothermal Reactions of Pyruvic Acid: Synthesis,Selection, and Self-Assembly of Amphiphilic Molecules

Selection and self-assembly of organic compounds in aqueous phases must have been a primary process leading to emergent molecular complexity and ultimately to the origin of life. Facile reactions of pyruvic acid under hydrothermal conditions produce a complex mixture of larger organic molecules, some of which are amphiphiles that readily self-assemble into cell-sized vesicular structures. Chemical characterization of major components of this mixture reveals similarities to the suite of organic compounds present in the Murchison carbonaceous chondrite, some of whose molecules also self-assemble into membranous vesicles. Physical properties of the products are thus relevant to understanding the prebiotic emergence of molecular complexity. These results suggest that a robust family of prebiotic reaction pathways produces similar products over a range of geochemical and astrochemical environments.     

The Fine Structure of Streptomyces coelicolor : I. The Cytoplasmic Membrane System

Colonies and spore suspensions of Streptomyces coelicolor were fixed by the method of Kellenberger, Ryter, and Séchaud (1958) and embedded in methacrylate or araldite. Thin sections were cut with an A. F. Huxley microtome and examined in a Siemens' Elmiskop I. At all stages of development the hyphae of Streptomyces coelicolor have an extensive membranous component in the cytoplasm. The membranes are continuous with the plasma membrane and have a variety of configurations at different places in the hyphae. Tubular structures, vesicles, and parallel stacks of membranes are seen. In some areas concentric layers of membranes form whorled structures which are particularly frequent in the region of developing cross-walls and within maturing spores. In the spores membranous structures often lie embedded in the nuclear material. In disintegrating hyphae the intracytoplasmic membranes round off into small vesicles and remain when the rest of the cytoplasmic structure has gone. In the absence of typical mitochondria and other cytoplasmic membranous structures it is possible that the membranous component of the cytoplasm of Streptomyces coelicolor may perform the functions of the endoplasmic reticulum and/or the mitochondria of higher cells.     

Cellular origin and ultrastructure of membranes induced during poliovirus infection.

Poliovirus RNA replicative complexes are associated with cytoplasmic membranous structures that accumulate during viral infection. These membranes were immunoisolated by using a monoclonal antibody against the viral nonstructural protein 2C. Biochemical analysis of the isolated membranes revealed that several organelles of the host cell (lysosomes, trans-Golgi stack and trans-Golgi network, and endoplasmic reticulum) contributed to the virus-induced membranous structures. Electron microscopy of infected cells preserved by high-pressure freezing revealed that the virus-induced membranes contain double lipid bilayers that surround apparently cytosolic material. Immunolabeling experiments showed that poliovirus proteins 2C and 3D were localized to the same membranes as the cellular markers tested. The morphological and biochemical data are consistent with the hypothesis that autophagy or a similar host process is involved in the formation of the poliovirus-induced membranes.     

Intra-cellular storage, transport and exocytosis of halogenated compounds in marine red alga Laurencia obtusa

The production of secondary metabolites in seaweed have been related to a capability to partition compounds into cellular specialized storage structures, like gland cells and the corps en cerise (CC) or cherry bodies. The possible mechanisms that bring these compounds to the thallus surface remain poorly understood. Therefore, the aim of this work is perform a characterization of the CC and determine the intra-cellular dynamics of halogenated compounds in Laurencia obtusa. The dynamics of CC and the mechanisms related to the intra-cellular transport of halogenated compounds were evaluated by using optical tweezers and time-lapse video microscopy. The CC were isolated and its elemental composition was characterized using X-ray microanalysis. The cellular distribution of halogenated compounds was also demonstrated by fluorescence microscopy. Three-dimensional reconstruction technique was used to provide a visualization of the structures that connect CC to cell periphery. As main findings, we confirmed that the halogenated compounds are mainly found in CC and also in vesicles distributed along the cytoplasm and within the chloroplasts. We demonstrated that CC is mechanically fixed to cell periphery by a stalk-like connection. A vesicle transport though membranous tubular connections was seen occurring from CC to cell wall region. We also demonstrated a process of cortical cell death event, resulting in degradation of CC. We suggested that the vesicle transportation along membranous tubular connections and cell death events are related to the mechanisms of halogenated compounds exudation to the thallus surface and consequently with defensive role against herbivores and fouling.     

Activity of membrane-bound acetylcholinesterases in the presence of benzene alcohol and concanavalin A]

UV-sensitivity of membrane-bound acetylcholinesterases in the presence of agents, which selectively modify lipid phase and integral proteins of erythrocytic membranes (benzene alcohol and concanavalin A), has been studied. It has been determined, that UV-irradiation of human erythrocytic membranes within the range of wavelengths 240-390 and 300-400 nm leads to differently directed changes of enzymatic activity, which are caused by different number of membranous chromophores of UV-light and by their different nature. The scheme of process, causing the photomodification of membranous acetylcholinesterases, has been suggested. It takes into consideration a contribution of several structural components of membranes in these processes. Authors have made the conclusion about the important role of microenvironment in processes of acetylcholinesterases functioning and about the possibility of purposeful regulation of its UV-sensitivity by introduction of exogenous agents, which modify structural state of closest "neighbours" of enzyme.     

Lipids and proteolipids in isolated subcellular membranes of rat brain cortex

The cerebral cortex of the rat was submitted to an extensive cell fractionation schedule and in the various fractions, protein, proteolipid protein, total phospholipids, cholesterol, galactolipids, plasmalogens, and gangliosides were determined. With increasing purification the different isolated membranous structures: i.e. myelin, nerve ending membranes, synaptic vesicles, mitochondria, and microsomes, show a definite biochemical specialization reflected in their lipid composition. The presence of gangliosides in some nerve ending membranes is confirmed, and the possible functional role of these acid glyco-lipids is discussed. The importance of proteolipids as structural components of membranes is recognized. The richness of these compounds in myelin is confirmed and a special localization in the nerve ending membranes is indicated. Analysis of the molar ratios of the different lipids and proteins in the isolated membranes demonstrates that each one has a specific pattern of molecular organization. This pattern is discussed in relation to the macromolecular structures revealed by electronmicroscopy and some of the molecular models postulated for cell membranes.     

Ultrastructure of the replication site in Taura syndrome virus (TSV)-infected cells

Taura syndrome virus (TSV) is a member of the family Dicistroviridae that infects Pacific white shrimp Litopenaeus vannamei (also called Penaeus vannamei), and its replication strategy is largely unknown. To identify the viral replication site within infected shrimp cells, the viral RNA was located in correlation with virus-induced membrane rearrangement. Ultrastructural changes in the infected cells, analyzed by transmission electron microscopy (TEM), included the induction and proliferation of intracellular vesicle-like membranes, while the intracytoplasmic inclusion bodies and pyknotic nuclei indicative of TSV infection were frequently seen. TSV plus-strand RNA, localized by electron microscopic in situ hybridization (EM-ISH) using TSV-specific cDNA probes, was found to be associated with the membranous structures. Moreover, TSV particles were observed in infected cells by TEM, and following EM-ISH, they were also seen in close association with the proliferating membranes. Taken together, our results suggest that the membranous vesicle-like structures carry the TSV RNA replication complex and that they are the site of nascent viral RNA synthesis. Further investigations on cellular origins and biochemical compositions of these membranous structures will elucidate the morphogenesis and propagation strategy of TSV.     

Membrane recognition by phospholipid-binding domains

Many different globular domains bind to the surfaces of cellular membranes, or to specific phospholipid components in these membranes, and this binding is often tightly regulated. Examples include pleckstrin homology and C2 domains, which are among the largest domain families in the human proteome. Crystal structures, binding studies and analyses of subcellular localization have provided much insight into how members of this diverse group of domains bind to membranes, what features they recognize and how binding is controlled. A full appreciation of these processes is crucial for understanding how protein localization and membrane topography and trafficking are regulated in cells.     

Role of membrane colchicine binding proteins in the transmission of prolactin message to casein genes in the rabbit mammary gland

Previous work demonstrated that tubulin binding drugs specifically inhibit the capacity of prolactin to initiate casein and DNA synthesis in the mammary cell. It was concluded that microtubules or other tubulin containing cellular structures were involved in the transmission of the prolactin message to genes. In the present work, it is shown that griseofulvin, an antimitotic drug which alters microtubule structure and function, does not prevent prolactin actions. Autoradiographic studies showed that [3H]colchicine binds preferentially to plasma and Golgi membranes in the mammary cell. Short term cultures of mammary explants with [3H]colchicine demonstrated that the labelled drug binds to membranous cellular structures which were isolated from explants at the end of the culture. Fractions containing plasma and Golgi membranes contained the highest amount of radioactivity. Solubilisation of the membranes by Triton X-100 dissociated the [3H]colchicine from the prolactin receptors as judged by a chromatography of the soluble fraction on a Sepharose 6 B column. On the column, the labelled colchicine remains associated with a molecular entity which may be free tubulin. In all cases, the binding of [3H]colchicine was greatly attenuated by an excess of unlabelled colchicine but was only slightly affected by the competition with lumicolchicine. These results suggest that mammary membranes contain tubulin and that binding of drugs to this molecule inhibits the generation of the prolactin second messengers eliciting the hormonal actions in the mammary cell. This also suggests that microtubules are probably not involved in the mechanism of prolactin action.     

Heat shock proteins and the biogenesis of cellular membranes

The successful function of cells is importantly contributed by lipid membranes that are more than a simple physical barrier. The major components of cellular membranes are lipids, in particular glycerophospholipids, that have the capacity to assemble spontaneously into vesicles containing a lipid bilayer after exposure to an aqueous milieu due to their amphiphilic characteristics. The lipid capacity to form vesicles and encapsulate substrates has been proposed as a fundamental event during the biogenesis of cells. However, the stability of small vesicles is compromised during their expansion into larger and more complex particles. Recent observations by (Cornell et al. Proc Natl Acad Sci U S A 116:17239–17244, 2019) have shown that the insertion of amino acids into rudimentary vesicles could play a stabilizing role that was critical to the formation of early cells. Fatty acids were likely substituted by glycerophospholipids and amino acids replaced by polypeptides during the evolution of protocells. Thus, archaic peptides displaying lipid-binding and membrane-penetrating capacities could have played a key function in the development of current cells. In this regard, heat shock proteins (HSP), particularly the Hsp70 (HSPA) and small HSP (HSPB) families, could have portrayed that role. Indeed, bacterial DnaK is closest in sequence to the earliest members of the Hsp70 family and inserts into lipid membranes spontaneously. Moreover, extensive studies by the Vigh group have shown that, certainly, Hsp70s stabilize membranes. Thus, the ability of ancestral HSP70s and small HSPs to associate with lipids and stabilize membranes could have been a fundamental event in the genesis of cells.     

Differential destabilization of membranes by tryptophan and phenylalanine during freezing: the roles of lipid composition and membrane fusion

The stability of cellular membranes during dehydration can be strongly influenced by the partitioning of amphiphilic solutes from the aqueous phase into the membranes. The effects of partitioning on membrane stability depend in a complex manner on the structural properties of the amphiphiles and on membrane lipid composition. Here, we have investigated the effects of the amphiphilic aromatic amino acids Trp and Phe on membrane stability during freezing. Both amino acids were cryotoxic to isolated chloroplast thylakoid membranes and to large unilamellar liposomes, but Trp had a much stronger effect than Phe. In liposomes, both amino acids induced solute leakage and membrane fusion during freezing. The presence of the chloroplast galactolipids monogalactosyldiacylglycerol or digalactosyldiacylglycerol in egg phosphatidylcholine (EPC) membranes reduced leakage from liposomes during freezing in the presence of up to 5 mM Trp, as compared to membranes composed of pure EPC. The presence of the nonbilayer-forming lipid phosphatidylethanolamine increased leakage. Membrane fusion followed a similar trend, but was dramatically reduced when the anthracycline antibiotic daunomycin was incorporated into the membranes. Daunomycin has been shown to stabilize the bilayer phase of membranes in the presence of nonbilayer lipids and was therefore expected to reduce fusion. Surprisingly, this had only a small influence on leakage. Collectively, these data indicate that Trp and Phe induce solute leakage from liposomes during freezing by a mechanism that is largely independent of fusion events.     

Biochemical aspects of the visual process. XXXVII. Evidence for lateral aggregation of rhodopsin molecules in phospholipase C-treated bovine photoreceptor membranes

Photoreceptor membranes derived from isolated bovine rod outer segments, are subjected to treatment with phospholipase C (Bacillus cereus). This results in varying degrees of hydrolysis of the membrane phospholipids into diglycerides and water soluble phosphate esters without loss of rhodopsin. Electron microscopic observations of thin sections and freeze-fractured preparations indicate extrusion of diglycerides from the membranes and their coalescence to lipid droplets, beginning at 20% hydrolysis of phospholipids. After 90% hydrolysis of phospholipids membranous structures are still present. The rhodopsin is located in these structures, presumably in the form of two-dimensional lateral aggregates. This explains the cross-fracturing of the membranous structures, regularly observed upon freeze-fracturing of the phospholipase-treated photoreceptor membranes.     

Differential destabilization of membranes by tryptophan and phenylalanine during freezing: the roles of lipid composition and membrane fusion

The stability of cellular membranes during dehydration can be strongly influenced by the partitioning of amphiphilic solutes from the aqueous phase into the membranes. The effects of partitioning on membrane stability depend in a complex manner on the structural properties of the amphiphiles and on membrane lipid composition. Here, we have investigated the effects of the amphiphilic aromatic amino acids Trp and Phe on membrane stability during freezing. Both amino acids were cryotoxic to isolated chloroplast thylakoid membranes and to large unilamellar liposomes, but Trp had a much stronger effect than Phe. In liposomes, both amino acids induced solute leakage and membrane fusion during freezing. The presence of the chloroplast galactolipids monogalactosyldiacylglycerol or digalactosyldiacylglycerol in egg phosphatidylcholine (EPC) membranes reduced leakage from liposomes during freezing in the presence of up to 5 mM Trp, as compared to membranes composed of pure EPC. The presence of the nonbilayer-forming lipid phosphatidylethanolamine increased leakage. Membrane fusion followed a similar trend, but was dramatically reduced when the anthracycline antibiotic daunomycin was incorporated into the membranes. Daunomycin has been shown to stabilize the bilayer phase of membranes in the presence of nonbilayer lipids and was therefore expected to reduce fusion. Surprisingly, this had only a small influence on leakage. Collectively, these data indicate that Trp and Phe induce solute leakage from liposomes during freezing by a mechanism that is largely independent of fusion events.