In vivo testing of gold nanoparticles using the Caenorhabditis elegans model organism

Gold nanoparticles (AuNPs) are present in many man-made products and cosmetics, and are also used by the food and medical industries. Tight regulations regarding the use of mammalian animals for product testing can hamper the study of the specific interactions between engineered nanoparticles and biological systems. Invertebrate models, such as the nematode Caenorhabditis elegans (C. elegans), can offer alternative approaches during the early phases of nanoparticle discovery. Here, we thoroughly evaluated the biodistribution of 11-nm and 150-nm citrate-capped AuNPs in the model organism C. elegans at multiple scales, moving from micrometric to nanometric resolution and from the organismal to cellular level. We confirmed that the nanoparticles were not able to cross the intestinal and dermal barriers. We investigated the effect of AuNPs on the survival and reproductive performance of C. elegans, and correlated these effects with the uptake of AuNPs in terms of their number, surface area, and metal mass. In general, exposure to 11-nm AuNPs resulted in a higher toxicity than the larger 150-nm AuNPs. NP aggregation inside C. elegans was determined using absorbance microspectroscopy, which allowed the plasmonic properties of AuNPs to be correlated with their confinement inside the intestinal lumen, where anatomical traits, acidic pH and the presence of biomolecules play an essential role on NP aggregation. Finally, quantitative PCR of selected molecular markers indicated that exposure to AuNPs did not significantly affect endocytosis and intestinal barrier integrity. Statement of significance This work highlights how the simple, yet information-rich, animal model C. elegans is ideally suited for preliminary screening of nanoparticles or chemicals mitigating most of the difficulties associated with mammalian animal models, namely the ethical issues, the high cost, and time constraints. This is of particular relevance to the cosmetic, food, and pharmaceutical industries, which all have to justify the use of animals, especially during the discovery, development and initial screening phases. This work provides a detailed and thorough analysis of 11-nm and 150-nm AuNPs at multiple levels of organization (the whole organism, organs, tissues, cells and molecules).Peer ReviewedPostprint (author's final draft

Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication

All positive strand RNA viruses are known to replicate their genomes in close association with intracellular membranes. In case of the hepatitis C virus (HCV), a member of the family Flaviviridae, infected cells contain accumulations of vesicles forming a membranous web (MW) that is thought to be the site of viral RNA replication. However, little is known about the biogenesis and three-dimensional structure of the MW. In this study we used a combination of immunofluorescence- and electron microscopy (EM)-based methods to analyze the membranous structures induced by HCV in infected cells. We found that the MW is derived primarily from the endoplasmic reticulum (ER) and contains markers of rough ER as well as markers of early and late endosomes, COP vesicles, mitochondria and lipid droplets (LDs). The main constituents of the MW are single and double membrane vesicles (DMVs). The latter predominate and the kinetic of their appearance correlates with kinetics of viral RNA replication. DMVs are induced primarily by NS5A whereas NS4B induces single membrane vesicles arguing that MW formation requires the concerted action of several HCV replicase proteins. Three-dimensional reconstructions identify DMVs as protrusions from the ER membrane into the cytosol, frequently connected to the ER membrane via a neck-like structure. In addition, late in infection multi-membrane vesicles become evident, presumably as a result of a stress-induced reaction. Thus, the morphology of the membranous rearrangements induced in HCV-infected cells resemble those of the unrelated picorna-, corona- and arteriviruses, but are clearly distinct from those of the closely related flaviviruses. These results reveal unexpected similarities between HCV and distantly related positive-strand RNA viruses presumably reflecting similarities in cellular pathways exploited by these viruses to establish their membranous replication factories

Pre-assembled Nuclear Pores Insert into the Nuclear Envelope during Early Development

SummaryNuclear pore complexes (NPCs) span the nuclear envelope (NE) and mediate nucleocytoplasmic transport. In metazoan oocytes and early embryos, NPCs reside not only within the NE, but also at some endoplasmic reticulum (ER) membrane sheets, termed annulate lamellae (AL). Although a role for AL as NPC storage pools has been discussed, it remains controversial whether and how they contribute to the NPC density at the NE. Here, we show that AL insert into the NE as the ER feeds rapid nuclear expansion in Drosophila blastoderm embryos. We demonstrate that NPCs within AL resemble pore scaffolds that mature only upon insertion into the NE. We delineate a topological model in which NE openings are critical for AL uptake that nevertheless occurs without compromising the permeability barrier of the NE. We finally show that this unanticipated mode of pore insertion is developmentally regulated and operates prior to gastrulation

ISWI is a RanGTP-dependent MAP required for chromosome segregation

Chromatin-remodeling factor ISWI is a microtubule-associated protein that contributes to chromosome segregation by stabilizing microtubules during anaphase

The Compartmentalized Bacteria of the Planctomycetes-Verrucomicrobia-Chlamydiae Superphylum Have Membrane Coat-Like Proteins

Compartmentalized bacteria have proteins that are structurally related to eukaryotic membrane coats, and one of these proteins localizes at the membrane of vesicles formed inside bacterial cells

Coordination of kinase and phosphatase activities by Lem4 enables nuclear envelope reassembly during mitosis

Resumen del trabajo presentado al 4th Spanish Worm Meeting, celebrado en Carmona (Sevilla) del 14 al 15 de marzo de 2013.-- et al.The nuclear envelope (NE) comprises inner and outer nuclear membranes that fuse at nuclear pore complexes. In metazoa the NE is broken down upon entry into mitosis and reformed upon mitotic exit; how this happens is not fully understood. The LEM domain protein family shares a ~40 amino acid domain, first identified in the proteins Lap2, Emerin and Man1. Most LEM proteins contain transmembrane domains, reside in the INM and interact with the nuclear lamina. One described function of the LEM domain is to interact with the essential and highly conserved chromatin-binding protein Barrier-to-autointegration factor (BAF). These BAF-LEM interactions serve as an important link between chromatin and the NE, both through the maintenance of nuclear architecture and during post-mitotic NE reassembly. Numerous studies have shown that NE breakdown (NEBD) and reformation are controlled by protein phosphorylation. Members of the Vaccinia-Related Kinase (VRK) family of mitotic kinases phosphorylate BAF in mitosis and meiosis; this modification strongly reduces the affinity of BAF for chromatin and slightly weakens its affinity for LEM proteins. Phosphorylation of BAF by VRK-1 is therefore proposed to be essential to break the link between chromatin, BAF and LEM proteins upon entry into mitosis. However the mechanism that permits BAF re-association with chromatin upon mitotic exit is unclear. Protein phosphatases regulate numerous processes during mitotic progression but their roles in mitotic exit are largely uncharacterized. A protein phosphatase 2A complex comprising PP2A-CA, PP2A-R1A and PP2A-B55α has been shown to regulate mitotic exit in human cells, although the target(s) of this complex are unknown. Here we show that a PP2A complex regulates BAF chromatin recruitment during mitotic exit and is required to enable BAFʼs essential function in NE assembly. In addition, we show that the uncharacterized LEM protein Lem4 is essential for BAF recruitment to chromatin upon mitotic exit in C. elegans and human cells. Lem4 depletion or mutation causes NE morphology defects and BAF hyperphosphorylation. In vivo and in vitro data show that BAF phosphorylation is dependent on VRK-1 and is counteracted by protein phosphatase 2A (PP2A). We propose an evolutionarily conserved model whereby Lem4 coordinates the control of BAF dephosphorylation by interacting with and inhibiting VRK-1 and recruiting a PP2A complex to BAF. This results in BAF dephosphorylation and chromatin recruitment, thereby facilitating NE assembly during mitotic exit.Peer reviewe

A bacterial tubulovesicular network

We report the presence of a membranous tubulovesicular network in the planctomycete bacterium Gemmata obscuriglobus. This endomembrane system interacts with membrane coat proteins and is capable of protein internalization and degradation. Taken together, this suggests that the planctomycetal bacterium could illuminate the emergence of complex endomembrane systems.D.A. and R.S.-M. are supported by European Molecular Biology Laboratory (EMBL); D.P.P. was supported by the Centre for Organismal Studies (COS), Heidelberg University.Peer Reviewe

Three-dimensional reconstruction of bacteria with a complex endomembrane system.

The division of cellular space into functionally distinct membrane-defined compartments has been one of the major transitions in the history of life. Such compartmentalization has been claimed to occur in members of the Planctomycetes, Verrucomicrobiae, and Chlamydiae bacterial superphylum. Here we have investigated the three-dimensional organization of the complex endomembrane system in the planctomycete bacteria Gemmata obscuriglobus. We reveal that the G. obscuriglobus cells are neither compartmentalized nor nucleated as none of the spaces created by the membrane invaginations are closed; instead, they are all interconnected. Thus, the membrane organization of G. obscuriglobus, and most likely all PVC members, is not different from, but an extension of, the "classical" Gram-negative bacterial membrane system. Our results have implications for our definition and understanding of bacterial cell organization, the genesis of complex structure, and the origin of the eukaryotic endomembrane system

CHD4 Is a RanGTP-Dependent MAP that Stabilizes Microtubules and Regulates Bipolar Spindle Formation

Background Production of the GTP-bound form of the Ran GTPase (RanGTP) around chromosomes induces spindle assembly by activating nuclear localization signal (NLS)-containing proteins. Several NLS proteins have been identified as spindle assembly factors, but the complexity of the process led us to search for additional proteins with distinct roles in spindle assembly. Results We identify a chromatin-remodeling ATPase, CHD4, as a RanGTP-dependent microtubule (MT)-associated protein (MAP). MT binding occurs via the region containing an NLS and chromatin-binding domains. In Xenopus egg extracts and cultured cells, CHD4 largely dissociates from mitotic chromosomes and partially localizes to the spindle. Immunodepletion of CHD4 from egg extracts significantly reduces the quantity of MTs produced around chromatin and prevents spindle assembly. CHD4 RNAi in both HeLa and Drosophila S2 cells induces defects in spindle assembly and chromosome alignment in early mitosis, leading to chromosome missegregation. Further analysis in egg extracts and in HeLa cells reveals that CHD4 is a RanGTP-dependent MT stabilizer. Moreover, the CHD4-containing NuRD complex promotes organization of MTs into bipolar spindles in egg extracts. Importantly, this function of CHD4 is independent of chromatin remodeling. Conclusions Our results uncover a new role for CHD4 as a MAP required for MT stabilization and involved in generating spindle bipolarity. © 2013 Elsevier Ltd