Envelope Assembly Research Articles

Insights into the Function of YciM, a Heat Shock Membrane Protein Required To Maintain Envelope Integrity in Escherichia coli

The cell envelope of Gram-negative bacteria is an essential organelle that is important for cell shape and protection from toxic compounds. Proteins involved in envelope biogenesis are therefore attractive targets for the design of new antibacterial agents. In a search for new envelope assembly factors, we screened a collection of Escherichia coli deletion mutants for sensitivity to detergents and hydrophobic antibiotics, a phenotype indicative of defects in the cell envelope. Strains lacking yciM were among the most sensitive strains of the mutant collection. Further characterization of yciM mutants revealed that they display a thermosensitive growth defect on low-osmolarity medium and that they have a significantly altered cell morphology. At elevated temperatures, yciM mutants form bulges containing cytoplasmic material and subsequently lyse. We also discovered that yciM genetically interacts with envC, a gene encoding a regulator of the activity of peptidoglycan amidases. Altogether, these results indicate that YciM is required for envelope integrity. Biochemical characterization of the protein showed that YciM is anchored to the inner membrane via its N terminus, the rest of the protein being exposed to the cytoplasm. Two CXXC motifs are present at the C terminus of YciM and serve to coordinate a redox-sensitive iron center of the rubredoxin type. Both the N-terminal membrane anchor and the C-terminal iron center of YciM are important for function.

Transcriptional characterisation of the negative effect exerted by a deficiency in type II signal peptidase on extracellular protein secretion in Streptomyces lividans

Bacterial lipoproteins are a specialised class of membrane proteins that represent a small percentage of the proteome of Gram-positive bacteria, yet these lipoproteins have been reported to play important roles in nutrient scavenging, cell envelope assembly, protein folding, environmental signalling, host cell adhesion and virulence. Upon translocation of lipoproteins, the type II signal peptidase (Lsp) cleaves the signal peptide, leaving the lipoproteins bound to the outer face of the cytoplasmic membrane by means of linking lipid molecule to their +1 cysteine residue. We have studied the role played by Lsp in Streptomyces lividans cellular metabolism, particularly, in secretory protein production, and found that the absence of functional Lsp, apparently produces a translocase blockage, diminishes the synthesis of secretory proteins and triggers a stringent response. These findings could be particularly relevant when optimising S. lividans for the overproduction of secretory proteins of industrial application.

PREEParing for Mitosis

Reporting in Developmental Cell, Schlaitz et al. (2013) show that endoplasmic reticulum (ER) membrane exclusion from the mitotic spindle is an active process requiring REEP membrane proteins. REEP protein depletion results in ER membrane retention on the spindle and chromosomes, leading to defects in chromosome segregation and nuclear envelope assembly.

N-glycosylation of the premembrane protein of Japanese encephalitis virus is critical for folding of the envelope protein and assembly of virus-like particles

Premembrane (prM) and envelope (E) proteins, the major structural proteins of Japanese encephalitis virus (JEV) each contain single potential N-glycosylation site. In this study, the role of N-glycosylation of these proteins on their folding and activity were investigated. Three mutant prM and/or E (prM-E) genes lacking N-glycosylation sites were generated by site-directed mutagenesis. The effects of the N-glycan on folding, secretion and cytotoxicity of mutant proteins were determined by comparison with their wild type (wt) counterparts. Removal of N-glycan from the prM protein resulted in a complete misfolding of the E protein and failure to form virus-like particles (VLPs). A similar removal of N-glycan from the E protein led to a low efficiency of its folding and VLPs formation. The secretion and cytotoxicity of the E protein was also markedly impaired in case the glycosylation sites in the prM or E or both proteins were removed. These results suggest that the N-glycosylation of the prM protein is critical to the folding of the E protein, which makes it pivotal in the cytotoxicity of JEV particles and their production.

Assembly of the Marburg virus envelope

The key player to assemble the filamentous Marburg virus particles is the matrix protein VP40 which orchestrates recruitment of nucleocapsid complexes and the viral glycoprotein GP to the budding sites at the plasma membrane. Here, VP40 induces the formation of the viral particles, determines their morphology and excludes cellular proteins from the virions. Budding takes place at filopodia in non-polarized cells and at the basolateral cell pole in polarized epithelial cells. Molecular basis of how VP40 exerts its multifunctional role in these different processes is currently under investigation. Here we summarize recent data on structure-function relationships of VP40 and GP in connection with their function in assembly. Questions concerning the complex particle assembly, budding and release remaining enigmatic are addressed. Cytoplasmic domains of viral surface proteins often serve as a connection to the viral matrix protein or as binding sites for further viral or cellular proteins. A cooperation of MARV GP and VP40 building up the viral envelope can be proposed and is discussed in more detail in this review, as the cytoplasmic domain of GP represents an obvious interaction candidate because of its localization adjacent to the VP40 layer. Interestingly, truncation of the short cytoplasmic domain of GP neither inhibited interaction with VP40 nor incorporation of GP into progeny viral particles. Based on reverse genetics we generated recombinant virions expressing a GP mutant without the cytoplasmic tail. Investigations revealed attenuation in virus growth and an obvious defect in entry. Further investigations showed that the truncation of the cytoplasmic domain of GP impaired the structural integrity of the ectodomain, whichconsequently had impact on entry steps downstream of virus binding. Our data indicated that changes in the cytoplasmic domain are relayed over the lipid membrane to alter the function of the ectodomain.

Acute Manipulation of Diacylglycerol Reveals Roles in Nuclear Envelope Assembly & Endoplasmic Reticulum Morphology

The functions and morphology of cellular membranes are intimately related and depend not only on their protein content but also on the repertoire of lipids that comprise them. In the absence of in vivo data on lipid asymmetry in endomembranes, it has been argued that motors, scaffolding proteins or integral membrane proteins rather than non-lamellar bilayer lipids such as diacylglycerol (DAG), are responsible for shaping of organelles, local membrane curvature and fusion. The effects of direct alteration of levels of such lipids remain predominantly uninvestigated. Diacylglycerol (DAG) is a well documented second messenger. Here we demonstrate two additional conserved functions of DAG: a structural role in organelle morphology, and a role in localised extreme membrane curvature required for fusion for which proteins alone are insufficient. Acute and inducible DAG depletion results in failure of the nuclear envelope (NE) to reform at mitosis and reorganisation of the ER into multi-lamellar sheets as revealed by correlative light and electron microscopy and 3D reconstructions. Remarkably, depleted cells divide without a complete NE, and unless rescued by 1,2 or 1,3 DAG soon die. Attenuation of DAG levels by enzyme microinjection into echinoderm eggs and embryos also results in alterations of ER morphology and nuclear membrane fusion. Our findings demonstrate that DAG is an in vivo modulator of organelle morphology in mammalian and echinoderm cells, indicating a fundamental role conserved across the deuterostome superphylum.

Chromatin-bound NLS proteins recruit membrane vesicles and nucleoporins for nuclear envelope assembly via importin-α/β

The mechanism for nuclear envelope (NE) assembly is not fully understood. Importin-β and the small GTPase Ran have been implicated in the spatial regulation of NE assembly process. Here we report that chromatin-bound NLS (nuclear localization sequence) proteins provide docking sites for the NE precursor membrane vesicles and nucleoporins via importin-α and -β during NE assembly in Xenopus egg extracts. We show that along with the fast recruitment of the abundant NLS proteins such as nucleoplasmin and histones to the demembranated sperm chromatin in the extracts, importin-α binds the chromatin NLS proteins rapidly. Meanwhile, importin-β binds cytoplasmic NE precursor membrane vesicles and nucleoporins. Through interacting with importin-α on the chromatin NLS proteins, importin-β targets the membrane vesicles and nucleoporins to the chromatin surface. Once encountering Ran-GTP on the chromatin generated by RCC1, importin-β preferentially binds Ran-GTP and releases the membrane vesicles and nucleoporins for NE assembly. NE assembly is disrupted by blocking the interaction between importin-α and NLS proteins with excess soluble NLS proteins or by depletion of importin-β from the extract. Our findings reveal a novel molecular mechanism for NE assembly in Xenopus egg extracts.

Virion-Associated Complement Regulator CD55 Is More Potent than CD46 in Mediating Resistance of Mumps Virus and Vesicular Stomatitis Virus to Neutralization

Enveloped viruses can incorporate host cell membrane proteins during the budding process. Here we demonstrate that mumps virus (MuV) and vesicular stomatitis virus (VSV) assemble to include CD46 and CD55, two host cell regulators which inhibit propagation of complement pathways through distinct mechanisms. Using viruses which incorporated CD46 alone, CD55 alone, or both CD46 and CD55, we have tested the relative contribution of these regulators in resistance to complement-mediated neutralization. Virion-associated CD46 and CD55 were biologically active, with VSV showing higher levels of activity of both cofactors, which promoted factor I-mediated cleavage of C3b into iC3b as well as decay-accelerating factor (DAF) activity against the C3 convertase, than MuV. Time courses of in vitro neutralization with normal human serum (NHS) showed that both regulators could delay neutralization, but viruses containing CD46 alone were neutralized faster and more completely than viruses containing CD55 alone. A dominant inhibitory role for CD55 was most evident for VSV, where virus containing CD55 alone was not substantially different in neutralization kinetics from virus harboring both regulators. Electron microscopy showed that VSV neutralization proceeded through virion aggregation followed by lysis, with virion-associated CD55 providing a delay in both aggregation and lysis more substantial than that conferred by CD46. Our results demonstrate the functional significance of incorporation of host cell factors during virion envelope assembly. They also define pathways of virus complement-mediated neutralization and suggest the design of more effective viral vectors.

Pan‐Selective Aptamers for the Family of Small GTPases

Pan‐Selective Aptamers for the Family of Small GTPases

Gag-Vpu cross talk modulating HIV-1 envelope incorporation and infectivity in cell-type dependent manner

HIV-1 Vpu plays important role in enhancing virus release and CD4 down-modulation. We investigated the effect of Vpu-Gag cross talk on envelope incorporation and assembly in different cell types including primary cells.

Temporal Control of Nuclear Envelope Assembly by Phosphorylation of Lamin B Receptor

The nuclear envelope of metazoans disassembles during mitosis and reforms in late anaphase after sister chromatids have well separated. The coordination of these mitotic events is important for genome stability, yet the temporal control of nuclear envelope reassembly is unknown. Although the steps of nuclear formation have been extensively studied in vitro using the reconstitution system from egg extracts, the temporal control can only be studied in vivo. Herein, we use time‐lapse microscopy to investigate this process in living HeLa cells. We demonstrate that Cdk1 activity prevents premature nuclear envelope assembly and that phosphorylation of the inner nuclear membrane protein lamin B receptor (LBR) by Cdk1 contributes to the temporal control. We further identify a region in the nucleoplasmic domain of LBR that inhibits premature chromatin binding of the protein. We propose that this inhibitory effect is partly mediated by Cdk1 phosphorylation. Furthermore, we show that the reduced chromatin‐binding ability of LBR together with Aurora B activity contribute to nuclear envelope breakdown. Our studies reveal for the first time a mechanism that controls the timing of nuclear envelope reassembly through modification of an integral nuclear membrane protein.

Cocaine hijacks sigma‐1 receptors from the ER to the nuclear membrane to interact with lamina A/C: Effect on MeCP2 gene expression

The sigma‐1 receptor (Sig‐1R) is an endoplasmic reticulum (ER)‐resident non‐opioid receptor that is expressed in the nervous system and can regulate cellular differentiation, neuroplasticity, and neuroprotection. Nuclear lamins in the nucleus are essential for the nuclear envelope assembly, DNA synthesis, and gene transcription. We tested here the hypothesis that cocaine, via Sig‐1Rs, may affect the gene expression of methyl CpG binding protein 2 (MeCP2) which relates to cocaine addiction. In glial and NG108‐15 cells, cocaine increases the nuclear envelope invaginations (NEI) and grooving, suggesting that the nuclear‐cytoplasmic exchange is enhanced by cocaine. Confocal microscopic data show that cocaine and another Sig‐1R agonist DHEAS cause the translocation of Sig‐1Rs from the ER into the nuclear membrane. After cocaine treatment, Sig‐1Rs localize mainly in the outer membrane of the nucleus and could increase the chance to interact with lamin A/C. Cocaine is known to cause addictive behavior by upregulating (MeCP2) in the brain. In rat primary cortical neurons, cocaine causes an increase in MeCP2. This effect is antagonized by Sig‐1R antagonist BD1063. Our results suggest that cocaine increases MeCP2 gene expression by translocating Sig‐1Rs into the nuclear outer membrane to increase the NEI and grooving to facilitate the Sig‐1R‐lamin A/C interaction to regulate the MeCP2 gene expression.

Interaction of the Profilaggrin N-Terminal Domain with Loricrin in Human Cultured Keratinocytes and Epidermis

The relationship between the two coexpressed differentiation markers, profilaggrin and loricrin, is not clear right now. In this study, we explored the interaction of profilaggrin N-terminal domain (PND) with loricrin in keratinocytes and epidermis. Confocal immunofluorescence microscopic analysis of human epidermis showed that PND colocalized with loricrin. Loricrin nucleofected into HaCaT cells colocalized with PND in the nucleus and cytoplasm. The PND localizes to both the nucleus and cytoplasm of epidermal granular layer cells. Nucleofected PND also colocalized with keratin 10 (K10) in the nucleus and cytoplasm. Immunoelectron microscopic analysis of human epidermis confirmed the findings in nucleofected keratinocytes. Yeast two-hybrid assays showed that the B domain of human and mouse PND interacted with loricrin. The glutathione S-transferase (GST) pull-down analysis using recombinant GST-PND revealed that PND interacted with loricrin and K10. Knockdown of PND in an organotypic skin culture model caused loss of filaggrin expression and a reduction in both the size and number of keratohyalin granules, as well as markedly reduced expression of loricrin. Considering that expression of PND is closely linked to keratinocyte terminal differentiation, we conclude that PND interacts with loricrin and K10 in vivo and that these interactions are likely to be relevant for cornified envelope assembly and subsequent epidermal barrier formation.

HIV-1 gp41 Transmembrane Domain Interacts with the Fusion Peptide: Implication in Lipid Mixing and Inhibition of Virus–Cell Fusion

Fusion of the human immunodeficiency virus (HIV) with target cells is mediated by the gp41 subunit of the envelope protein. Mutation and deletion studies within the transmembrane domain (TMD) of intact gp41 influenced its fusion activity. In addition, current models suggest that the TMD is in proximity with the fusion peptide (FP) at the late fusion stages, but there are no direct experimental data to support this hypothesis. Here, we investigated the TMD focusing on two regions: the N-terminal containing the GxxxG motif and the C-terminal containing the GLRI motif, which is conserved among the TMDs of HIV and the T-cell receptor. Studies utilizing the ToxR expression system combined with synthetic peptides and their fluorescent analogues derived from TMD revealed that the GxxxG motif is important for TMD self-association, whereas the C-terminal region is for its heteroassociation with FP. Functionally, all three TMD peptides induced lipid mixing that was enhanced significantly upon mixing with FP. Furthermore, the TMD peptides inhibited virus-cell fusion apparently through their interaction with their endogenous counterparts. Notably, the R2E mutant (in the GLRI) was significantly less potent than the two others. Overall, our findings provide experimental evidence that HIV-1 TMD contributes to membrane assembly and function of the HIV-1 envelope. Owing to similarities between functional domains within viruses, these findings suggest that the TMDs and FPs may contribute similarly in other viruses as well.

Dissection of the NUP107 nuclear pore subcomplex reveals a novel interaction with spindle assembly checkpoint protein MAD1 inCaenorhabditis elegans

Nuclear pore complexes consist of several subcomplexes. The NUP107 complex is important for nucleocytoplasmic transport, nuclear envelope assembly, and kinetochore function. However, the underlying molecular mechanisms and the roles of individual complex members remain elusive. We report the first description of a genetic disruption of NUP107 in a metazoan. Caenorhabditis elegans NUP107/npp-5 mutants display temperature-dependent lethality. Surprisingly, NPP-5 is dispensable for incorporation of most nucleoporins into nuclear pores and for nuclear protein import. In contrast, NPP-5 is essential for proper kinetochore localization of NUP133/NPP-15, another NUP107 complex member, whereas recruitment of NUP96/NPP-10C and ELYS/MEL-28 is NPP-5 independent. We found that kinetochore protein NUF2/HIM-10 and Aurora B/AIR-2 kinase are less abundant on mitotic chromatin upon NPP-5 depletion. npp-5 mutants are hypersensitive to anoxia, suggesting that the spindle assembly checkpoint (SAC) is compromised. Indeed, NPP-5 interacts genetically and physically with SAC protein MAD1/MDF-1, whose nuclear envelope accumulation requires NPP-5. Thus our results strengthen the emerging connection between nuclear pore proteins and chromosome segregation.

Bacteriophage φ6—Structure Investigated by Fluorescence Stokes Shift Spectroscopy

The Stokes shift of tryptophan (Trp) fluorescence from layers of the lipid-containing bacteriophage φ6 is compared to determine the relative effect of the layers on virus hydrophobicity. In the inner most layer, the empty procapsid (PC) which contains 80-90% of the virion Trp residues, λ(max) = 339.8 nm. The PC emission is substantially more redshifted than the other φ6 layers and nearer to that of the Pseudomonad host cell than the other φ6 layers. The Trp emission from the nucleocapsid (NC) with λ(max) = 337.4 nm, is blueshifted by 2.4 nm relative to the PC although the number of Trp in the NC is identical to the PC. This shift represents an increase in Trp hydrophobicity, likely a requirement for the maintenance of A-form doubled-stranded RNA. Fluorescence from the completely assembled virion indicates it is in a considerably more hydrophobic environment with λ(max) = 330.9 nm. Density measurements show that the water content in the NC does not change during envelope assembly, therefore the blueshifted φ6 emission suggests that the envelope changes the PC environment, probably via the P8 layer. This change in hydrophobicity likely arises from charge redistribution or envelope-induced structural changes in the PC proteins.

The induction of a nucleoplasmic reticulum by prelamin A accumulation requires CTP:phosphocholine cytidylyltransferase-α.

Farnesylated prelamin A accumulates when the final endoproteolytic maturation of the protein fails to occur and causes a dysmorphic nuclear phenotype; however, the morphology and mechanisms of biogenesis of these changes remain unclear. We show here that acute prelamin A accumulation after reduction in the activity of the ZMPSTE24 endoprotease by short interfering RNA knockdown, results in the generation of a complex nucleoplasmic reticulum that depends for its formation on the enzyme CTP:phosphocholine-cytidylyltransferase-α (CCT-α, also known as choline-phosphate cytidylyltransferase A). This structure can form during interphase, confirming that it is independent of mitosis and therefore not a consequence of disordered nuclear envelope assembly. Serial-section dual-axis electron tomography reveals that these invaginations can take two forms: one in which the inner nuclear membrane infolds alone with an inter membrane space interior, and the other in which an invagination of both nuclear membranes occurs, enclosing a cytoplasmic core. Both types of invagination can co-exist in one nucleus and both are frequently studded with nuclear pore complexes (NPC), which reduces NPC abundance on the nuclear surface.

Cell autonomous and systemic factors in progeria development

Progeroid laminopathies are accelerated aging syndromes caused by defects in nuclear envelope proteins. Accordingly, mutations in the LMNA gene and functionally related genes have been described to cause HGPS (Hutchinson-Gilford progeria syndrome), MAD (mandibuloacral dysplasia) or RD (restrictive dermopathy). Functional studies with animal and cellular models of these syndromes have facilitated the identification of the molecular alterations and regulatory pathways involved in progeria development. We have recently described a novel regulatory pathway involving miR-29 and p53 tumour suppressor which has provided valuable information on the molecular components orchestrating the response to nuclear damage stress. Furthermore, by using progeroid mice deficient in ZMPSTE24 (zinc metalloprotease STE24 homologue) involved in lamin A maturation, we have demonstrated that, besides these abnormal cellular responses to stress, dysregulation of the somatotropic axis is responsible for some of the alterations associated with progeria. Consistent with these observations, pharmacological restoration of the somatotroph axis in these mice delays the onset of their progeroid features, significantly extending their lifespan and supporting the importance of systemic alterations in progeria progression. Finally, we have very recently identified a novel progeroid syndrome with distinctive features from HGPS and MAD, which we have designated NGPS (Néstor-Guillermo progeria syndrome) (OMIM #614008). This disorder is caused by a mutation in BANF1, a gene encoding a protein with essential functions in the assembly of the nuclear envelope, further illustrating the importance of the nuclear lamina integrity for human health and providing additional support to the study of progeroid syndromes as a valuable source of information on human aging.

Genetic Interaction Maps in Escherichia coli Reveal Functional Crosstalk among Cell Envelope Biogenesis Pathways

As the interface between a microbe and its environment, the bacterial cell envelope has broad biological and clinical significance. While numerous biosynthesis genes and pathways have been identified and studied in isolation, how these intersect functionally to ensure envelope integrity during adaptive responses to environmental challenge remains unclear. To this end, we performed high-density synthetic genetic screens to generate quantitative functional association maps encompassing virtually the entire cell envelope biosynthetic machinery of Escherichia coli under both auxotrophic (rich medium) and prototrophic (minimal medium) culture conditions. The differential patterns of genetic interactions detected among >235,000 digenic mutant combinations tested reveal unexpected condition-specific functional crosstalk and genetic backup mechanisms that ensure stress-resistant envelope assembly and maintenance. These networks also provide insights into the global systems connectivity and dynamic functional reorganization of a universal bacterial structure that is both broadly conserved among eubacteria (including pathogens) and an important target.

Investigation of the resistance welding of multilayers aluminum-coated polymer complexes used as envelopes of vacuum insulation panels

Vacuum Insulation Panels (VIPs) are thermal insulators consisting of a core material enveloped by a high barrier film or envelope required to maintain the vacuum.Multilayered aluminum-coated polymer complex are the common barrier envelopes of VIPs used as high performance thermal insulation in building applications. The multilayer complex is sealed to maintain the vacuum for a long period of time. From a mechanical and barrier properties standpoint, sealed edges represent the weak point of the envelope assembly. This paper investigates the influence of sealing parameters on the properties of the weld. T-peel and tensile tests in combination with Optic Microscopy observations were used to characterize the heat seals. A minimum seal initiation temperature of 115°C was identified for the studied complex. Peel strength increased sharply to reaching a maximum value at (140°C–5s), which considered as optimum temperature–time couple. Above and beyond these values, a gradual decrease was observed and microscopic images indicate the appearance of roll generated by the expelled polymer mass from the center to the edges of the weld which strongly impacts the morphology of the welded area.