Academic literature on the topic 'Cell envelope biogenesi'

To elucidate the evolution of cyanobacterial envelopes and the relation between gene content and environmental adaptation, cell envelope structures and components of unicellular and filamentous cyanobacteria were analyzed in comparative genomics. Hundreds of envelope biogenesis genes were divided into 5 major groups and annotated according to their conserved domains and phylogenetic profiles. Compared to unicellular species, the gene numbers of filamentous cyanobacteria expanded due to genome enlargement effect, but only few gene families amplified disproportionately, such as those encoding waaG and glycosyl transferase 2. Comparison of envelope genes among various species suggested that the significant variance of certain cyanobacterial envelope biogenesis genes should be the response to their environmental adaptation, which might be also related to the emergence of filamentous shapes with some new functions.

Chloroplasts are of prokaryotic origin with a double-membrane envelope separating plastid metabolism from the cytosol. Envelope membrane proteins integrate chloroplasts with the cell, but envelope biogenesis mechanisms remain elusive. We show that maize defective kernel5 (dek5) is critical for envelope biogenesis. Amyloplasts and chloroplasts are larger and reduced in number in dek5 with multiple ultrastructural defects. The DEK5 protein is homologous to rice SSG4, Arabidopsis thaliana EMB2410/TIC236, and Escherichia coli tamB. TamB functions in bacterial outer membrane biogenesis. DEK5 is localized to the envelope with a topology analogous to TamB. Increased levels of soluble sugars in dek5 developing endosperm and elevated osmotic pressure in mutant leaf cells suggest defective intracellular solute transport. Proteomics and antibody-based analyses show dek5 reduces levels of Toc75 and chloroplast envelope transporters. Moreover, dek5 chloroplasts reduce inorganic phosphate uptake with at least an 80% reduction relative to normal chloroplasts. These data suggest that DEK5 functions in plastid envelope biogenesis to enable transport of metabolites and proteins.

Melinjo seeds (Gnetum gnemon L.) have many benefits, that it is necessary to know its morphological and anatomical characters. This study aimed to determine differences in morphological characters and anatomical development of melinjo seeds at four seed maturity stages. The morphological observation was carried out based on the quantitative and organoleptic characteristics of the melinjo seeds: outer envelope, size, and the color of the middle envelope. Seed development was anatomically observed in slides prepared with a non-embedding method using a sliding microtome then observed through a microscope. The outer seed envelope has a green to blackish-red color in the final stage, while the seed middle envelope has a light-brown to dark-brown in the final stage. The inner seed envelope is thin, non-rigid, and attached on the outside of the endosperm. The seeds' length ranges from ±1,5 cm until ±2,25 cm at the end-stage; seeds width are 1 cm – 1,18 cm; seeds diameter are 1 cm – 1,16 cm. The anatomical development showed tissue thickening and differentiation. The middle envelope is getting thicker: 318,84 μm to 397,29 μm. Endosperm tissue cells undergo cell compaction as the seeds ripen. At the same time, embryonic tissue differentiation forms hypocotyl, epicotyl, and cotyledon.

Vaccinia virus, the prototype of the Poxviridae, is a large DNA virus which replicates in the cytoplasm of the host cell. The assembly pathway of vaccinia virus displays several unique features, such as the production of two structurally distinct, infectious forms. One of these, termed intracellular naked virus (INV), remains cells associated while the other, termed extracellular enveloped virus (EEV), is released from the cell. In addition, it has long been believed that INVs acquire their lipid envelopes by a unique example of de novo membrane biogenesis. To examine the structure and assembly of vaccinia virus we have used immunoelectron microscopy using antibodies to proteins of different subcellular compartments as well as a phospholipid analysis of purified INV and EEV. Our data are not consistent with the de novo model of viral membrane synthesis but rather argue that the vaccinia virus DNA becomes enwrapped by a membrane cisterna derived from the intermediate compartment between the ER and the Golgi stacks, thus acquiring two membranes in one step. Phospholipid analysis of purified INV supports its derivation from an early biosynthetic compartment. This unique assembly process is repeated once more when the INV becomes enwrapped by an additional membrane cisterna, in agreement with earlier reports. The available data suggest that after fusion between the outer envelope and the plasma membrane, mature EEV is released from the cell.

We obtained a monoclonal antibody (RL13) that identifies three integral membrane proteins specific to the nuclear envelope of rat liver, a major 75-kD polypeptide and two more minor components of 68 and 55 kD. Immunogold labeling of isolated nuclear envelopes demonstrates that these antigens are localized specifically to the inner nuclear membrane, and that the RL13 epitope occurs on the inner membrane's nucleoplasmic surface where the nuclear lamina is found. When nuclear envelopes are extracted with solutions containing nonionic detergent and high salt to solubilize nuclear membranes and pore complexes, most of these integral proteins remain associated with the insoluble lamina. Since the polypeptides recognized by RL13 are relatively abundant, they may function as lamina attachment sites in the inner nuclear membrane. Major cross-reacting antigens are found by immunoblotting and immunofluorescence microscopy in all rat cells examined. Therefore, these integral proteins are biochemical markers for the inner nuclear membrane and will be useful models for studying nuclear membrane biogenesis.

Following infection of BHK-21 cells with Herpes simplex virus type 1 (HSV-1), progeny nucleocapsids in the nucleus acquire a glycoprotein-rich envelope by budding through host-cell nuclear membranes. To investigate the nature of the glycoprotein products assembled in the virion at the nuclear envelope, infected cells were pulse-labeled with [3H]-mannose, an oligosaccharidal core sugar, or [3H]-fucose, a terminal sugar. After various chase periods, the incorporation of these sugars was monitored by electron microscope radioautography. The results show that HSV glycoproteins accumulate very rapidly in nuclear membranes, where they exist only as core-glycosylated precursors, i.e., containing [3H]-mannose but not [3H]-fucose. [3H]-fucose grains are seen mainly over Golgi membranes and over virions located in the Golgi and in other cytoplasmic vesicular structures. Our data support a model where addition of terminal sugars (e.g., fucose) to HSV-1 glycoprotein precursors can occur at the surface of newly enveloped viral particles as the virions themselves egress from the cell via the Golgi apparatus.

The outer envelope of vaccinia virus extracellular virions is derived from intracellular membranes that, at late times in infection, are enriched in several virus-encoded proteins. Although palmitoylation is common in vaccinia virus envelope proteins, little is known about the role of palmitoylation in the biogenesis of the enveloped virus. We have studied the palmitoylation of B5, a 42 kDa type I transmembrane glycoprotein comprising a large ectodomain and a short (17 aa) cytoplasmic tail. Mutation of two cysteine residues located in the cytoplasmic tail in close proximity to the transmembrane domain abrogated palmitoylation of the protein. Virus mutants expressing non-palmitoylated versions of B5 and/or lacking most of the cytoplasmic tail were isolated and characterized. Cell-to-cell virus transmission and extracellular virus formation were only slightly affected by those mutations. Notably, B5 versions lacking palmitate showed decreased interactions with proteins A33 and F13, but were still incorporated into the virus envelope. Expression of mutated B5 by transfection into uninfected cells showed that both the cytoplasmic tail and palmitate have a role in the intracellular transport of B5. These results indicate that the C-terminal portion of protein B5, while involved in protein transport and in protein–protein interactions, is broadly dispensable for the formation and egress of infectious extracellular virus and for virus transmission.

Corynebacteriales are Actinobacteria that possess an atypical didermic cell envelope. One of the principal features of this cell envelope is the presence of a large complex made up of peptidoglycan, arabinogalactan and mycolic acids. This covalent complex constitutes the backbone of the cell wall and supports an outer membrane, called mycomembrane in reference to the mycolic acids that are its major component. The biosynthesis of the cell envelope of Corynebacteriales has been extensively studied, in particular because it is crucial for the survival of important pathogens such as Mycobacterium tuberculosis and is therefore a key target for anti-tuberculosis drugs. In this study, we explore the biogenesis of the cell envelope of Corynebacterium glutamicum, a non-pathogenic Corynebacteriales, which can tolerate dramatic modifications of its cell envelope as important as the loss of its mycomembrane. For this purpose, we used a genetic approach based on genome-wide transposon mutagenesis. We developed a highly effective immunological test based on the use of anti-cell wall antibodies that allowed us to rapidly identify bacteria exhibiting an altered cell envelope. A very large number (10,073) of insertional mutants were screened by means of this test, and 80 were finally selected, representing 55 different loci. Bioinformatics analyses of these loci showed that approximately 60% corresponded to genes already characterized, 63% of which are known to be directly involved in cell wall processes, and more specifically in the biosynthesis of the mycoloyl-arabinogalactan-peptidoglycan complex. We identified 22 new loci potentially involved in cell envelope biogenesis, 76% of which encode putative cell envelope proteins. A mutant of particular interest was further characterized and revealed a new player in mycolic acid metabolism. Because a large proportion of the genes identified by our study is conserved in Corynebacteriales, the library described here provides a new resource of genes whose characterization could lead to a better understanding of the biosynthesis of the envelope components of these bacteria.

The emergence of multidrug-resistant strains of Gram-negative pathogens that rapidly spread in the clinic is of great concern, since the range of antibiotics still effective against these organisms is limited and will continue to diminish. Therefore, the identification of novel and unexplored drug targets is an urgent need. Gram-negative bacteria possess an outer membrane (OM) with highly selective permeability properties due to its asymmetric structure with LPS in the outer leaflet and phospholipids in the inner leaflet. Dissecting the biogenesis of the OM and gaining insights into the multiprotein machineries that assemble this structure is vital if we want to succeed in developing novel antibiotic compounds that can target these machineries. This thesis focuses on the machinery that transports lipopolysaccharide (LPS) to the cell surface: the LPS transport (Lpt) machinery. In Escherichia coli, the Lpt system is composed of seven essential proteins spanning the cell envelope: the ABC transporter LptB2FGC powers the LPS extraction from the inner membrane (IM) and its transport along the periplasmic bridge, comprising LptA, to the OM LptDE translocon, which assembles LPS on the cell surface. Due to its vital role in cell physiology, the Lpt system represents a good target for the development of antibiotics with an innovative mechanism of action. Encouragingly, two promising inhibitors of this machinery have been discovered: murepavadin, which is currently in preclinical development, and thanatin. The research project of this thesis focuses on two main topics: elucidating the mechanism behind thanatin’s antibacterial activity, and the characterization of a mutant six-component Lpt machinery that is functional without LptC. Thanatin is a host-defence antimicrobial peptide recently shown to cause defects in membrane assembly and to bind to the N-terminal β-strand of LptA in vitro (Vetterli et al., 2018). Since this region is involved in both LptA dimerization and interaction with LptC, we implemented the Bacterial Adenylate Cyclase Two-Hybrid (BACTH) system to detect these interactions in the periplasm and probe which is the target of thanatin. With this technique, we found that thanatin targets both interactions and has a stronger inhibitory effect on the LptC-LptA interaction (Moura et al., 2020: https://doi.org/10.3389/fmicb.2020.00909). Further demonstrating a direct effect upon the LPS transport, we observed in thanatin-treated cells the degradation of LptA and the accumulation of LPS decorated with colanic acid (Moura et al., 2020), both of which have been previously reported to be indicative of LPS transport defects (Sperandeo et al., 2008, 2011). We further explored how thanatin affects the integrity of the cell envelope and observed that it induces promoters regulated by envelope-specific stress response systems (unpublished data). Although all seven Lpt proteins have been shown to be essential, viable mutants lacking LptC but carrying suppressor mutations at the residue R212 in the periplasmic domain of LptF were isolated by our group (Benedet et al., 2016). Interestingly, LptC was recently proposed to have a regulatory role on the LptB2FGC transporter by modulating its ATPase activity (Owens et al., 2019; Li et al., 2019), thus adding to the mystery of how the suppressor mutants can survive without LptC. In the second part of the project, we elucidated how the cell can bypass the presence of LptC and its regulatory role in the machinery by performing a biochemical characterization of the most representative suppressor mutant (manuscript ready for submission). Moreover, by analysing the interaction networks around the residue R212 of LptF, we also formulated a putative mechanism adopted by the Lpt transporter to regulate LPS transfer from LptB2FGC to LptA.

In Gram-negative bacteria, the three-layered cell envelope, including the cell wall, outer and inner membranes, is essential for cell survival in the changing, and often hostile environments. Conserved in all prokaryotes, the cell wall is incredibly thin, yet it functions to prevent osmotic lysis in diluted conditions. Based on observations obtained by genetic and chemical perturbations, time-lapse live cell imaging, quantitative imaging and statistical analysis, Part I of this dissertation explores the molecular and physical events leading to cell lysis induced by division-specific beta-lactams. We found that such lysis requires the complete assembly of all essential components of the cell division apparatus and the subsequent recruitment of hydrolytic amidases. We propose that division-specific beta-lactams lyze cells by inhibiting FtsI (PBP3) without perturbing the normal assembly of the cell division machinery and the consequent activation of cell wall hydrolases. On the other hand, we demonstrated that cell lysis by beta-lactams proceeds through four physical phases: elongation, bulge formation, bulge stagnation and lysis. Bulge formation dynamics is determined by the specific perturbation of the cell wall and outer membrane plays an independent role in stabilizing the bulge once it is formed. The stabilized bulge delays lysis, and allows escape and recovery upon drug removal. Asymmetrical in structure and unique to Gram-negative bacteria, outer membrane prevents the passage of many hydrophobic, toxic compounds. Together with inner membrane and the cell wall, three layers of the Gram-negative cell envelope must be well coordinated throughout the cell cycle to allow elongation and division. Part II of this dissertation explores the essentiality of the LPS layer, the outer leaflet of the outer membrane. Using a conditional mutant severely defective in LPS transport, we found that mutations in the initiation phase of fatty acid synthesis suppress cells defective in LPS transport. The suppressor cells are remarkably small with a 70% reduction in cell volume and a 50 % reduction in growth rate. They are also blind to nutrient excess with respect to cell size control. We propose a model where fatty acid synthesis regulates cell size in response to nutrient availability, thereby influencing growth rate.
Chemistry and Chemical Biology

The three layered Gram-negative bacteria envelope consists of an inner membrane (IM), the periplasm‐containing peptidoglycan (PG), and an asymmetric outer membrane (OM) decorated with lipopolysaccharide (LPS) in the outer leaflet. Growth and assembly of cell envelope is orchestrated by action of dedicated protein machineries which span the entire envelope and whose coordinated activity guarantees proper envelope stiffness. Defects in biogenesis in any of these layers compromise the whole cell integrity and lead to cell death. In this thesis we show that Escherichia coli remodels the PG structure by increasing the level of 3-3 crosslinks produced by LD – Transpeptidases (LDTs), to avoid cell lysis when the LPS transport to the OM is disrupted. E. coli codes for six LDTs (LdtA-F): LdtA, LdtB, and LdtC covalently attach Lpp to PG while LdtD and LdtE introduce 3-3crosslinks. LdtF has no LD-Transpeptidase (LD-Tpase) activity but enhances the enzymatic activity of LdtD and LdtE. Our data outlines a major contribution of LdtD in PG remodelling and suggest that LdtD works in concert with the PG synthase PBP1B, its activator LpoB and the DD-CPase PBP6a to form a dedicated PG repair machine that runs a PG remodeling program to counteract damages to the OM. We also show that the lysis phenotype and morphological defects seen in mutants with an impaired LPS transport and lacking ldtF, are rescued and suppressed, respectively, by the loss of YgeR an uncharacterized lipoprotein predicted to be OM anchored. YgeR belongs to the family of LytM-domain factors which are hydrolases or hydrolase regulators implicated in PG remodeling/turnover. Important PG hydrolases are amidases which promote PG septal splitting and daughter cell separation. Our biochemical data reveal that YgeR is an amidase regulator able to activate AmiA, AmiB and AmiC the three amidases encoded by E. coli. We also show that YgeR binds purified PG and physically interacts with the amidase AmiC. Our biochemical analyses are complemented by in vivo data showing that YgeR preferentially activates AmiC and that it does it through its LytM domain. Altogether, our results point out an unexplored protective role of the 3-3 crosslinks in PG to overcome severe OM biogenesis defects and propose YgeR as a novel amidase activator whose action seems required upon envelope stress.

Chez les eucaryotes, les pores nucléaires (NPCs), ancrés dans l’enveloppe nucléaire (EN), régulent les échanges nucléocytoplasmiques. Ces complexes, très conservés, sont composés d’une trentaine de protéines appelées nucléoporines (Nups) présentes en multiples copies au sein de chaque NPC. Chez la levure S. cerevisiae, seules quatre Nups, dont la protéine Pom33, possèdent des domaines transmembranaires. Une étude réalisée en amont de ce projet a permis de caractériser Pom33 et de montrer que le mutant pom33∆ est viable et ne présente pas de défaut apparent de transport nucléocytoplasmique mais se caractérise par un défaut de distribution des NPCs. Pom33 joue également un rôle dans l’assemblage des pores nucléaires au sein de l’EN (biogenèse de novo des NPCs). POM33 appartient à une famille de gènes très conservés. Il possède un paralogue chez S. cerevisiae, PER33, qui code pour une protéine localisée majoritairement au réticulum endoplasmique et minoritairement aux NPCs et qui n’est pas impliquée dans la biogenèse des NPCs. Chez les mammifères, il n’existe qu’un homologue de Pom33/Per33, TMEM33. Dans le cadre de ce doctorat, nous nous sommes demandés quels étaient les déterminants contribuant à l’adressage spécifique de Pom33 au niveau des NPCs et à sa fonction dans la biogenèse de ces structures. La purification de Pom33-ProtA, suivie de spectrométrie de masse, nous a permis d’identifier un nouveau partenaire de Pom33, le facteur d’import Kap123. Des approches in vitro ont montré une interaction directe entre Kap123 et le domaine C-terminal (CTD) de Pom33, qui est perturbée en présence de RanGTP. Par ailleurs, des prédictions in silico ont révélé la présence dans ce domaine CTD de deux hélices amphipathiques, conservées chez l’humain. Des analyses par dichroïsme circulaire et flottaisons ont confirmé la capacité du CTD à s’organiser en hélice en présence de membranes lipidiques et à interagir préférentiellement avec les membranes très courbées. L’expression d’une version mutée de Pom33-CTD, incapable de se lier aux membranes et couplée à la GFP, a révélé la capacité de ce domaine à agir comme un NLS, importé spécifiquement dans le noyau par Kap123. Alors que la délétion du domaine CTD affecte l’adressage de Pom33 aux NPCs et provoque un défaut de distribution des NPCs, la mutation des résidus basiques impliqués dans l’interaction avec Kap123 ou des résidus permettant sa liaison aux membranes lipidiques ne récapitule pas ce phénotype. En revanche, la perte combinée de ces deux déterminants affecte l’adressage de Pom33 aux NPCs et provoque un défaut de distribution des NPCs ainsi qu'une interaction génétique avec le mutant nup133∆, impliqué dans la biogenèse de novo des NPCs. Les résultats obtenus lors de cette étude indiquent donc que l’adressage de Pom33 est un mécanisme actif et multifactoriel, qui met en jeu au moins deux déterminants dans son domaine CTD. Ces données indiquent également un rôle de ce domaine dans la biogenèse de novo des NPCs, qui pourrait néanmoins n’être qu’un effet indirect de son rôle dans l’adressage de Pom33 aux NPCs. Au cours de cette étude, nous avons également mis en évidence d’autres partenaires potentiels de Pom33, en particulier Myo2, une localisation de Pom33 au niveau du bourgeon lors de la division et une interaction génétique entre POM33 et KAP123. Ces observations préliminaires ouvrent de nouvelles pistes de réflexion quant au rôle de Pom33 lors de la division cellulaire
In eukaryotic cells, nucleocytoplasmic exchanges take place through the nuclear pores complexes (NPCs). These conserved macromolecular assemblies are embedded in the nuclear envelope (NE) and composed of ~30 distinct proteins called nucleoporins (Nups), each presents in multiple copies. In the budding yeast Sacharomyces cerevisiae, there are only four transmembrane Nups, including Pom33. A previous study leds to the characterization of Pom33 and revealed that pom33∆ mutant cells, although viable and without apparent alteration in nucleocytoplasmic transport, display NPCs distribution defect. Pom33 also contributes to the biogenesis of NPCs into the intact NE (de novo biogenesis). Pom33 is highly conserved among species and has a paralogue in S. cerevisiae, Per33, which can associate with NPCs but is mainly localized at the endoplasmic reticulum (ER) and NE. Unlike Pom33, Per33 is not involved in NPCs distribution and biogenesis. In mammalian cells, there is a unique homologue of Pom33/Per33, named TMEM33. In the context of this thesis, we aimed to identify the determinants involved in the specific targeting of Pom33 to NPCs and in its function in pore biogenesis. To characterize these determinants, we first performed affinity-purification experiments followed by mass spectrometry analyses. This identified a novel Pom33 partner, the nuclear import factor Kap123. In vitro experiments revealed a direct interaction between Pom33 C-terminal domain (CTD) and Kap123 that involves positively-charged residues within Pom33-CTD and is altered in the presence of Ran-GTP. Moreover, in silico analyses predicted the presence of two evolutionarily-conserved amphipathic ~-helices within Pom33-CTD. Circular dichroism studies and liposome co-floatation assays confirmed that this CTD domain is able to fold into ~-helices in the presence of liposomes and revealed its preferential binding to highly curved lipid membranes. When expressed in yeast, under conditions abolishing Pom33-CTD membrane association, Pom33-CTD behaves as a Kap123-dependent nuclear localization domain. While deletion of Pom33 C-terminal domain (Pom33-∆CTD-GFP) impairs Pom33 NPC targeting and stability and leads to a NPC distribution phenotype, mutants affecting either Kap123 binding or the amphipathic properties of the ~-helices do not display any detectable defect. However, combined impairment of lipid and Kap123 binding affects Pom33 targeting to NPCs and leads to an altered NPC distribution and a genetic interaction with the deletion of NUP133, a gene coding for a nucleoporin involved in NPCs biogenesis. Together, these results indicate that Pom33 targeting to NPCs is an active and multifactorial process that requires at least two determinants within its CTD. They also suggest a role of Pom33-CTD in the de novo NPCs biogenesis process, which could however only be an indirect consequence of its requirement for Pom33 targeting to NPCs. Our mass spectrometry analysis also identified other partners of Pom33, in particular Myo2, a molecular motor required for the cell cycle-regulated transport of various organelles and proteins and for correct alignment of the spindle during mitosis. Our studies also revealed a specific localization of Pom33 at the bud tip during mitosis and a genetic interaction between POM33 and KAP123. Taken together, these preliminary observations open new perspectives regarding additional functions of Pom33 during cell division

Extracellular vesicles (EVs) are a heterogeneous group of endogenous nanoscale vesicles that are secreted by various cell types. Based on their biogenesis and size distribution, EVs can be broadly classified as exosomes and microvesicles. Exosomes are enveloped by lipid bilayers with a size of 30–150 nm in diameter, which contain diverse biomolecules, including lipids, proteins and nucleic acids. Exosomes transport their bioactive cargoes from original cells to recipient cells, thus play crucial roles in mediating intercellular communication. Breast cancer is the most common malignancy among women and remains a major health problem worldwide, diagnostic strategies and therapies aimed at breast cancer are still limited. Growing evidence shows that exosomes are involved in the pathogenesis of breast cancer, including tumorigenesis, invasion and metastasis. Here, we provide a straightforward overview of exosomes and highlight the role of exosomes in the pathogenesis of breast cancer, moreover, we discuss the potential application of exosomes as biomarkers and therapeutic tools in breast cancer diagnostics and therapeutics.