101 results on '"Pypaert M"'
Search Results
2. Transport through the yeast endocytic pathway occurs through morphologically distinct compartments and requires an active secretory pathway and Sec18p/N-ethylmaleimide-sensitive fusion protein.
- Author
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Hicke, L, primary, Zanolari, B, additional, Pypaert, M, additional, Rohrer, J, additional, and Riezman, H, additional
- Published
- 1997
- Full Text
- View/download PDF
3. The Golgi stack reassembles during telophase before arrival of proteins transported from the endoplasmic reticulum
- Author
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Souter, E, primary, Pypaert, M, additional, and Warren, G, additional
- Published
- 1993
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4. Mitotic Golgi clusters are not tubular endosomes
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Pypaert, M., primary, Nilsson, T., additional, Berger, E.G., additional, and Warren, G., additional
- Published
- 1993
- Full Text
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5. Overlapping distribution of two glycosyltransferases in the Golgi apparatus of HeLa cells.
- Author
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Nilsson, T, primary, Pypaert, M, additional, Hoe, M H, additional, Slusarewicz, P, additional, Berger, E G, additional, and Warren, G, additional
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- 1993
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6. Mitotic cytosol inhibits invagination of coated pits in broken mitotic cells.
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Pypaert, M, primary, Mundy, D, additional, Souter, E, additional, Labbé, J C, additional, and Warren, G, additional
- Published
- 1991
- Full Text
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7. Role for sialic acid in the formation of tight lysosome-derived vacuoles during Trypanosoma cruzi invasion
- Author
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Lopez, M., Huynh, C., Andrade, L. O., Pypaert, M., and Andrews, N. W.
- Published
- 2002
- Full Text
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8. In vitro import of proteins into mitochondria of Trypanosoma brucei and Leishmania tarentolae.
- Author
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Hauser, R, Pypaert, M, Häusler, T, Horn, E K, and Schneider, A
- Abstract
In eukaryotic evolution, the earliest branch of organisms to have mitochondria are the trypanosomatids. Their mitochondrial biogenesis not only includes import of most proteins, but also, unlike in other organisms, import of the whole set of tRNAs. In order to investigate these processes, we devised novel procedures for the isolation of mitochondria from two trypanosomatid species: Trypanosoma brucei and Leishmania tarentolae. Isotonic cell lysis followed by equilibrium density centrifugation in Nycodenz gradients yielded mitochondrial fractions exhibiting a membrane potential. Furthermore, we have used these fractions to reconstitute import of mitochondrial matrix proteins in vitro. Energy-dependent uptake of an artificial precursor protein, containing a trypanosomal presequence attached to mouse dihydrofolate reductase and of yeast mitochondrial alcohol dehydrogenase could be demonstrated. The presequences of both proteins were processed in T. brucei whereas only the trypanosomal one was cleaved in L. tarentolae. Trypsin pretreatment abolished the ability of the mitochondria to import proteins, indicating the involvement of proteinaceous components at the surface of mitochondria.
- Published
- 1996
9. Formation of coated vesicles from coated pits in broken A431 cells.
- Author
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Smythe, E, Pypaert, M, Lucocq, J, and Warren, G
- Abstract
Biochemical and morphological techniques were used to demonstrate the early steps in the endocytosis of transferrin in broken A431 cells. After binding 125I-transferrin, the cells were broken by scraping and then warmed. 125I-transferrin became inaccessible to exogenous anti-transferrin antibody providing a measure of the internalization process. Parallel morphological experiments using transferrin coupled to horseradish peroxidase confirmed internalization in broken cells. The process was characterized and compared with endocytosis in intact cells and showed many similar features. The system was used to show that both the appearance of new coated pits and the scission of coated pits to form coated vesicles were dependent on the addition of cytosol and ATP whereas invagination of pits was dependent on neither.
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- 1989
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10. AMP-activated protein kinase mediates ischemic glucose uptake and prevents postischemic cardiac dysfunction, apoptosis, and injury
- Author
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Russell Rd, R. R., Li, J., Coven, D. L., Pypaert, M., Zechner, C., Palmeri, M., Giordano, F. J., Mu, J., Morris Birnbaum, and Young, L. H.
11. Matrix protein 2 of influenza A virus blocks autophagosome fusion with lysosomes
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Gannagé, M, Dormann, D, Albrecht, R, Dengjel, J, Torossi, T, Rämer, P C, Lee, M, Strowig, T, Arrey, F, Conenello, G, Pypaert, M, Andersen, J, García-Sastre, A, and Münz, C
- Subjects
3. Good health
12. Matrix protein 2 of influenza A virus blocks autophagosome fusion with lysosomes.
- Author
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Gannagé M, Dormann D, Albrecht R, Dengjel J, Torossi T, Rämer PC, Lee M, Strowig T, Arrey F, Conenello G, Pypaert M, Andersen J, García-Sastre A, and Münz C
- Subjects
- Animals, Cell Line, Dogs, Epithelial Cells virology, Humans, Lysosomes ultrastructure, Mice, Microscopy, Confocal, Microscopy, Electron, Transmission, Phagosomes ultrastructure, Apoptosis, Autophagy, Influenza A virus pathogenicity, Lysosomes metabolism, Phagosomes metabolism, Viral Matrix Proteins physiology, Virulence Factors physiology
- Abstract
Influenza A virus is an important human pathogen causing significant morbidity and mortality every year and threatening the human population with epidemics and pandemics. Therefore, it is important to understand the biology of this virus to develop strategies to control its pathogenicity. Here, we demonstrate that influenza A virus inhibits macroautophagy, a cellular process known to be manipulated by diverse pathogens. Influenza A virus infection causes accumulation of autophagosomes by blocking their fusion with lysosomes, and one viral protein, matrix protein 2, is necessary and sufficient for this inhibition of autophagosome degradation. Macroautophagy inhibition by matrix protein 2 compromises survival of influenza virus-infected cells but does not influence viral replication. We propose that influenza A virus, which also encodes proapoptotic proteins, is able to determine the death of its host cell by inducing apoptosis and also by blocking macroautophagy.
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- 2009
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13. The bilobe structure of Trypanosoma brucei contains a MORN-repeat protein.
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Morriswood B, He CY, Sealey-Cardona M, Yelinek J, Pypaert M, and Warren G
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- Amino Acid Sequence, Animals, Gene Silencing, Genes, Essential, Genes, Protozoan, Microbial Viability, Molecular Sequence Data, RNA, Small Interfering genetics, RNA, Small Interfering metabolism, Sequence Alignment, Golgi Apparatus chemistry, Protozoan Proteins analysis, Trypanosoma brucei brucei chemistry
- Abstract
The Golgi of the kinetoplastid parasite Trypanosoma brucei is closely apposed to a bilobe structure containing TbCentrin2 and TbCentrin4 in procyclic cells. However, both are additionally localized to the basal bodies. Here we report the characterization of a membrane occupation and recognition nexus (MORN)-repeat protein, TbMORN1, present at the bilobe but not at the basal body. The anterior part of the TbMORN1 structure partially overlapped with the flagellar attachment zone while the posterior part overlapped with the flagellar pocket. Depletion studies using RNAi showed that there was a modest growth inhibition in procyclic cells but lethality in bloodstream cells, showing that it is an essential protein in the bloodstream form of the organism. TbMORN1 appears to be a useful marker for the bilobe in T. brucei.
- Published
- 2009
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14. Targeting of the GTPase Irgm1 to the phagosomal membrane via PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) promotes immunity to mycobacteria.
- Author
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Tiwari S, Choi HP, Matsuzawa T, Pypaert M, and MacMicking JD
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- Animals, Cells, Cultured, GTP-Binding Proteins genetics, Immunity, Innate, Interferon-gamma physiology, Intracellular Membranes metabolism, Lysosomes metabolism, Macrophages immunology, Macrophages metabolism, Macrophages microbiology, Mice, Mycobacterium tuberculosis immunology, Phosphatidylinositol 3-Kinases metabolism, Protein Binding, Protein Structure, Secondary, Protein Transport physiology, SNARE Proteins metabolism, Signal Transduction, GTP-Binding Proteins metabolism, Mycobacterium tuberculosis physiology, Phagosomes metabolism, Phosphatidylinositol Phosphates metabolism
- Abstract
Vertebrate immunity to infection enlists a newly identified family of 47-kilodalton immunity-related GTPases (IRGs). One IRG in particular, Irgm1, is essential for macrophage host defense against phagosomal pathogens, including Mycobacterium tuberculosis (Mtb). Here we show that Irgm1 targets the mycobacterial phagosome through lipid-mediated interactions with phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P(2)) and PtdIns(3,4,5)P(3). An isolated Irgm1 amphipathic helix conferred lipid binding in vitro and in vivo. Substitutions in this region blocked phagosome recruitment and failed to complement the antimicrobial defect in Irgm1(-/-) macrophages. Removal of PtdIns(3,4,5)P(3) or inhibition of class I phosphatidylinositol-3-OH kinase (PI(3)K) mimicked this effect in wild-type cells. Cooperation between Irgm1 and PI(3)K further facilitated the engagement of Irgm1 with its fusogenic effectors at the site of infection, thereby ensuring pathogen-directed responses during innate immunity.
- Published
- 2009
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15. CD2 distinguishes two subsets of human plasmacytoid dendritic cells with distinct phenotype and functions.
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Matsui T, Connolly JE, Michnevitz M, Chaussabel D, Yu CI, Glaser C, Tindle S, Pypaert M, Freitas H, Piqueras B, Banchereau J, and Palucka AK
- Subjects
- B7-1 Antigen analysis, Cell Proliferation, Cytotoxicity, Immunologic, Dendritic Cells immunology, Humans, Interleukin-12 Subunit p40 analysis, Neoplasms immunology, Phenotype, T-Lymphocytes cytology, T-Lymphocytes immunology, CD2 Antigens, Dendritic Cells cytology
- Abstract
Plasmacytoid dendritic cells (pDCs) are key regulators of antiviral immunity. They rapidly secrete IFN-alpha and cross-present viral Ags, thereby launching adaptive immunity. In this study, we show that activated human pDCs inhibit replication of cancer cells and kill them in a contact-dependent fashion. Expression of CD2 distinguishes two pDC subsets with distinct phenotype and function. Both subsets secrete IFN-alpha and express granzyme B and TRAIL. CD2(high) pDCs uniquely express lysozyme and can be found in tonsils and in tumors. Both subsets launch recall T cell responses. However, CD2(high) pDCs secrete higher levels of IL12p40, express higher levels of costimulatory molecule CD80, and are more efficient in triggering proliferation of naive allogeneic T cells. Thus, human blood pDCs are composed of subsets with specific phenotype and functions.
- Published
- 2009
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16. Role of an ancestral d-bifunctional protein containing two sterol-carrier protein-2 domains in lipid uptake and trafficking in Toxoplasma.
- Author
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Lige B, Jayabalasingham B, Zhang H, Pypaert M, and Coppens I
- Subjects
- 17-Hydroxysteroid Dehydrogenases genetics, 17-Hydroxysteroid Dehydrogenases metabolism, Amino Acid Sequence, Animals, Carrier Proteins chemistry, Carrier Proteins genetics, Cell Line, Cholesterol metabolism, Humans, Hydro-Lyases genetics, Hydro-Lyases metabolism, Membrane Lipids metabolism, Molecular Sequence Data, Peroxisomal Multifunctional Protein-2, Peroxisomes metabolism, Phosphatidylcholines metabolism, Protein Processing, Post-Translational, Protozoan Proteins genetics, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Sequence Alignment, Toxoplasma cytology, Zellweger Syndrome metabolism, Biological Transport physiology, Carrier Proteins metabolism, Lipid Metabolism, Protozoan Proteins metabolism, Sterols metabolism, Toxoplasma metabolism
- Abstract
The inability to synthesize cholesterol is universal among protozoa. The intracellular pathogen Toxoplasma depends on host lipoprotein-derived cholesterol to replicate in mammalian cells. Mechanisms of cholesterol trafficking in this parasite must be important for delivery to proper organelles. We characterized a unique d-bifunctional protein variant expressed by Toxoplasma consisting of one N-terminal d-3-hydroxyacyl-CoA dehydrogenase domain fused to two tandem sterol carrier protein-2 (SCP-2) domains. This multidomain protein undergoes multiple cleavage steps to release free SCP-2. The most C-terminal SCP-2 carries a PTS1 that directs the protein to vesicles before processing. Abrogation of this signal results in SCP-2 accumulation in the cytoplasm. Cholesterol specifically binds to parasite SCP-2 but with 10-fold lower affinity than phosphatidylcholine. In mammalian cells and Toxoplasma, the two parasite SCP-2 domains promote the circulation of various lipids between organelles and to the surface. Compared with wild-type parasites, TgHAD-2SCP-2-transfected parasites replicate faster and show enhanced uptake of cholesterol and oleate, which are incorporated into neutral lipids that accumulate at the basal end of Toxoplasma. This work provides the first evidence that the lipid transfer capability of an ancestral eukaryotic SCP-2 domain can influence the lipid metabolism of an intracellular pathogen to promote its multiplication in mammalian cells.
- Published
- 2009
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17. An internal domain of Exo70p is required for actin-independent localization and mediates assembly of specific exocyst components.
- Author
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Hutagalung AH, Coleman J, Pypaert M, and Novick PJ
- Subjects
- Amino Acid Sequence, Cell Membrane metabolism, Models, Molecular, Molecular Sequence Data, Mutation, Protein Structure, Tertiary, Protein Subunits chemistry, Protein Subunits genetics, Protein Subunits metabolism, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins chemistry, Saccharomyces cerevisiae Proteins genetics, Secretory Pathway physiology, Vesicular Transport Proteins, rho GTP-Binding Proteins genetics, rho GTP-Binding Proteins metabolism, Actins metabolism, Exocytosis physiology, Saccharomyces cerevisiae cytology, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism
- Abstract
The exocyst consists of eight rod-shaped subunits that align in a side-by-side manner to tether secretory vesicles to the plasma membrane in preparation for fusion. Two subunits, Sec3p and Exo70p, localize to exocytic sites by an actin-independent pathway, whereas the other six ride on vesicles along actin cables. Here, we demonstrate that three of the four domains of Exo70p are essential for growth. The remaining domain, domain C, is not essential but when deleted, it leads to synthetic lethality with many secretory mutations, defects in exocyst assembly of exocyst components Sec5p and Sec6p, and loss of actin-independent localization. This is analogous to a deletion of the amino-terminal domain of Sec3p, which prevents an interaction with Cdc42p or Rho1p and blocks its actin-independent localization. The two mutations are synthetically lethal, even in the presence of high copy number suppressors that can bypass complete deletions of either single gene. Although domain C binds Rho3p, loss of the Exo70p-Rho3p interaction does not account for the synthetic lethal interactions or the exocyst assembly defects. The results suggest that either Exo70p or Sec3p must associate with the plasma membrane for the exocyst to function as a vesicle tether.
- Published
- 2009
- Full Text
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18. Hypoxia-inducible factor-dependent degeneration, failure, and malignant transformation of the heart in the absence of the von Hippel-Lindau protein.
- Author
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Lei L, Mason S, Liu D, Huang Y, Marks C, Hickey R, Jovin IS, Pypaert M, Johnson RS, and Giordano FJ
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- Animals, Capillaries growth & development, Cell Transformation, Neoplastic genetics, Cell Transformation, Neoplastic metabolism, Cell Transformation, Neoplastic pathology, ErbB Receptors metabolism, Gene Deletion, Gene Transfer Techniques, Heart Failure metabolism, Heart Neoplasms genetics, Heart Neoplasms metabolism, Heart Neoplasms pathology, Hypoxia metabolism, Hypoxia-Inducible Factor 1, alpha Subunit metabolism, Lipid Metabolism genetics, Lipids analysis, Mice, Mice, Knockout, Myocardium chemistry, Neovascularization, Physiologic genetics, Phosphorylation, Proto-Oncogene Proteins c-met metabolism, ras Proteins metabolism, Heart Failure genetics, Heart Failure pathology, Hypoxia genetics, Hypoxia pathology, Hypoxia-Inducible Factor 1, alpha Subunit genetics, Myocardium metabolism, Myocardium pathology, Von Hippel-Lindau Tumor Suppressor Protein genetics
- Abstract
Hypoxia-inducible transcription factor 1 (HIF-1) and HIF-2alpha regulate the expression of an expansive array of genes associated with cellular responses to hypoxia. Although HIF-regulated genes mediate crucial beneficial short-term biological adaptations, we hypothesized that chronic activation of the HIF pathway in cardiac muscle, as occurs in advanced ischemic heart disease, is detrimental. We generated mice with cardiac myocyte-specific deletion of the von Hippel-Lindau protein (VHL), an essential component of an E3 ubiquitin ligase responsible for suppressing HIF levels during normoxia. These mice were born at expected frequency and thrived until after 3 months postbirth, when they developed severe progressive heart failure and premature death. VHL-null hearts developed lipid accumulation, myofibril rarefaction, altered nuclear morphology, myocyte loss, and fibrosis, features seen for various forms of human heart failure. Further, nearly 50% of VHL(-/-) hearts developed malignant cardiac tumors with features of rhabdomyosarcoma and the capacity to metastasize. As compelling evidence for the mechanistic contribution of HIF-1alpha, the concomitant deletion of VHL and HIF-1alpha in the heart prevented this phenotype and restored normal longevity. These findings strongly suggest that chronic activation of the HIF pathway in ischemic hearts is maladaptive and contributes to cardiac degeneration and progression to heart failure.
- Published
- 2008
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19. Repair of injured plasma membrane by rapid Ca2+-dependent endocytosis.
- Author
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Idone V, Tam C, Goss JW, Toomre D, Pypaert M, and Andrews NW
- Subjects
- Animals, Bacterial Toxins toxicity, Calcium metabolism, Cell Line, Cell Membrane drug effects, Cell Membrane ultrastructure, Cell Membrane Structures drug effects, Cell Membrane Structures metabolism, Cell Membrane Structures ultrastructure, Epithelial Cells drug effects, Epithelial Cells ultrastructure, HeLa Cells, Humans, Immunity, Innate physiology, Rats, Reaction Time physiology, Sterols metabolism, Streptolysins toxicity, Time Factors, Wound Healing physiology, Calcium Signaling physiology, Cell Membrane metabolism, Endocytosis physiology, Epithelial Cells metabolism, Membrane Proteins metabolism
- Abstract
Ca2+ influx through plasma membrane lesions triggers a rapid repair process that was previously shown to require the exocytosis of lysosomal organelles (Reddy, A., E. Caler, and N. Andrews. 2001. Cell. 106:157-169). However, how exocytosis leads to membrane resealing has remained obscure, particularly for stable lesions caused by pore-forming proteins. In this study, we show that Ca2+-dependent resealing after permeabilization with the bacterial toxin streptolysin O (SLO) requires endocytosis via a novel pathway that removes SLO-containing pores from the plasma membrane. We also find that endocytosis is similarly required to repair lesions formed in mechanically wounded cells. Inhibition of lesion endocytosis (by sterol depletion) inhibits repair, whereas enhancement of endocytosis through disruption of the actin cytoskeleton facilitates resealing. Thus, endocytosis promotes wound resealing by removing lesions from the plasma membrane. These findings provide an important new insight into how cells protect themselves not only from mechanical injury but also from microbial toxins and pore-forming proteins produced by the immune system.
- Published
- 2008
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20. Bcl-xL induces Drp1-dependent synapse formation in cultured hippocampal neurons.
- Author
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Li H, Chen Y, Jones AF, Sanger RH, Collis LP, Flannery R, McNay EC, Yu T, Schwarzenbacher R, Bossy B, Bossy-Wetzel E, Bennett MV, Pypaert M, Hickman JA, Smith PJ, Hardwick JM, and Jonas EA
- Subjects
- Animals, Cells, Cultured, Hippocampus cytology, Mitochondria metabolism, Rats, Synaptic Transmission, Dynamins physiology, Hippocampus metabolism, Synapses, bcl-X Protein physiology
- Abstract
Maturation of neuronal synapses is thought to involve mitochondria. Bcl-xL protein inhibits mitochondria-mediated apoptosis but may have other functions in healthy adult neurons in which Bcl-xL is abundant. Here, we report that overexpression of Bcl-xL postsynaptically increases frequency and amplitude of spontaneous miniature synaptic currents in rat hippocampal neurons in culture. Bcl-xL, overexpressed either pre or postsynaptically, increases synapse number, the number and size of synaptic vesicle clusters, and mitochondrial localization to vesicle clusters and synapses, likely accounting for the changes in miniature synaptic currents. Conversely, knockdown of Bcl-xL or inhibiting it with ABT-737 decreases these morphological parameters. The mitochondrial fission protein, dynamin-related protein 1 (Drp1), is a GTPase known to localize to synapses and affect synaptic function and structure. The effects of Bcl-xL appear mediated through Drp1 because overexpression of Drp1 increases synaptic markers, and overexpression of the dominant-negative dnDrp1-K38A decreases them. Furthermore, Bcl-xL coimmunoprecipitates with Drp1 in tissue lysates, and in a recombinant system, Bcl-xL protein stimulates GTPase activity of Drp1. These findings suggest that Bcl-xL positively regulates Drp1 to alter mitochondrial function in a manner that stimulates synapse formation.
- Published
- 2008
- Full Text
- View/download PDF
21. Par3 functions in the biogenesis of the primary cilium in polarized epithelial cells.
- Author
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Sfakianos J, Togawa A, Maday S, Hull M, Pypaert M, Cantley L, Toomre D, and Mellman I
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- Amino Acid Sequence, Animals, Binding Sites, Cell Membrane metabolism, Cell Membrane ultrastructure, Cells, Cultured, Cilia ultrastructure, Dogs, Epithelial Cells ultrastructure, Humans, Membrane Proteins chemistry, Membrane Proteins metabolism, Models, Biological, Molecular Sequence Data, PDZ Domains, Sequence Alignment, Cilia metabolism, Epithelial Cells metabolism, Membrane Proteins physiology
- Abstract
Par3 is a PDZ protein important for the formation of junctional complexes in epithelial cells. We have identified an additional role for Par3 in membrane biogenesis. Although Par3 was not required for maintaining polarized apical or basolateral membrane domains, at the apical surface, Par3 was absolutely essential for the growth and elongation of the primary cilium. The activity reflected its ability to interact with kinesin-2, the microtubule motor responsible for anterograde transport of intraflagellar transport particles to the tip of the growing cilium. The Par3 binding partners Par6 and atypical protein kinase C interacted with the ciliary membrane component Crumbs3 and we show that the PDZ binding motif of Crumbs3 was necessary for its targeting to the ciliary membrane. Thus, the Par complex likely serves as an adaptor that couples the vectorial movement of at least a subset of membrane proteins to microtubule-dependent transport during ciliogenesis.
- Published
- 2007
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22. Internalization, intracellular trafficking, and biodistribution of monoclonal antibody 806: a novel anti-epidermal growth factor receptor antibody.
- Author
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Perera RM, Zoncu R, Johns TG, Pypaert M, Lee FT, Mellman I, Old LJ, Toomre DK, and Scott AM
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- Animals, Antibodies, Monoclonal immunology, Antibody Specificity, Biological Transport, Cell Line, Tumor, Clathrin-Coated Vesicles metabolism, Endocytosis, Endosomes metabolism, Epitopes immunology, Humans, Immunoconjugates pharmacokinetics, Lysosomes metabolism, Mice, Mice, Inbred BALB C, Mice, Nude, Tissue Distribution, Transfection, Antibodies, Monoclonal pharmacokinetics, ErbB Receptors immunology, Neoplasm Proteins immunology
- Abstract
Overexpression of the epidermal growth factor receptor (EGFR) in epithelial tumors is associated with poor prognosis and is the target for a number of cancer therapeutics. Monoclonal antibody (mAb) 806 is a novel anti-EGFR antibody with significant therapeutic efficacy in tumor models when used as a single agent, and displays synergistic antitumor activity in combination with other EGFR therapeutics. Unlike other EGFR antibodies, mAb 806 is selective for tumor cells and does not bind to normal tissue, making it an ideal candidate for generation of radioisotope or toxin conjugates. Ideally, antibodies suited to these therapeutic applications must bind to and actively internalize their cognate receptor. We investigated the intracellular trafficking of fluorescently tagged mAb 806 in live cells and analyzed its biodistribution in a tumor xenografted nude mouse model. Following binding to EGFR, mAb 806 was internalized through dynamin-dependent, clathrin-mediated endocytosis. Internalized mAb 806 localized to early endosomes and subsequently trafficked to and accumulation in lysosomal compartments. Furthermore, biodistribution analysis in nude mice showed specific uptake and retention of radiolabeled mAb 806 to human tumor xenografts. These results highlight the potential use of mAb 806 for generation of conjugates suitable for diagnostic and therapeutic use in patients with EGFR-positive malignancies.
- Published
- 2007
- Full Text
- View/download PDF
23. RNAi screen in Drosophila cells reveals the involvement of the Tom complex in Chlamydia infection.
- Author
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Derré I, Pypaert M, Dautry-Varsat A, and Agaisse H
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- Animals, Chlamydia physiology, Chlamydia Infections metabolism, Drosophila, Fluorescent Antibody Technique, Guinea Pigs, HeLa Cells, Humans, Image Processing, Computer-Assisted, Inclusion Bodies microbiology, Inclusion Bodies ultrastructure, Microscopy, Electron, Transmission, Mitochondria microbiology, Mitochondria ultrastructure, Mitochondrial Precursor Protein Import Complex Proteins, Carrier Proteins metabolism, Chlamydia pathogenicity, Chlamydia Infections genetics, Host-Parasite Interactions genetics, RNA Interference
- Abstract
Chlamydia spp. are intracellular obligate bacterial pathogens that infect a wide range of host cells. Here, we show that C. caviae enters, replicates, and performs a complete developmental cycle in Drosophila SL2 cells. Using this model system, we have performed a genome-wide RNA interference screen and identified 54 factors that, when depleted, inhibit C. caviae infection. By testing the effect of each candidate's knock down on L. monocytogenes infection, we have identified 31 candidates presumably specific of C. caviae infection. We found factors expected to have an effect on Chlamydia infection, such as heparansulfate glycosaminoglycans and actin and microtubule remodeling factors. We also identified factors that were not previously described as involved in Chlamydia infection. For instance, we identified members of the Tim-Tom complex, a multiprotein complex involved in the recognition and import of nuclear-encoded proteins to the mitochondria, as required for C. caviae infection of Drosophila cells. Finally, we confirmed that depletion of either Tom40 or Tom22 also reduced C. caviae infection in mammalian cells. However, C. trachomatis infection was not affected, suggesting that the mechanism involved is C. caviae specific.
- Published
- 2007
- Full Text
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24. The transcription factor XBP-1 is essential for the development and survival of dendritic cells.
- Author
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Iwakoshi NN, Pypaert M, and Glimcher LH
- Subjects
- Animals, Cell Survival, Cells, Cultured, DNA-Binding Proteins deficiency, DNA-Binding Proteins genetics, Dendritic Cells metabolism, Endoplasmic Reticulum immunology, Endoplasmic Reticulum metabolism, Lymphocytes immunology, Lymphocytes metabolism, Mice, Mice, Knockout, Microscopy, Electron, Regulatory Factor X Transcription Factors, Sensitivity and Specificity, Transcription Factors deficiency, Transcription Factors genetics, X-Box Binding Protein 1, Cell Differentiation immunology, DNA-Binding Proteins immunology, DNA-Binding Proteins metabolism, Dendritic Cells cytology, Dendritic Cells immunology, Transcription Factors immunology, Transcription Factors metabolism
- Abstract
Dendritic cells (DCs) play a critical role in the initiation, maintenance, and resolution of an immune response. DC survival is tightly controlled by extracellular stimuli such as cytokines and Toll-like receptor (TLR) signaling, but the intracellular events that translate such extracellular stimuli into life or death for the DC remain poorly understood. The endoplasmic reticulum (ER) stress, or unfolded protein response (UPR), is a signaling pathway that is activated when unfolded proteins accumulate in the ER. The most conserved arm of the UPR involves IRE1alpha, an ER transmembrane kinase and endoribonuclease that activates the transcription factor XBP-1 to maintain ER homeostasis and prevent activation of cell death pathways caused by sustained ER stress. We report that XBP-1 is essential for DC development and survival. Lymphoid chimeras lacking XBP-1 possessed decreased numbers of both conventional and plasmacytoid DCs with reduced survival both at baseline and in response to TLR signaling. Overexpression of XBP-1 in hematopoietic progenitors rescued and enhanced DC development. Remarkably, in contrast to other cell types we have examined, the XBP-1 pathway was constitutively activated in immature DCs.
- Published
- 2007
- Full Text
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25. Expression of the voltage-gated sodium channel NaV1.5 in the macrophage late endosome regulates endosomal acidification.
- Author
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Carrithers MD, Dib-Hajj S, Carrithers LM, Tokmoulina G, Pypaert M, Jonas EA, and Waxman SG
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- Acids metabolism, Cell Membrane chemistry, Cells, Cultured, Endosomes chemistry, Endosomes drug effects, Humans, Hydrogen-Ion Concentration, Lipopolysaccharides pharmacology, Macrophages ultrastructure, NAV1.5 Voltage-Gated Sodium Channel, NAV1.6 Voltage-Gated Sodium Channel, Nerve Tissue Proteins analysis, Nerve Tissue Proteins metabolism, Phagosomes chemistry, Phagosomes metabolism, Sodium Channels analysis, Sodium Channels genetics, Sodium Channels metabolism, Tetrodotoxin pharmacology, Veratridine pharmacology, Endosomes metabolism, Macrophages immunology, Phagocytosis, Sodium Channels physiology
- Abstract
Voltage-gated sodium channels expressed on the plasma membrane activate rapidly in response to changes in membrane potential in cells with excitable membranes such as muscle and neurons. Macrophages also require rapid signaling mechanisms as the first line of defense against invasion by microorganisms. In this study, our goal was to examine the role of intracellular voltage-gated sodium channels in macrophage function. We demonstrate that the cardiac voltage-gated sodium channel, NaV1.5, is expressed on the late endosome, but not the plasma membrane, in a human monocytic cell line, THP-1, and primary human monocyte-derived macrophages. Although the neuronal channel, NaV1.6, is also expressed intracellularly, it has a distinct subcellular localization. In primed cells, NaV1.5 regulates phagocytosis and endosomal pH during LPS-mediated endosomal acidification. Activation of the endosomal channel causes sodium efflux and decreased intraendosomal pH. These results demonstrate a functionally relevant intracellular voltage-gated sodium channel and reveal a novel mechanism to regulate macrophage endosomal acidification.
- Published
- 2007
- Full Text
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26. v-SNARE cellubrevin is required for basolateral sorting of AP-1B-dependent cargo in polarized epithelial cells.
- Author
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Fields IC, Shteyn E, Pypaert M, Proux-Gillardeaux V, Kang RS, Galli T, and Fölsch H
- Subjects
- Adaptor Protein Complex 1 genetics, Adaptor Protein Complex beta Subunits genetics, Animals, Cell Line, Cell Membrane metabolism, Cell Polarity drug effects, Dogs, Epithelial Cells cytology, Humans, Membrane Fusion drug effects, Membrane Fusion physiology, Metalloendopeptidases pharmacology, Protein Transport drug effects, Protein Transport physiology, Qa-SNARE Proteins genetics, Qa-SNARE Proteins metabolism, Receptors, LDL metabolism, SNARE Proteins genetics, Tetanus Toxin pharmacology, Vesicle-Associated Membrane Protein 3 genetics, Adaptor Protein Complex 1 metabolism, Adaptor Protein Complex beta Subunits metabolism, Cell Polarity physiology, Endosomes metabolism, Epithelial Cells metabolism, SNARE Proteins metabolism, Vesicle-Associated Membrane Protein 3 metabolism
- Abstract
The epithelial cell-specific adaptor complex AP-1B is crucial for correct delivery of many transmembrane proteins from recycling endosomes to the basolateral plasma membrane. Subsequently, membrane fusion is dependent on the formation of complexes between SNARE proteins located at the target membrane and on transport vesicles. Although the t-SNARE syntaxin 4 has been localized to the basolateral membrane, the v-SNARE operative in the AP-1B pathway remained unknown. We show that the ubiquitously expressed v-SNARE cellubrevin localizes to the basolateral membrane and to recycling endosomes, where it colocalizes with AP-1B. Furthermore, we demonstrate that cellubrevin coimmunoprecipitates preferentially with syntaxin 4, implicating this v-SNARE in basolateral fusion events. Cleavage of cellubrevin with tetanus neurotoxin (TeNT) results in scattering of AP-1B localization and missorting of AP-1B-dependent cargos, such as transferrin receptor and a truncated low-density lipoprotein receptor, LDLR-CT27. These data suggest that cellubrevin and AP-1B cooperate in basolateral membrane trafficking.
- Published
- 2007
- Full Text
- View/download PDF
27. Retroviruses can establish filopodial bridges for efficient cell-to-cell transmission.
- Author
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Sherer NM, Lehmann MJ, Jimenez-Soto LF, Horensavitz C, Pypaert M, and Mothes W
- Subjects
- Animals, Avian Proteins genetics, Avian Proteins metabolism, CD4 Antigens genetics, CD4 Antigens metabolism, COS Cells, Cell Line, Cell Line, Tumor, Chlorocebus aethiops, Endocytosis physiology, Eukaryotic Cells metabolism, HIV-1 physiology, Humans, Luminescent Proteins genetics, Luminescent Proteins metabolism, Microscopy, Electron, Scanning, Microscopy, Fluorescence, Mutation, Pseudopodia ultrastructure, Receptors, CXCR4 genetics, Receptors, CXCR4 metabolism, Receptors, Virus genetics, Receptors, Virus metabolism, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, TRPV Cation Channels genetics, TRPV Cation Channels metabolism, Viral Envelope Proteins genetics, Viral Envelope Proteins metabolism, Cell Communication physiology, Eukaryotic Cells virology, Pseudopodia virology, Retroviridae physiology
- Abstract
The spread of retroviruses between cells is estimated to be 2-3 orders of magnitude more efficient when cells can physically interact with each other. The underlying mechanism is largely unknown, but transfer is believed to occur through large-surface interfaces, called virological or infectious synapses. Here, we report the direct visualization of cell-to-cell transmission of retroviruses in living cells. Our results reveal a mechanism of virus transport from infected to non-infected cells, involving thin filopodial bridges. These filopodia originate from non-infected cells and interact, through their tips, with infected cells. A strong association of the viral envelope glycoprotein (Env) in an infected cell with the receptor molecules in a target cell generates a stable bridge. Viruses then move along the outer surface of the filopodial bridge toward the target cell. Our data suggest that retroviruses spread by exploiting an inherent ability of filopodia to transport ligands from cell to cell.
- Published
- 2007
- Full Text
- View/download PDF
28. Excitatory local circuits and their implications for olfactory processing in the fly antennal lobe.
- Author
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Shang Y, Claridge-Chang A, Sjulson L, Pypaert M, and Miesenböck G
- Subjects
- Acetylcholine physiology, Animals, Brain physiology, Drosophila Proteins genetics, Drosophila Proteins physiology, Nerve Net physiology, Olfactory Receptor Neurons physiology, Receptors, Odorant physiology, Synapses physiology, Drosophila physiology, Neurons, Afferent physiology, Odorants, Smell
- Abstract
Conflicting views exist of how circuits of the antennal lobe, the insect equivalent of the olfactory bulb, translate input from olfactory receptor neurons (ORNs) into projection-neuron (PN) output. Synaptic connections between ORNs and PNs are one-to-one, yet PNs are more broadly tuned to odors than ORNs. The basis for this difference in receptive range remains unknown. Analyzing a Drosophila mutant lacking ORN input to one glomerulus, we show that some of the apparent complexity in the antennal lobe's output arises from lateral, interglomerular excitation of PNs. We describe a previously unidentified population of cholinergic local neurons (LNs) with multiglomerular processes. These excitatory LNs respond broadly to odors but exhibit little glomerular specificity in their synaptic output, suggesting that PNs are driven by a combination of glomerulus-specific ORN afferents and diffuse LN excitation. Lateral excitation may boost PN signals and enhance their transmission to third-order neurons in a mechanism akin to stochastic resonance.
- Published
- 2007
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29. Aging-associated reductions in AMP-activated protein kinase activity and mitochondrial biogenesis.
- Author
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Reznick RM, Zong H, Li J, Morino K, Moore IK, Yu HJ, Liu ZX, Dong J, Mustard KJ, Hawley SA, Befroy D, Pypaert M, Hardie DG, Young LH, and Shulman GI
- Subjects
- AMP-Activated Protein Kinase Kinases, AMP-Activated Protein Kinases, Aminoimidazole Carboxamide analogs & derivatives, Aminoimidazole Carboxamide pharmacology, Animals, Guanidines administration & dosage, Guanidines pharmacology, Male, Mitochondria drug effects, Physical Conditioning, Animal, Propionates administration & dosage, Propionates pharmacology, Rats, Rats, Inbred F344, Ribonucleotides pharmacology, Aging, Mitochondria enzymology, Multienzyme Complexes metabolism, Protein Serine-Threonine Kinases metabolism
- Abstract
Recent studies have demonstrated a strong relationship between aging-associated reductions in mitochondrial function, dysregulated intracellular lipid metabolism, and insulin resistance. Given the important role of the AMP-activated protein kinase (AMPK) in the regulation of fat oxidation and mitochondrial biogenesis, we examined AMPK activity in young and old rats and found that acute stimulation of AMPK-alpha(2) activity by 5'-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) and exercise was blunted in skeletal muscle of old rats. Furthermore, mitochondrial biogenesis in response to chronic activation of AMPK with beta-guanidinopropionic acid (beta-GPA) feeding was also diminished in old rats. These results suggest that aging-associated reductions in AMPK activity may be an important contributing factor in the reduced mitochondrial function and dysregulated intracellular lipid metabolism associated with aging.
- Published
- 2007
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- View/download PDF
30. The tetraspanin CD9 mediates lateral association of MHC class II molecules on the dendritic cell surface.
- Author
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Unternaehrer JJ, Chow A, Pypaert M, Inaba K, and Mellman I
- Subjects
- Animals, Antigen Presentation, Antigens, CD chemistry, B-Lymphocytes immunology, B7-1 Antigen analysis, B7-2 Antigen analysis, Histocompatibility Antigens Class II analysis, Membrane Glycoproteins chemistry, Mice, Mice, Inbred C57BL, Tetraspanin 29, Antigens, CD physiology, Dendritic Cells immunology, Histocompatibility Antigens Class II chemistry, Membrane Glycoproteins physiology
- Abstract
We have found that MHC class II (MHC II) molecules exhibit a distinctive organization on the dendritic cell (DC) plasma membrane. Both in DC lysates and on the surface of living cells, I-A and I-E molecules engaged in lateral interactions not observed on other antigen-presenting cells such as B blasts. Because DCs and B blasts express MHC II at comparable surface densities, the interaction was not due to simple mass action. Instead, it reflected the selective expression of the tetraspanin CD9 at the DC surface. I-A and I-E molecules coprecipitated with each other and with CD9. The association of heterologous MHC II molecules was abrogated in DCs from CD9(-/-) mice. Conversely, expression of exogenous CD9 in B cells induced MHC II interactions. CD9 is thus necessary for the association of heterologous MHC II, a specialization that would facilitate the formation of MHC II multimers expected to enhance T cell receptor stimulation by DCs.
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- 2007
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31. Antigen-loading compartments for major histocompatibility complex class II molecules continuously receive input from autophagosomes.
- Author
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Schmid D, Pypaert M, and Münz C
- Subjects
- B-Lymphocytes immunology, B-Lymphocytes metabolism, CD4-Positive T-Lymphocytes immunology, CD4-Positive T-Lymphocytes metabolism, Cell Line, Dendritic Cells immunology, Dendritic Cells metabolism, Enzyme-Linked Immunosorbent Assay, Epithelial Cells immunology, Epithelial Cells metabolism, Histocompatibility Antigens Class II metabolism, Humans, Immunohistochemistry, Microscopy, Confocal, Microscopy, Electron, Transmission, Phagosomes metabolism, RNA, Small Interfering, Reverse Transcriptase Polymerase Chain Reaction, Transfection, Antigen Presentation immunology, Autophagy immunology, Histocompatibility Antigens Class II immunology, Lymphocyte Activation immunology, Phagosomes immunology
- Abstract
Major histocompatibility complex (MHC) class II molecules present products of lysosomal proteolysis to CD4(+) T cells. Although extracellular antigen uptake is considered to be the main source of MHC class II ligands, a few intracellular antigens have been described to gain access to MHC class II loading after macroautophagy. However, the general relevance and efficacy of this pathway is unknown. Here we demonstrated constitutive autophagosome formation in MHC class II-positive cells, including dendritic, B, and epithelial cells. The autophagosomes continuously fuse with multivesicular MHC class II-loading compartments. This pathway was of functional relevance, because targeting of the influenza matrix protein 1 to autophagosomes via fusion to the autophagosome-associated protein Atg8/LC3 led to strongly enhanced MHC class II presentation to CD4(+) T cell clones. We suggest that macroautophagy constitutively and efficiently delivers cytosolic proteins for MHC class II presentation and can be harnessed for improved helper T cell stimulation.
- Published
- 2007
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32. Surface expression of MHC class II in dendritic cells is controlled by regulated ubiquitination.
- Author
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Shin JS, Ebersold M, Pypaert M, Delamarre L, Hartley A, and Mellman I
- Subjects
- Amino Acid Sequence, Animals, Antigens, Differentiation, B-Lymphocyte metabolism, Cell Differentiation, Dendritic Cells cytology, Endocytosis, Lysosomes metabolism, Mice, Mice, Inbred C57BL, Molecular Sequence Data, Ubiquitin deficiency, Ubiquitin genetics, Cell Membrane metabolism, Dendritic Cells metabolism, Gene Expression Regulation, Histocompatibility Antigens Class II metabolism, Ubiquitin metabolism
- Abstract
Dendritic cells have a unique function in the immune response owing to their ability to stimulate immunologically naive T lymphocytes. In response to microbial and inflammatory stimuli, dendritic cells enhance their capacity for antigen presentation by a process of terminal differentiation, termed maturation. The conversion of immature to mature dendritic cells is accompanied by a marked cellular reorganization, including the redistribution of major histocompatibility complex class II molecules (MHC II) from late endosomal and lysosomal compartments to the plasma membrane and the downregulation of some forms of endocytosis, which has been thought to slow the clearance of MHC II from the surface. The relative extent to which these or other mechanisms contribute to the regulation of surface MHC II remains unclear, however. Here we find that the MHC II beta-chain cytoplasmic tail is ubiquitinated in mouse immature dendritic cells. Although only partly required for the sequestration of MHC II in multivesicular bodies, this modification is essential for endocytosis. Notably, ubiquitination of MHC II ceased upon maturation, resulting in the accumulation of MHC II at the cell surface. Dendritic cells thus exhibit a unique ability to regulate MHC II surface expression by selectively controlling MHC II ubiquitination.
- Published
- 2006
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33. Global aggregation of newly translated proteins in an Escherichia coli strain deficient of the chaperonin GroEL.
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Chapman E, Farr GW, Usaite R, Furtak K, Fenton WA, Chaudhuri TK, Hondorp ER, Matthews RG, Wolf SG, Yates JR, Pypaert M, and Horwich AL
- Subjects
- Chaperonin 60 genetics, Escherichia coli isolation & purification, Escherichia coli Proteins metabolism, Inclusion Bodies metabolism, Methyltransferases metabolism, Mutation genetics, Phenotype, Proteomics, Solubility, Substrate Specificity, Temperature, Time Factors, Transcription, Genetic genetics, Chaperonin 60 metabolism, Escherichia coli genetics, Escherichia coli metabolism, Protein Biosynthesis genetics
- Abstract
In a newly isolated temperature-sensitive lethal Escherichia coli mutant affecting the chaperonin GroEL, we observed wholesale aggregation of newly translated proteins. After temperature shift, transcription, translation, and growth slowed over two to three generations, accompanied by filamentation and accretion (in approximately 2% of cells) of paracrystalline arrays containing mutant chaperonin complex. A biochemically isolated inclusion body fraction contained the collective of abundant proteins of the bacterial cytoplasm as determined by SDS/PAGE and proteolysis/MS analyses. Pulse-chase experiments revealed that newly made proteins, but not preexistent ones, were recruited to this insoluble fraction. Although aggregation of "stringent" GroEL/GroES-dependent substrates may secondarily produce an "avalanche" of aggregation, the observations raise the possibility, supported by in vitro refolding experiments, that the widespread aggregation reflects that GroEL function supports the proper folding of a majority of newly translated polypeptides, not just the limited number indicated by interaction studies and in vitro experiments.
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- 2006
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34. Modulation of cell adhesion and motility in the immune system by Myo1f.
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Kim SV, Mehal WZ, Dong X, Heinrich V, Pypaert M, Mellman I, Dembo M, Mooseker MS, Wu D, and Flavell RA
- Subjects
- Actins metabolism, Animals, CD18 Antigens metabolism, Cell Degranulation, Chemotaxis, Leukocyte, Colony Count, Microbial, Cytoplasmic Granules metabolism, Exocytosis, Ligands, Listeria monocytogenes growth & development, Listeriosis microbiology, Mice, Mice, Knockout, Myosin Type I deficiency, Myosin Type I genetics, N-Formylmethionine Leucyl-Phenylalanine, Neutrophil Activation, Neutrophils immunology, Cell Adhesion, Cell Movement, Immunity, Innate, Listeriosis immunology, Myosin Type I physiology, Neutrophils physiology
- Abstract
Although class I myosins are known to play a wide range of roles, the physiological function of long-tailed class I myosins in vertebrates remains elusive. We demonstrated that one of these proteins, Myo1f, is expressed predominantly in the mammalian immune system. Cells from Myo1f-deficient mice exhibited abnormally increased adhesion and reduced motility, resulting from augmented exocytosis of beta2 integrin-containing granules. Also, the cortical actin that co-localizes with Myo1f was reduced in Myo1f-deficient cells. In vivo, Myo1f-deficient mice showed increased susceptibility to infection by Listeria monocytogenes and an impaired neutrophil response. Thus, Myo1f directs immune cell motility and innate host defense against infection.
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- 2006
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35. Ca2+ and synaptotagmin VII-dependent delivery of lysosomal membrane to nascent phagosomes.
- Author
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Czibener C, Sherer NM, Becker SM, Pypaert M, Hui E, Chapman ER, Mothes W, and Andrews NW
- Subjects
- Animals, Cell Membrane chemistry, Cell Membrane metabolism, Cells, Cultured, Lysosomal-Associated Membrane Protein 1 metabolism, Lysosomes metabolism, Mice, Mice, Knockout, Synaptotagmins genetics, Calcium metabolism, Lysosomal Membrane Proteins metabolism, Macrophages metabolism, Phagocytosis, Phagosomes metabolism, Synaptotagmins metabolism
- Abstract
Synaptotagmin (Syt) VII is a ubiquitously expressed member of the Syt family of Ca2+ sensors. It is present on lysosomes in several cell types, where it regulates Ca2+-dependent exocytosis. Because [Ca2+]i and exocytosis have been associated with phagocytosis, we investigated the phagocytic ability of macrophages from Syt VII-/- mice. Syt VII-/- macrophages phagocytose normally at low particle/cell ratios but show a progressive inhibition in particle uptake under high load conditions. Complementation with Syt VII rescues this phenotype, but only when functional Ca2+-binding sites are retained. Reinforcing a role for Syt VII in Ca2+-dependent phagocytosis, particle uptake in Syt VII-/- macrophages is significantly less dependent on [Ca2+]i. Syt VII is concentrated on peripheral domains of lysosomal compartments, from where it is recruited to nascent phagosomes. Syt VII recruitment is rapidly followed by the delivery of Lamp1 to phagosomes, a process that is inhibited in Syt VII-/- macrophages. Thus, Syt VII regulates the Ca2+-dependent mobilization of lysosomes as a supplemental source of membrane during phagocytosis.
- Published
- 2006
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- View/download PDF
36. Radixin is required to maintain apical canalicular membrane structure and function in rat hepatocytes.
- Author
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Wang W, Soroka CJ, Mennone A, Rahner C, Harry K, Pypaert M, and Boyer JL
- Subjects
- Animals, Cell Membrane drug effects, Cell Membrane ultrastructure, Cells, Cultured, Fluoresceins pharmacokinetics, Fluorescent Dyes pharmacokinetics, Hepatocytes drug effects, Hepatocytes ultrastructure, Immunoblotting, In Vitro Techniques, Microscopy, Electron, Rats, Cell Membrane metabolism, Cytoskeletal Proteins genetics, Gene Expression, Hepatocytes metabolism, Membrane Proteins genetics, RNA, Small Interfering genetics
- Abstract
Background & Aims: Ezrin-radixin-moesin proteins are cross-linkers between the plasma membrane and actin filaments. Radixin, the dominant ezrin-radixin-moesin protein in hepatocytes, has been reported to selectively tether multidrug-resistance-associated protein 2 to the apical canalicular membrane. However, it remains to be determined if this is its primary function., Methods: An adenovirus-mediated short interfering RNA (siRNA) was used to down-regulate radixin expression in collagen sandwich-cultured rat hepatocytes and morphologic and functional changes were characterized quantitatively., Results: In control cultures, an extensive bile canalicular network developed with properly localized apical and basolateral transporters that provided for functional excretion of fluorescent cholephiles into the bile canalicular lumina. siRNA-induced suppression of radixin was associated with a marked reduction in the canalicular membrane structure as observed by differential interference contrast microscopy and F-actin staining, in contrast to control cells exposed to adenovirus encoding scrambled siRNA. Indirect immunofluorescence showed that apical transporters (multidrug-resistance-associated protein 2, bile salt export pump, and multidrug-resistance protein 1) dissociated from their normal location at the apical membrane and were found largely associated with Rab11-containing endosomes. Localization of the basolateral membrane transporter, organic anion transporting polypeptide 2 (Oatp2), was not affected. Consistent with this dislocation of apical transporters, the biliary excretion of glutathione-methylfluorescein and cholylglycylamido-fluorescein was decreased significantly in the radixin-deficient cells, but not in the control siRNA cells., Conclusions: Radixin is essential for maintaining the polarized targeting and/or retaining of canalicular membrane transporters and is a critical determinant of the overall structure and function of the apical membrane of hepatocytes.
- Published
- 2006
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37. mBet3p is required for homotypic COPII vesicle tethering in mammalian cells.
- Author
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Yu S, Satoh A, Pypaert M, Mullen K, Hay JC, and Ferro-Novick S
- Subjects
- Animals, Brefeldin A pharmacology, COS Cells, Carrier Proteins metabolism, Chlorocebus aethiops, Endoplasmic Reticulum ultrastructure, Golgi Apparatus ultrastructure, HeLa Cells, Humans, Protein Transport drug effects, COP-Coated Vesicles metabolism, Vesicular Transport Proteins metabolism
- Abstract
TRAPPI is a large complex that mediates the tethering of COPII vesicles to the Golgi (heterotypic tethering) in the yeast Saccharomyces cerevisiae. In mammalian cells, COPII vesicles derived from the transitional endoplasmic reticulum (tER) do not tether directly to the Golgi, instead, they appear to tether to each other (homotypic tethering) to form vesicular tubular clusters (VTCs). We show that mammalian Bet3p (mBet3p), which is the most highly conserved TRAPP subunit, resides on the tER and adjacent VTCs. The inactivation of mBet3p results in the accumulation of cargo in membranes that colocalize with the COPII coat. Furthermore, using an assay that reconstitutes VTC biogenesis in vitro, we demonstrate that mBet3p is required for the tethering and fusion of COPII vesicles to each other. Consistent with the proposal that mBet3p is required for VTC biogenesis, we find that ERGIC-53 (VTC marker) and Golgi architecture are disrupted in siRNA-treated mBet3p-depleted cells. These findings imply that the TRAPPI complex is essential for VTC biogenesis.
- Published
- 2006
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38. Rtn1p is involved in structuring the cortical endoplasmic reticulum.
- Author
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De Craene JO, Coleman J, Estrada de Martin P, Pypaert M, Anderson S, Yates JR 3rd, Ferro-Novick S, and Novick P
- Subjects
- Endoplasmic Reticulum metabolism, Gene Deletion, Intracellular Membranes metabolism, Membrane Proteins analysis, Membrane Proteins genetics, Membrane Transport Proteins, Organelles metabolism, Organelles ultrastructure, Proteomics, SEC Translocation Channels, Saccharomyces cerevisiae chemistry, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins analysis, Saccharomyces cerevisiae Proteins genetics, Secretory Vesicles metabolism, Endoplasmic Reticulum ultrastructure, Membrane Proteins metabolism, Saccharomyces cerevisiae ultrastructure, Saccharomyces cerevisiae Proteins metabolism
- Abstract
The endoplasmic reticulum (ER) contains both cisternal and reticular elements in one contiguous structure. We identified rtn1Delta in a systematic screen for yeast mutants with altered ER morphology. The ER in rtn1Delta cells is predominantly cisternal rather than reticular, yet the net surface area of ER is not significantly changed. Rtn1-green fluorescent protein (GFP) associates with the reticular ER at the cell cortex and with the tubules that connect the cortical ER to the nuclear envelope, but not with the nuclear envelope itself. Rtn1p overexpression also results in an altered ER structure. Rtn proteins are found on the ER in a wide range of eukaryotes and are defined by two membrane-spanning domains flanking a conserved hydrophilic loop. Our results suggest that Rtn proteins may direct the formation of reticulated ER. We independently identified Rtn1p in a proteomic screen for proteins associated with the exocyst vesicle tethering complex. The conserved hydophilic loop of Rtn1p binds to the exocyst subunit Sec6p. Overexpression of this loop results in a modest accumulation of secretory vesicles, suggesting impaired exocyst function. The interaction of Rtn1p with the exocyst at the bud tip may trigger the formation of a cortical ER network in yeast buds.
- Published
- 2006
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39. Toxoplasma gondii sequesters lysosomes from mammalian hosts in the vacuolar space.
- Author
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Coppens I, Dunn JD, Romano JD, Pypaert M, Zhang H, Boothroyd JC, and Joiner KA
- Subjects
- Animals, Antigens, Protozoan metabolism, Cell Line, Cell Membrane metabolism, Chlorocebus aethiops, Endosomes metabolism, Endosomes ultrastructure, Lipids, Liposomes metabolism, Lysosomes ultrastructure, Microtubules metabolism, Protozoan Proteins metabolism, Toxoplasma cytology, Vacuoles ultrastructure, Lysosomes metabolism, Toxoplasma metabolism, Vacuoles metabolism
- Abstract
The intracellular compartment harboring Toxoplasma gondii satisfies the parasite's nutritional needs for rapid growth in mammalian cells. We demonstrate that the parasitophorous vacuole (PV) of T. gondii accumulates material coming from the host mammalian cell via the exploitation of the host endo-lysosomal system. The parasite actively recruits host microtubules, resulting in selective attraction of endo-lysosomes to the PV. Microtubule-based invaginations of the PV membrane serve as conduits for the delivery of host endo-lysosomes within the PV. These tubular conduits are decorated by a parasite coat, including the tubulogenic protein GRA7, which acts like a garrote that sequesters host endocytic organelles in the vacuolar space. These data define an unanticipated process allowing the parasite intimate and concentrated access to a diverse range of low molecular weight components produced by the endo-lysosomal system. More generally, they identify a unique mechanism for unidirectional transport and sequestration of host organelles.
- Published
- 2006
- Full Text
- View/download PDF
40. CHMP5 is essential for late endosome function and down-regulation of receptor signaling during mouse embryogenesis.
- Author
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Shim JH, Xiao C, Hayden MS, Lee KY, Trombetta ES, Pypaert M, Nara A, Yoshimori T, Wilm B, Erdjument-Bromage H, Tempst P, Hogan BL, Mellman I, and Ghosh S
- Subjects
- Activin Receptors, Type I genetics, Activin Receptors, Type I metabolism, Amino Acid Sequence, Animals, Carrier Proteins genetics, Cell Line, Cells, Cultured, Down-Regulation, Embryo, Mammalian metabolism, Embryonic Development genetics, Endocytosis genetics, Endocytosis physiology, Endosomal Sorting Complexes Required for Transport, Gene Expression Regulation, Developmental genetics, Histocompatibility Antigens Class II metabolism, Horseradish Peroxidase metabolism, Intracellular Signaling Peptides and Proteins metabolism, Lysosomes metabolism, Mice, Mice, Inbred C57BL, Mice, Knockout, Mice, Transgenic, Molecular Sequence Data, NIH 3T3 Cells, Phenotype, Phosphorylation, Protein Serine-Threonine Kinases, RNA, Small Interfering genetics, Receptor, Transforming Growth Factor-beta Type I, Receptor, Transforming Growth Factor-beta Type II, Receptors, Transforming Growth Factor beta genetics, Receptors, Transforming Growth Factor beta metabolism, Sequence Homology, Amino Acid, Signal Transduction genetics, Stem Cells metabolism, Transfection, Carrier Proteins physiology, Embryonic Development physiology, Endosomes physiology, Signal Transduction physiology
- Abstract
Charged MVB protein 5 (CHMP5) is a coiled coil protein homologous to the yeast Vps60/Mos10 gene and other ESCRT-III complex members, although its precise function in either yeast or mammalian cells is unknown. We deleted the CHMP5 gene in mice, resulting in a phenotype of early embryonic lethality, reflecting defective late endosome function and dysregulation of signal transduction. Chmp5-/- cells exhibit enlarged late endosomal compartments that contain abundant internal vesicles expressing proteins that are characteristic of late endosomes and lysosomes. This is in contrast to ESCRT-III mutants in yeast, which are defective in multivesicular body (MVB) formation. The degradative capacity of Chmp5-/- cells was reduced, and undigested proteins from multiple pathways accumulated in enlarged MVBs that failed to traffic their cargo to lysosomes. Therefore, CHMP5 regulates late endosome function downstream of MVB formation, and the loss of CHMP5 enhances signal transduction by inhibiting lysosomal degradation of activated receptors.
- Published
- 2006
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41. Mutants in trs120 disrupt traffic from the early endosome to the late Golgi.
- Author
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Cai H, Zhang Y, Pypaert M, Walker L, and Ferro-Novick S
- Subjects
- Coat Protein Complex I metabolism, Endosomes genetics, Endosomes ultrastructure, Golgi Apparatus genetics, Golgi Apparatus ultrastructure, Guanine Nucleotide Exchange Factors metabolism, Membrane Proteins genetics, Microscopy, Electron, Transmission, Mutation, Protein Subunits genetics, Protein Subunits physiology, Protein Transport, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae ultrastructure, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins physiology, Vesicular Transport Proteins genetics, Endosomes metabolism, Golgi Apparatus metabolism, Membrane Proteins physiology, Saccharomyces cerevisiae metabolism, Vesicular Transport Proteins physiology
- Abstract
Transport protein particle (TRAPP), a large complex that mediates membrane traffic, is found in two forms (TRAPPI and -II). Both complexes share seven subunits, whereas three subunits (Trs130p, -120p, and -65p) are specific to TRAPPII. Previous studies have shown that mutations in the TRAPPII-specific gene trs130 block traffic through or from the Golgi. Surprisingly, we report that mutations in trs120 do not block general secretion. Instead, trs120 mutants accumulate aberrant membrane structures that resemble Berkeley bodies and disrupt the traffic of proteins that recycle through the early endosome. Mutants defective in recycling also display a defect in the localization of coat protein I (COPI) subunits, implying that Trs120p may participate in a COPI-dependent trafficking step on the early endosomal pathway. Furthermore, we demonstrate that Trs120p largely colocalizes with the late Golgi marker Sec7p. Our findings imply that Trs120p is required for vesicle traffic from the early endosome to the late Golgi.
- Published
- 2005
- Full Text
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42. Peculiarities of host cholesterol transport to the unique intracellular vacuole containing Toxoplasma.
- Author
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Sehgal A, Bettiol S, Pypaert M, Wenk MR, Kaasch A, Blader IJ, Joiner KA, and Coppens I
- Subjects
- Animals, Carrier Proteins physiology, Caveolin 1 physiology, Cell Membrane metabolism, Chlorocebus aethiops, Cholesterol Esters metabolism, Cholesterol, LDL metabolism, Gene Expression Profiling, Toxoplasma, Vero Cells, Cholesterol metabolism, Toxoplasmosis metabolism, Vacuoles metabolism, Vacuoles parasitology
- Abstract
The intracellular protozoan Toxoplasma gondii is auxotrophic for low-density lipoprotein (LDL)-derived cholesterol (C). We previously showed that T. gondii scavenges this essential lipid from host endolysosomal compartments and that C delivery to the parasitophorous vacuole (PV) does not require transit through host Golgi or endoplasmic reticulum. In this study, we explore the itinerary of C from the host endolysosomes to the PV. Labeled C incorporated into LDL is rapidly detected in intravacuolar parasites and partially esterified by the parasites. In contrast to diverse mammalian organelles, the post-endolysosomal transfer of C to the PV does not involve the host plasma membrane as an intermediate. Nevertheless, the PV membrane is accessible to extracellular sterol acceptors, suggesting C trafficking from intracellular parasites to host plasma membrane. C movement to the PV requires temperatures permissive for vesicular transport, metabolic energy and functional microtubules. Host caveolae vesicles and the sterol carrier protein-2 do not participate in this process. Proteolytic treatment of purified PV or free parasites abolishes C acquisition by the parasites. Altogether, these results support a vesicular transport system from host endolysosomes to the PV, and a requirement for PV membrane and parasite plasma membrane proteins in C delivery to T. gondii.
- Published
- 2005
- Full Text
- View/download PDF
43. Reduced mitochondrial density and increased IRS-1 serine phosphorylation in muscle of insulin-resistant offspring of type 2 diabetic parents.
- Author
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Morino K, Petersen KF, Dufour S, Befroy D, Frattini J, Shatzkes N, Neschen S, White MF, Bilz S, Sono S, Pypaert M, and Shulman GI
- Subjects
- Biopsy, Blood Glucose metabolism, Blotting, Western, Body Mass Index, Body Weight, DNA, Mitochondrial metabolism, Family Health, Female, Gene Expression Regulation, Glucose Clamp Technique, Glucose Tolerance Test, Humans, Hyperinsulinism, Immunoprecipitation, Insulin metabolism, Insulin Receptor Substrate Proteins, Lipids chemistry, Male, Microscopy, Electron, Microscopy, Electron, Transmission, Mitochondria metabolism, Muscles pathology, Phosphorylation, Protein Serine-Threonine Kinases metabolism, RNA, Messenger metabolism, Reverse Transcriptase Polymerase Chain Reaction, Signal Transduction, Time Factors, Transcription, Genetic, Triglycerides metabolism, Diabetes Mellitus, Type 2 genetics, Diabetes Mellitus, Type 2 metabolism, Insulin Resistance, Mitochondria pathology, Phosphoproteins metabolism, Serine chemistry
- Abstract
To further explore the nature of the mitochondrial dysfunction and insulin resistance that occur in the muscle of young, lean, normoglycemic, insulin-resistant offspring of parents with type 2 diabetes (IR offspring), we measured mitochondrial content by electron microscopy and insulin signaling in muscle biopsy samples obtained from these individuals before and during a hyperinsulinemic-euglycemic clamp. The rate of insulin-stimulated muscle glucose uptake was approximately 60% lower in the IR offspring than the control subjects and was associated with an approximately 60% increase in the intramyocellular lipid content as assessed by H magnetic resonance spectroscopy. Muscle mitochondrial density was 38% lower in the IR offspring. These changes were associated with a 50% increase in IRS-1 Ser312 and IRS-1 Ser636 phosphorylation and an approximately 60% reduction in insulin-stimulated Akt activation in the IR offspring. These data provide new insights into the earliest defects that may be responsible for the development of type 2 diabetes and support the hypothesis that reductions in mitochondrial content result in decreased mitochondrial function, which predisposes IR offspring to intramyocellular lipid accumulation, which in turn activates a serine kinase cascade that leads to defects in insulin signaling and action in muscle.
- Published
- 2005
- Full Text
- View/download PDF
44. Golgi duplication in Trypanosoma brucei requires Centrin2.
- Author
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He CY, Pypaert M, and Warren G
- Subjects
- Animals, Calcium-Binding Proteins physiology, Golgi Apparatus physiology, Protozoan Proteins physiology, Trypanosoma brucei brucei physiology
- Abstract
Centrins are highly conserved components of the centrosome, which in the parasitic protozoan T. brucei comprises the basal body and nucleates the flagellum used for locomotion. Here, we found TbCentrin2 in an additional bi-lobed structure near to the Golgi apparatus. One lobe was associated with the old Golgi, and the other became associated with the newly forming Golgi as the cell grew. Depletion of TbCentrin1 inhibited duplication of the basal body, whereas depletion of TbCentrin2 also inhibited duplication of the Golgi. Thus, a Centrin2-containing structure distinct from the basal body appears to mark the site for new Golgi assembly.
- Published
- 2005
- Full Text
- View/download PDF
45. Quantitative and dynamic assessment of the contribution of the ER to phagosome formation.
- Author
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Touret N, Paroutis P, Terebiznik M, Harrison RE, Trombetta S, Pypaert M, Chow A, Jiang A, Shaw J, Yip C, Moore HP, van der Wel N, Houben D, Peters PJ, de Chastellier C, Mellman I, and Grinstein S
- Subjects
- Animals, Cell Differentiation physiology, Cell Line, Cell Membrane ultrastructure, Dendritic Cells metabolism, Dendritic Cells ultrastructure, Endoplasmic Reticulum ultrastructure, Endosomes metabolism, Endosomes ultrastructure, Lysosomes metabolism, Lysosomes ultrastructure, Macrophages metabolism, Macrophages ultrastructure, Membrane Fusion physiology, Mice, Microscopy, Electron, Transmission, Phagosomes ultrastructure, Cell Membrane metabolism, Endoplasmic Reticulum metabolism, Phagocytosis physiology, Phagosomes metabolism
- Abstract
Phagosomes were traditionally thought to originate from an invagination and scission of the plasma membrane to form a distinct intracellular vacuole. An alternative model implicating the endoplasmic reticulum (ER) as a major component of nascent and maturing phagosomes was recently proposed (Gagnon et al., 2002). To reconcile these seemingly disparate hypotheses, we used a combination of biochemical, fluorescence imaging, and electron microscopy techniques to quantitatively and dynamically assess the contribution of the plasmalemma and of the ER to phagosome formation and maturation. We could not verify even a transient physical continuity between the ER and the plasma membrane, nor were we able to detect a significant contribution of the ER to forming or maturing phagosomes in either macrophages or dendritic cells. Instead, our data indicate that the plasma membrane is the main constituent of nascent and newly formed phagosomes, which are progressively remodeled by fusion with endosomal and eventually lysosomal compartments as phagosomes mature into acidic, degradative organelles.
- Published
- 2005
- Full Text
- View/download PDF
46. Actin- and myosin-driven movement of viruses along filopodia precedes their entry into cells.
- Author
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Lehmann MJ, Sherer NM, Marks CB, Pypaert M, and Mothes W
- Subjects
- Animals, Avian Leukosis Virus drug effects, Avian Leukosis Virus ultrastructure, Cell Line, Cell Membrane metabolism, Cell Membrane ultrastructure, Cell Membrane virology, Cytochalasin D pharmacology, Heterocyclic Compounds, 4 or More Rings pharmacology, Humans, Leukemia Virus, Murine drug effects, Leukemia Virus, Murine ultrastructure, Mice, Microscopy, Electron, Pseudopodia ultrastructure, Pseudopodia virology, Actins physiology, Avian Leukosis Virus physiology, Leukemia Virus, Murine physiology, Myosins physiology, Pseudopodia physiology
- Abstract
Viruses have often been observed in association with the dense microvilli of polarized epithelia as well as the filopodia of nonpolarized cells, yet whether interactions with these structures contribute to infection has remained unknown. Here we show that virus binding to filopodia induces a rapid and highly ordered lateral movement, "surfing" toward the cell body before cell entry. Virus cell surfing along filopodia is mediated by the underlying actin cytoskeleton and depends on functional myosin II. Any disruption of virus cell surfing significantly reduces viral infection. Our results reveal another example of viruses hijacking host machineries for efficient infection by using the inherent ability of filopodia to transport ligands to the cell body.
- Published
- 2005
- Full Text
- View/download PDF
47. Host cell lipids control cholesteryl ester synthesis and storage in intracellular Toxoplasma.
- Author
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Nishikawa Y, Quittnat F, Stedman TT, Voelker DR, Choi JY, Zahn M, Yang M, Pypaert M, Joiner KA, and Coppens I
- Subjects
- Amino Acid Sequence, Animals, Binding Sites, Cell Membrane metabolism, Cells, Cultured, Cricetinae, Cricetulus, Endoplasmic Reticulum enzymology, Humans, Isoenzymes genetics, Isoenzymes metabolism, Mice, Microscopy, Immunoelectron, Molecular Sequence Data, Mutation, Palmitates metabolism, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae metabolism, Sterol O-Acyltransferase genetics, Toxoplasma enzymology, Toxoplasma genetics, Cholesterol Esters biosynthesis, Lipids physiology, Sterol O-Acyltransferase metabolism, Toxoplasma metabolism
- Abstract
The intracellular protozoan Toxoplasma gondii lacks a de novo mechanism for cholesterol synthesis and therefore must scavenge this essential lipid from the host environment. In this study, we demonstrated that T. gondii diverts cholesterol from low-density lipoproteins for cholesteryl ester synthesis and storage in lipid bodies. We identified and characterized two isoforms of acyl-CoA:cholesterol acyltransferase (ACAT)-related enzymes, designated TgACAT1alpha and TgACAT1beta in T. gondii. Both proteins are coexpressed in the parasite, localized to the endoplasmic reticulum and participate in cholesteryl ester synthesis. In contrast to mammalian ACAT, TgACAT1alpha and TgACAT1beta preferentially incorporate palmitate into cholesteryl esters and present a broad sterol substrate affinity. Mammalian ACAT-deficient cells transfected with either TgACAT1alpha or TgACAT1beta are restored in their capability of cholesterol esterification. TgACAT1alpha produces steryl esters and forms lipid bodies after transformation in a Saccharomyces cerevisiae mutant strain lacking neutral lipids. In addition to their role as ACAT substrates, host fatty acids and low-density lipoproteins directly serve as Toxoplasma ACAT activators by stimulating cholesteryl ester synthesis and lipid droplet biogenesis. Free fatty acids significantly increase TgACAT1alpha mRNA levels. Selected cholesterol esterification inhibitors impair parasite growth by rapid disruption of plasma membrane. Altogether, these studies indicate that host lipids govern neutral lipid synthesis in Toxoplasma and that interference with mechanisms of host lipid storage is detrimental to parasite survival in mammalian cells.
- Published
- 2005
- Full Text
- View/download PDF
48. Vesicles carry most exocyst subunits to exocytic sites marked by the remaining two subunits, Sec3p and Exo70p.
- Author
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Boyd C, Hughes T, Pypaert M, and Novick P
- Subjects
- Actin Cytoskeleton metabolism, Cell Membrane metabolism, Cell Surface Extensions metabolism, Cell Surface Extensions ultrastructure, Fluorescence Recovery After Photobleaching, Immunohistochemistry, Macromolecular Substances metabolism, Microscopy, Electron, Transmission, Protein Transport physiology, Saccharomyces cerevisiae ultrastructure, Secretory Vesicles ultrastructure, Vesicular Transport Proteins, Exocytosis physiology, Membrane Fusion physiology, Saccharomyces cerevisiae metabolism, Saccharomyces cerevisiae Proteins metabolism, Secretory Vesicles metabolism
- Abstract
Exocytosis in the budding yeast Saccharomyces cerevisiae occurs at discrete domains of the plasma membrane. The protein complex that tethers incoming vesicles to sites of secretion is known as the exocyst. We have used photobleaching recovery experiments to characterize the dynamic behavior of the eight subunits that make up the exocyst. One subset (Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, and Exo84p) exhibits mobility similar to that of the vesicle-bound Rab family protein Sec4p, whereas Sec3p and Exo70p exhibit substantially more stability. Disruption of actin assembly abolishes the ability of the first subset of subunits to recover after photobleaching, whereas Sec3p and Exo70p are resistant. Immunogold electron microscopy and epifluorescence video microscopy indicate that all exocyst subunits, except for Sec3p, are associated with secretory vesicles as they arrive at exocytic sites. Assembly of the exocyst occurs when the first subset of subunits, delivered on vesicles, joins Sec3p and Exo70p on the plasma membrane. Exocyst assembly serves to both target and tether vesicles to sites of exocytosis.
- Published
- 2004
- Full Text
- View/download PDF
49. TROSPA, an Ixodes scapularis receptor for Borrelia burgdorferi.
- Author
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Pal U, Li X, Wang T, Montgomery RR, Ramamoorthi N, Desilva AM, Bao F, Yang X, Pypaert M, Pradhan D, Kantor FS, Telford S, Anderson JF, and Fikrig E
- Subjects
- Amino Acid Sequence, Animals, Antibodies, Bacterial, Bacterial Vaccines, Base Sequence, Cloning, Molecular, Gene Expression Regulation, Host-Parasite Interactions, Intestines microbiology, Ixodes microbiology, Mice, Mice, Inbred C3H, Molecular Sequence Data, Receptors, Cell Surface genetics, Receptors, Cell Surface immunology, Receptors, Cell Surface isolation & purification, Recombinant Proteins metabolism, Antigens, Surface metabolism, Bacterial Outer Membrane Proteins metabolism, Borrelia burgdorferi pathogenicity, Ixodes metabolism, Lipoproteins metabolism
- Abstract
The Lyme disease agent Borrelia burgdorferi naturally persists in a cycle that primarily involves ticks and mammals. We have now identified a tick receptor (TROSPA) that is required for spirochetal colonization of Ixodes scapularis. B. burgdorferi outer surface protein A, which is abundantly expressed on spirochetes within the arthropod and essential for pathogen adherence to the vector, specifically bound to TROSPA. TROSPA mRNA levels in ticks increased following spirochete infestation and decreased in response to engorgement, events that are temporally linked to B. burgdorferi entry into and egress from the vector. The blockade of TROSPA by TROSPA antisera or by the repression of TROSPA expression via RNA interference reduced B. burgdorferi adherence to the I. scapularis gut in vivo, thereby preventing efficient colonization of the vector and subsequently reducing pathogen transmission to the mammalian host. Identification of an I. scapularis receptor for B. burgdorferi is the first step toward elucidating arthropod ligands that are required for survival of spirochetes in nature.
- Published
- 2004
- Full Text
- View/download PDF
50. Recycling endosomes can serve as intermediates during transport from the Golgi to the plasma membrane of MDCK cells.
- Author
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Ang AL, Taguchi T, Francis S, Fölsch H, Murrells LJ, Pypaert M, Warren G, and Mellman I
- Subjects
- Adaptor Protein Complex 1 metabolism, Animals, Cell Line, Dogs, Membrane Glycoproteins metabolism, Microscopy, Fluorescence, Microscopy, Video, Transferrin, Viral Envelope Proteins metabolism, Cell Membrane metabolism, Endocytosis, Endosomes physiology, Golgi Apparatus metabolism, Protein Transport
- Abstract
The AP-1B clathrin adaptor complex is responsible for the polarized transport of many basolateral membrane proteins in epithelial cells. Localization of AP-1B to recycling endosomes (REs) along with other components (exocyst subunits and Rab8) involved in AP-1B-dependent transport suggested that RE might be an intermediate between the Golgi and the plasma membrane. Although the involvement of endosomes in the secretory pathway has long been suspected, we now present direct evidence using four independent methods that REs play a role in basolateral transport in MDCK cells. Newly synthesized AP-1B-dependent cargo, vesicular stomatitis virus glycoprotein G (VSV-G), was found by video microscopy, immunoelectron microscopy, and cell fractionation to enter transferrin-positive REs within a few minutes after exit from the trans-Golgi network. Although transient, RE entry appears essential because enzymatic inactivation of REs blocked VSV-G delivery to the cell surface. Because an apically targeted VSV-G mutant behaved similarly, these results suggest that REs not only serve as an intermediate but also as a common site for polarized sorting on the endocytic and secretory pathways.
- Published
- 2004
- Full Text
- View/download PDF
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