Human annexin A6 interacts with influenza a virus protein M2 and negatively modulates infection

Copyright © 2012, American Society for Microbiology. All Rights ReservedThe influenza A virus M2 ion channel protein has the longest cytoplasmic tail (CT) among the three viral envelope proteins and is well conserved between different viral strains. It is accessible to the host cellular machinery after fusion with the endosomal membrane and during the trafficking, assembly, and budding processes. We hypothesized that identification of host cellular interactants of M2 CT could help us to better understand the molecular mechanisms regulating the M2-dependent stages of the virus life cycle. Using yeast two-hybrid screening with M2 CT as bait, a novel interaction with the human annexin A6 (AnxA6) protein was identified, and their physical interaction was confirmed by coimmunoprecipitation assay and a colocalization study of virus-infected human cells. We found that small interfering RNA (siRNA)-mediated knockdown of AnxA6 expression significantly increased virus production, while its overexpression could reduce the titer of virus progeny, suggesting a negative regulatory role for AnxA6 during influenza A virus infection. Further characterization revealed that AnxA6 depletion or overexpression had no effect on the early stages of the virus life cycle or on viral RNA replication but impaired the release of progeny virus, as suggested by delayed or defective budding events observed at the plasma membrane of virus-infected cells by transmission electron microscopy. Collectively, this work identifies AnxA6 as a novel cellular regulator that targets and impairs the virus budding and release stages of the influenza A virus life cycle.This work was supported by the Research Fund for the Control of Infectious Disease (project 09080892) of the Hong Kong Government, the Area of Excellence Scheme of the University Grants Committee (grant AoE/M-12/-06 of the Hong Kong Special Administrative Region, China), the French Ministry of Health, the RESPARI Pasteur Network

Optimizing Nervous System-Specific Gene Targeting with Cre Driver Lines: Prevalence of Germline Recombination and Influencing Factors.

The Cre-loxP system is invaluable for spatial and temporal control of gene knockout, knockin, and reporter expression in the mouse nervous system. However, we report varying probabilities of unexpected germline recombination in distinct Cre driver lines designed for nervous system-specific recombination. Selective maternal or paternal germline recombination is showcased with sample Cre lines. Collated data reveal germline recombination in over half of 64 commonly used Cre driver lines, in most cases with a parental sex bias related to Cre expression in sperm or oocytes. Slight differences among Cre driver lines utilizing common transcriptional control elements affect germline recombination rates. Specific target loci demonstrated differential recombination; thus, reporters are not reliable proxies for another locus of interest. Similar principles apply to other recombinase systems and other genetically targeted organisms. We hereby draw attention to the prevalence of germline recombination and provide guidelines to inform future research for the neuroscience and broader molecular genetics communities

Functional Phenotypic Rescue of Caenorhabditis elegans Neuroligin-Deficient Mutants by the Human and Rat NLGN1 Genes

Neuroligins are cell adhesion proteins that interact with neurexins at the synapse. This interaction may contribute to differentiation, plasticity and specificity of synapses. In humans, single mutations in neuroligin encoding genes lead to autism spectrum disorder and/or mental retardation. Caenorhabditis elegans mutants deficient in nlg-1, an orthologue of human neuroligin genes, have defects in different behaviors. Here we show that the expression of human NLGN1 or rat Nlgn1 cDNAs in C. elegans nlg-1 mutants rescues the fructose osmotic strength avoidance and gentle touch response phenotypes. Two specific point mutations in NLGN3 and NLGN4 genes, involved in autistic spectrum disorder, were further characterized in this experimental system. The R451C allele described in NLGN3, was analyzed with both human NLGN1 (R453C) and worm NLG-1 (R437C) proteins, and both were not functional in rescuing the osmotic avoidance behavior and the gentle touch response phenotype. The D396X allele described in NLGN4, which produces a truncated protein, was studied with human NLGN1 (D432X) and they did not rescue any of the behavioral phenotypes analyzed. In addition, RNAi feeding experiments measuring gentle touch response in wild type strain and worms expressing SID-1 in neurons (which increases the response to dsRNA), both fed with bacteria expressing dsRNA for nlg-1, provided evidence for a postsynaptic in vivo function of neuroligins both in muscle cells and neurons, equivalent to that proposed in mammals. This finding was further confirmed generating transgenic nlg-1 deficient mutants expressing NLG-1 under pan-neuronal (nrx-1) or pan-muscular (myo-3) specific promoters. All these results suggest that the nematode could be used as an in vivo model for studying particular synaptic mechanisms with proteins orthologues of humans involved in pervasive developmental disorders

NOD2 regulates hematopoietic cell function during graft-versus-host disease

Nucleotide-binding oligomerization domain 2 (NOD2) polymorphisms are independent risk factors for Crohn's disease and graft-versus-host disease (GVHD). In Crohn's disease, the proinflammatory state resulting from NOD2 mutations have been associated with a loss of antibacterial function of enterocytes such as paneth cells. NOD2 has not been studied in experimental allogeneic bone marrow transplantation (allo-BMT). Using chimeric recipients with NOD2−/− hematopoietic cells, we demonstrate that NOD2 deficiency in host hematopoietic cells exacerbates GVHD. We found that proliferation and activation of donor T cells was enhanced in NOD-deficient allo-BMT recipients, suggesting that NOD2 plays a role in the regulation of host antigen-presenting cells (APCs). Next, we used bone marrow chimeras in an experimental colitis model and observed again that NOD2 deficiency in the hematopoietic cells results in increased intestinal inflammation. We conclude that NOD2 regulates the development of GVHD through its inhibitory effect on host APC function

Caveolin-2 associates with intracellular chlamydial inclusions independently of caveolin-1

BACKGROUND: Lipid raft domains form in plasma membranes of eukaryotic cells by the tight packing of glycosphingolipids and cholesterol. Caveolae are invaginated structures that form in lipid raft domains when the protein caveolin-1 is expressed. The Chlamydiaceae are obligate intracellular bacterial pathogens that replicate entirely within inclusions that develop from the phagocytic vacuoles in which they enter. We recently found that host cell caveolin-1 is associated with the intracellular vacuoles and inclusions of some chlamydial strains and species, and that entry of those strains depends on intact lipid raft domains. Caveolin-2 is another member of the caveolin family of proteins that is present in caveolae, but of unknown function. METHODS: We utilized a caveolin-1 negative/caveolin-2 positive FRT cell line and laser confocal immunofluorescence techniques to visualize the colocalization of caveolin-2 with the chlamydial inclusions. RESULTS: We show here that in infected HeLa cells, caveolin-2, as well as caveolin-1, colocalizes with inclusions of C. pneumoniae (Cp), C. caviae (GPIC), and C. trachomatis serovars E, F and K. In addition, caveolin-2 also associates with C. trachomatis serovars A, B and C, although caveolin-1 did not colocalize with these organisms. Moreover, caveolin-2 appears to be specifically, or indirectly, associated with the pathogens at the inclusion membranes. Using caveolin-1 deficient FRT cells, we show that although caveolin-2 normally is not transported out of the Golgi in the absence of caveolin-1, it nevertheless colocalizes with chlamydial inclusions in these cells. However, our results also show that caveolin-2 did not colocalize with UV-irradiated Chlamydia in FRT cells, suggesting that in these caveolin-1 negative cells, pathogen viability and very likely pathogen gene expression are necessary for the acquisition of caveolin-2 from the Golgi. CONCLUSION: Caveolin-2 associates with the chlamydial inclusion independently of caveolin-1. The function of caveolin-2, either in the uninfected cell or in the chlamydial developmental cycle, remains to be elucidated. Nevertheless, this second caveolin protein can now be added to the small number of host proteins that are associated with the inclusions of this obligate intracellular pathogen

Dysbindin Promotes the Post-Endocytic Sorting of G Protein-Coupled Receptors to Lysosomes

BackgroundDysbindin, a cytoplasmic protein long known to function in the biogenesis of specialized lysosome-related organelles (LROs), has been reported to reduce surface expression of D2 dopamine receptors in neurons. Dysbindin is broadly expressed, and dopamine receptors are members of the large family of G protein-coupled receptors (GPCRs) that function in diverse cell types. Thus we asked if dysbindin regulates receptor number in non-neural cells, and further investigated the cellular basis of this regulation.Methodology/principal findingsWe used RNA interference to deplete endogenous dysbindin in HEK293 and HeLa cells, then used immunochemical and biochemical methods to assess expression and endocytic trafficking of epitope-tagged GPCRs. Dysbindin knockdown up-regulated surface expression of D2 receptors compared to D1 receptors, as reported previously in neurons. This regulation was not mediated by a change in D2 receptor endocytosis. Instead, dysbindin knockdown specifically reduced the subsequent trafficking of internalized D2 receptors to lysosomes. This distinct post-endocytic sorting function explained the minimal effect of dysbindin depletion on D1 receptors, which recycle efficiently and traverse the lysosomal pathway to only a small degree. Moreover, dysbindin regulated the delta opioid receptor, a more distantly related GPCR that is also sorted to lysosomes after endocytosis. Dysbindin was not required for lysosomal trafficking of all signaling receptors, however, as its depletion did not detectably affect down-regulation of the EGF receptor tyrosine kinase. Dysbindin co-immunoprecipitated with GASP-1 (or GPRASP-1), a cytoplasmic protein shown previously to modulate lysosomal trafficking of D2 dopamine and delta opioid receptors by direct interaction, and with HRS that is a core component of the conserved ESCRT machinery mediating lysosome biogenesis and sorting.Conclusions/significanceThese results identify a distinct, and potentially widespread function of dysbindin in promoting the sorting of specific GPCRs to lysosomes after endocytosis

Autism spectrum disorder is related to endoplasmic reticulum stress induced by mutations in the synaptic cell adhesion molecule, CADM1

Autism spectrum disorder (ASD) is a neurodevelopmental disorder with an unknown molecular pathogenesis. A recent molecular focus has been the mutated neuroligin 3, neuroligin 3(R451C), in gain-of-function studies and for its role in induced impairment of synaptic function, but endoplasmic reticulum (ER) stress induced by mutated molecules also deserves investigation. We previously found two missense mutations, H246N and Y251S, in the gene-encoding synaptic cell adhesion molecule-1 (CADM1) in ASD patients, including cleavage of the mutated CADM1 and its intracellular accumulation. In this study, we found that the mutated CADM1 showed slightly reduced homophilic interactions in vitro but that most of its interactions persist. The mutated CADM1 also showed morphological abnormalities, including shorter dendrites, and impaired synaptogenesis in neurons. Wild-type CADM1 was partly localized to the ER of C2C5 cells, whereas mutated CADM1 mainly accumulated in the ER despite different sensitivities toward 4-phenyl butyric acid with chemical chaperone activity and rapamycin with promotion activity for degradation of the aggregated protein. Modeling analysis suggested a direct relationship between the mutations and the conformation alteration. Both mutated CADM1 and neuroligin 3(R451C) induced upregulation of C/EBP-homologous protein (CHOP), an ER stress marker, suggesting that in addition to the trafficking impairment, this CHOP upregulation may also be involved in ASD pathogenesis

Syndecans Reside in Sphingomyelin-Enriched Low-Density Fractions of the Plasma Membrane Isolated from a Parathyroid Cell Line

BACKGROUND: Heparan sulfate proteoglycans (HSPGs) are one of the basic constituents of plasma membranes. Specific molecular interactions between HSPGs and a number of extracellular ligands have been reported. Mechanisms involved in controlling the localization and abundance of HSPG on specific domains on the cell surface, such as membrane rafts, could play important regulatory roles in signal transduction. METHODOLOGY/PRINCIPAL FINDINGS: Using metabolic radiolabeling and sucrose-density gradient ultracentrifugation techniques, we identified [(35)S]sulfate-labeled macromolecules associated with detergent-resistant membranes (DRMs) isolated from a rat parathyroid cell line. DRM fractions showed high specific radioactivity ([(35)S]sulfate/mg protein), implying the specific recruitment of HSPGs to the membrane rafts. Identity of DRM-associated [(35)S]sulfate-labeled molecules as HSPGs was confirmed by Western blotting with antibodies that recognize heparan sulfate (HS)-derived epitope. Analyses of core proteins by SDS-PAGE revealed bands with an apparent MW of syndecan-4 (30-33 kDa) and syndecan-1 (70 kDa) suggesting the presence of rafts with various HSPG species. DRM fractions enriched with HSPGs were characterized by high sphingomyelin content and found to only partially overlap with the fractions enriched in ganglioside GM1. HSPGs could be also detected in DRMs even after prior treatment of cells with heparitinase. CONCLUSIONS/SIGNIFICANCE: Both syndecan-1 and syndecan-4 have been found to specifically associate with membrane rafts and their association seemed independent of intact HS chains. Membrane rafts in which HSPGs reside were also enriched with sphingomyelin, suggesting their possible involvement in FGF signaling. Further studies, involving proteomic characterization of membrane domains containing HSPGs might improve our knowledge on the nature of HSPG-ligand interactions and their role in different signaling platforms

Structural Basis for Variant-Specific Neuroligin-Binding by α-Neurexin

Neurexins (Nrxs) are presynaptic membrane proteins with a single membrane-spanning domain that mediate asymmetric trans-synaptic cell adhesion by binding to their postsynaptic receptor neuroligins. α-Nrx has a large extracellular region comprised of multiple copies of laminin, neurexin, sex-hormone-binding globulin (LNS) domains and epidermal growth factor (EGF) modules, while that of β-Nrx has but a single LNS domain. It has long been known that the larger α-Nrx and the shorter β-Nrx show distinct binding behaviors toward different isoforms/variants of neuroligins, although the underlying mechanism has yet to be elucidated. Here, we describe the crystal structure of a fragment corresponding to the C-terminal one-third of the Nrx1α ectodomain, consisting of LNS5-EGF3-LNS6. The 2.3 Å-resolution structure revealed the presence of a domain configuration that was rigidified by inter-domain contacts, as opposed to the more common flexible “beads-on-a-string” arrangement. Although the neuroligin-binding site on the LNS6 domain was completely exposed, the location of the α-Nrx specific LNS5-EGF3 segment proved incompatible with the loop segment inserted in the B+ neuroligin variant, which explains the variant-specific neuroligin recognition capability observed in α-Nrx. This, combined with a low-resolution molecular envelope obtained by a single particle reconstruction performed on negatively stained full-length Nrx1α sample, allowed us to derive a structural model of the α-Nrx ectodomain. This model will help us understand not only how the large α-Nrx ectodomain is accommodated in the synaptic cleft, but also how the trans-synaptic adhesion mediated by α- and β-Nrxs could differentially affect synaptic structure and function

Synaptic proximity enables NMDAR signalling to promote brain metastasis.

Metastasis-the disseminated growth of tumours in distant organs-underlies cancer mortality. Breast-to-brain metastasis (B2BM) is a common and disruptive form of cancer and is prevalent in the aggressive basal-like subtype, but is also found at varying frequencies in all cancer subtypes. Previous studies revealed parameters of breast cancer metastasis to the brain, but its preference for this site remains an enigma. Here we show that B2BM cells co-opt a neuronal signalling pathway that was recently implicated in invasive tumour growth, involving activation by glutamate ligands of N-methyl-D-aspartate receptors (NMDARs), which is key in model systems for metastatic colonization of the brain and is associated with poor prognosis. Whereas NMDAR activation is autocrine in some primary tumour types, human and mouse B2BM cells express receptors but secrete insufficient glutamate to induce signalling, which is instead achieved by the formation of pseudo-tripartite synapses between cancer cells and glutamatergic neurons, presenting a rationale for brain metastasis.This work was principally supported by grants from the Swiss National Science Foundation and the European Research Council, and by a gift from the Biltema Foundation that was administered by the ISREC Foundation, Lausanne, Switzerland