RESUMO
We report the first results of a search for leptophobic dark matter (DM) from the Coherent-CAPTAIN-Mills (CCM) liquid argon (LAr) detector. An engineering run with 120 photomultiplier tubes (PMTs) and 17.9×10^{20} protons on target (POT) was performed in fall 2019 to study the characteristics of the CCM detector. The operation of this 10-ton detector was strictly light based with a threshold of 50 keV and used coherent elastic scattering off argon nuclei to detect DM. Despite only 1.5 months of accumulated luminosity, contaminated LAr, and nonoptimized shielding, CCM's first engineering run has already achieved sensitivity to previously unexplored parameter space of light dark matter models with a baryonic vector portal. With an expected background of 115 005 events, we observe 115 005+16.5 events which is compatible with background expectations. For a benchmark mediator-to-DM mass ratio of m_{V_{B}}/m_{χ}=2.1, DM masses within the range 9 MeVâ²m_{χ}â²50 MeV are excluded at 90% C. L. in the leptophobic model after applying the Feldman-Cousins test statistic. CCM's upgraded run with 200 PMTs, filtered LAr, improved shielding, and 10 times more POT will be able to exclude the remaining thermal relic density parameter space of this model, as well as probe new parameter space of other leptophobic DM models.
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We present constraints on the existence of weakly interacting massive particles (WIMPs) from an 11 kg d target exposure of the DAMIC experiment at the SNOLAB underground laboratory. The observed energy spectrum and spatial distribution of ionization events with electron-equivalent energies >200 eV_{ee} in the DAMIC CCDs are consistent with backgrounds from natural radioactivity. An excess of ionization events is observed above the analysis threshold of 50 eV_{ee}. While the origin of this low-energy excess requires further investigation, our data exclude spin-independent WIMP-nucleon scattering cross sections σ_{χ-n} as low as 3×10^{-41} cm^{2} for WIMPs with masses m_{χ} from 7 to 10 GeV c^{-2}. These results are the strongest constraints from a silicon target on the existence of WIMPs with m_{χ}<9 GeV c^{-2} and are directly relevant to any dark matter interpretation of the excess of nuclear-recoil events observed by the CDMS silicon experiment in 2013.
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We report direct-detection constraints on light dark matter particles interacting with electrons. The results are based on a method that exploits the extremely low levels of leakage current of the DAMIC detector at SNOLAB of 2-6×10^{-22} A cm^{-2}. We evaluate the charge distribution of pixels that collect <10e^{-} for contributions beyond the leakage current that may be attributed to dark matter interactions. Constraints are placed on so-far unexplored parameter space for dark matter masses between 0.6 and 100 MeV c^{-2}. We also present new constraints on hidden-photon dark matter with masses in the range 1.2-30 eV c^{-2}.
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We present direct detection constraints on the absorption of hidden-photon dark matter with particle masses in the range 1.2-30 eV c^{-2} with the DAMIC experiment at SNOLAB. Under the assumption that the local dark matter is entirely constituted of hidden photons, the sensitivity to the kinetic mixing parameter κ is competitive with constraints from solar emission, reaching a minimum value of 2.2×10^{-14} at 17 eV c^{-2}. These results are the most stringent direct detection constraints on hidden-photon dark matter in the galactic halo with masses 3-12 eV c^{-2} and the first demonstration of direct experimental sensitivity to ionization signals <12 eV from dark matter interactions.
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Shigella flexneri, the causative agent of bacillary dysentery, has the ability to enter nonphagocytic cells. The interferon (IFN) family of cytokines was found to inhibit Shigella invasion of cultured epithelial cells. We show here that IFN-alpha inhibits a Src-dependent signaling cascade triggered by Shigella that leads to the reorganization of the host cell cytoskeleton. Immunofluorescence studies showed that IFN-alpha inhibits Shigella-induced actin polymerization required for bacterial entry into cells. Phosphorylation of cortactin, a Src-substrate specifically tyrosyl-phosphorylated during Shigella entry, was inhibited by IFN-alpha. Overexpression of a dominant interfering form of pp60c-src led to inhibition of Shigella-induced cytoskeletal rearrangements and decreased cortactin phosphorylation indicating a role for Src in Shigella entry. Also, Shigella uptake in cells that expressed constitutively active Src was unaffected by IFN-alpha treatment. We conclude that Src kinase activity is necessary for Shigella invasion of epithelial cells and that IFN-alpha inhibits this Src-dependent signaling pathway.
Assuntos
Citoesqueleto/metabolismo , Células Epiteliais/microbiologia , Interferon-alfa/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Shigella flexneri/metabolismo , Actinas/metabolismo , Cortactina , Células Epiteliais/metabolismo , Células HeLa , Humanos , Proteínas dos Microfilamentos/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Transdução de Sinais/fisiologia , Tirosina/metabolismoRESUMO
Microtubules facilitate the maturation of phagosomes by favoring their interactions with endocytic compartments. Here, we show that phagosomes move within cells along tracks of several microns centrifugally and centripetally in a pH- and microtubule-dependent manner. Phagosome movement was reconstituted in vitro and required energy, cytosol and membrane proteins of this organelle. The activity or presence of these phagosome proteins was regulated as the organelle matured, with "late" phagosomes moving threefold more frequently than "early" ones. The majority of moving phagosomes were minus-end directed; the remainder moved towards microtubule plus-ends and a small subset moved bi-directionally. Minus-end movement showed pharmacological characteristics expected for dyneins, was inhibited by immunodepletion of cytoplasmic dynein and could be restored by addition of cytoplasmic dynein. Plus-end movement displayed pharmacological properties of kinesin, was inhibited partially by immunodepletion of kinesin and fully by addition of an anti-kinesin IgG. Immunodepletion of dynactin, a dynein-activating complex, inhibited only minus-end directed motility. Evidence is provided for a dynactin-associated kinase required for dynein-mediated vesicle transport. Movement in both directions was inhibited by peptide fragments from kinectin (a putative kinesin membrane receptor), derived from the region to which a motility-blocking antibody binds. Polypeptide subunits from these microtubule-based motility factors were detected on phagosomes by immunoblotting or immunoelectron microscopy. This is the first study using a single in vitro system that describes the roles played by kinesin, kinectin, cytoplasmic dynein, and dynactin in the microtubule-mediated movement of a purified membrane organelle.
Assuntos
Microtúbulos/metabolismo , Fagossomos/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Transporte Biológico/fisiologia , Células Cultivadas/química , Células Cultivadas/metabolismo , Células Cultivadas/ultraestrutura , Citosol/química , Citosol/enzimologia , Complexo Dinactina , Dineínas/metabolismo , Concentração de Íons de Hidrogênio , Rim/citologia , Cinesinas/metabolismo , Látex , Macrófagos/citologia , Macrófagos/metabolismo , Macrófagos/ultraestrutura , Proteínas de Membrana/metabolismo , Camundongos , Microesferas , Proteínas Associadas aos Microtúbulos/metabolismo , Fagossomos/química , Fagossomos/efeitos dos fármacos , Fosfotransferases/metabolismo , Ratos , Receptores de Superfície Celular/metabolismoRESUMO
A novel mouse homeobox-containing gene, Nkx-2.2, has been isolated. Nkx-2.2 is a member of a family of genes whose homeodomains are homologous to that of the Drosophila NK-2 gene. Nkx-2.2 transcripts are found in localized domains of the brain during mouse embryogenesis. Nkx-2.2 expression in the brain abuts and partially overlaps with the expression domains of two other related homeobox-containing genes, TTF-1 and Dlx. The expression domains of the three genes in the developing prosencephalon coincide with anatomical boundaries, particularly apparent in the diencephalon. This result raises the possibility that these genes may specify regional differentiation of the developing diencephalon into its anatomically and functionally defined subregions. Nkx-2.2 may be involved in specifying diencephalic neuromeric boundaries.
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Regulação da Expressão Gênica/genética , Genes Homeobox/genética , Prosencéfalo/embriologia , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Mapeamento Cromossômico , DNA/genética , Diencéfalo/embriologia , Diencéfalo/fisiologia , Regulação da Expressão Gênica/fisiologia , Genes Homeobox/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Morfogênese/fisiologia , Hibridização de Ácido Nucleico , Prosencéfalo/fisiologia , Transcrição Gênica/genéticaRESUMO
BACKGROUND: The transport of macromolecules between the nucleus and cytoplasm is an energy-dependent process. Substrates are translocated across the nuclear envelope through nuclear pore complexes (NPCs). Translocation requires nucleocytoplasmic transport receptors of the importin beta family, which interact both with the NPC and, either directly or via an adaptor, with the transport substrate. Although certain receptors have recently been shown to cross the NPC in an energy-independent manner, translocation of substrate-receptor complexes through the NPC has generally been regarded as an energy-requiring step. RESULTS: We describe an in vitro system that is based on permeabilised cells and supports nuclear export mediated by leucine-rich nuclear export signals. In this system, export is dependent on exogenous CRM1/Exportin1 - a nuclear export receptor - the GTPase Ran and nucleotide triphosphates (NTPs), and is further stimulated by Ran-binding protein 1 (RanBP1) and nuclear transport factor 2 (NTF2). Unexpectedly, non-hydrolysable NTP analogues completely satisfy the NTP requirements for a single-round of CRM1-mediated translocation of protein substrates across the NPC. Similarly, single transportin-mediated nuclear protein import events are shown not to require hydrolysable NTPs and to occur in the absence of the Ran GTPase. CONCLUSIONS: Our data show that, contrary to expectation and prior conclusions, the translocation of substrate-receptor complexes across the NPC in either direction occurs in the absence of NTP hydrolysis and is thus energy independent. The energy needed to drive substrate transport against a concentration gradient is supplied at the step of receptor recycling in the cytoplasm.
Assuntos
Proteínas de Transporte/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Carioferinas , Membrana Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático , Receptores Citoplasmáticos e Nucleares , Proteína ran de Ligação ao GTP , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Guanosina Difosfato/metabolismo , Células HeLa , Humanos , Microscopia de Fluorescência , Proteínas Recombinantes de Fusão/metabolismo , Proteína Exportina 1RESUMO
Several changes in cell morphology take place during the capping of surface receptors in Entamoeba histolytica. The amoebae develop the uroid, an appendage formed by membrane invaginations, which accumulates ligand-receptor complexes resulting from the capping process. Membrane shedding is particularly active in the uroid region and leads to the elimination of accumulated ligands. This appendage has been postulated to participate in parasitic defense mechanisms against the host immune response, because it eliminates complement and specific antibodies bound to the amoeba surface. The involvement of myosin II in the capping process of surface receptors has been suggested by experiments showing that drugs that affect myosin II heavy-chain phosphorylation prevent this activity. To understand the role of this mechanoenzyme in surface receptor capping, a myosin II dominant negative strain was constructed. This mutant is the first genetically engineered cytoskeleton-deficient strain of E. histolytica. It was obtained by overexpressing the light meromyosin domain, which is essential for myosin II filament formation. E. histolytica overexpressing light meromyosin domain displayed a myosin II null phenotype characterized by abnormal movement, failure to form the uroid, and failure to undergo the capping process after treatment with concanavalin A. In addition, the amoebic cytotoxic capacities of the transfectants on human colon cells was dramatically reduced, indicating a role for cytoskeleton in parasite pathogenicity.
Assuntos
Entamoeba histolytica/metabolismo , Entamoeba histolytica/patogenicidade , Subfragmentos de Miosina/metabolismo , Miosinas/fisiologia , Animais , Antígenos Glicosídicos Associados a Tumores/metabolismo , Células CACO-2 , Sobrevivência Celular , Entamoeba histolytica/genética , Humanos , Mutagênese , Miosinas/genética , Fenótipo , Receptores de Superfície Celular/metabolismo , Transfecção , VirulênciaRESUMO
Using an excimer laser working in the far ultraviolet and cutting fresh corneas of human cadaver eyes, we were able to demonstrate that there is an ideal fluence (joule/cm2/pulse) that allows a maximum tissue ablation depth per shot. Above the threshold of 1 J/cm2/pulse the depth of tissue ablated does not increase and both thermal and mechanical damage appears at the cut edges.
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Córnea/cirurgia , Terapia a Laser , Córnea/patologia , Endotélio/patologia , Humanos , Microscopia Eletrônica , Miopia/cirurgiaRESUMO
We present a method whereby the geometric registration of a series of ion microscopic images is performed by applying a two-step procedure. After applying a global linear transformation that corrects for geometric differences, a non-linear elastic transform is used in order to match local properties and structures of the images. Transformation parameters are computed on the basis of shape-specific points in the images. Distortion correction is achieved by relating ion images to the optical image of the same field and by using the two-step algorithm to register the images. This methodology is evaluated on synthetic misaligned objects and on thyroid tissue images.
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Processamento de Imagem Assistida por Computador , Microscopia/métodos , Glândula Tireoide/anatomia & histologia , Animais , Humanos , RatosRESUMO
A method has been developed in order to digitally correlate ion and optical microscopic images of the same sample areas. Serial cross-sections of human thyroid tissue were analyzed by secondary ion mass microscopy and by light microscopy. The resulting chemical and immunochemical map images were superimposed and correlated by means of a two-pass registration algorithm which allows to correct for geometrical distortions introduced by the ion microscope. Results are presented for the study of thyroglobulin chemical modification in pathological thyroid tissue that demonstrates heterogeneous molecular activity.
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Tireoglobulina/análise , Glândula Tireoide/ultraestrutura , Humanos , Técnicas Imunológicas , EnxofreRESUMO
Entamoeba histolytica is a protozoan parasite that causes amoebic dysentery in humans. The disease is prevalent worldwide. Infection with E. histolytica results in invasion of the intestine by the parasite, followed by tissue damage and inflammation. During this invasive process, parasites kill and phagocytose human epithelial cells, immune cells and erythrocytes. Expression of amoebic pathogenicity requires a dynamic cytoskeleton that allows movement, tissue penetration and changes in parasite morphology. Myosin IB is a member of the myosin I family of motor proteins. Studies conducted both with Dictyostelium discoideum, a non-pathogenic amoeba, and with the yeast Saccharomyces cerevisiae indicate the involvement of myosin IB in cellular processes including movement, phagocytosis and endocytosis. Recently, we isolated the gene encoding myosin IB from E. histolytica. Thus, we decided to analyze the role of myosin IB in pathogenesis of amoeba. Using a specific anti-myosin IB antibody, this protein was localized in cell regions including the pseudopod, vesicles and underneath the plasma membrane. When E. histolytica was activated for erythrophagocytosis, myosin IB was markedly recruited to both the phagocytic cup and around internalized phagosomes. To analyze the role of myosin IB in phagocytosis, a strain overexpressing the myosin IB gene was constructed. This strain synthesizes threefold more myosin IB than the wild-type strain. Challenge of the transfected cell line with erythrocytes showed that these amoebae were deficient in erythrophagocytosis mainly in the uptake step, suggesting a role for myosin IB in the pathogenic activity of a human parasite.
Assuntos
Entamoeba/metabolismo , Eritrócitos/fisiologia , Miosinas/fisiologia , Fagocitose , 3,3'-Diaminobenzidina/farmacologia , Animais , Movimento Celular , Células Cultivadas , Clonagem Molecular , Simulação por Computador , Citoesqueleto/metabolismo , Escherichia coli/metabolismo , Imunofluorescência , Hemoglobinas/metabolismo , Humanos , Microscopia Confocal , Modelos Genéticos , Miosinas/imunologia , Testes de Precipitina , Análise de Sequência de DNA , Espectrofotometria , Fatores de Tempo , TransfecçãoRESUMO
Analytical ion microscopy is a powerful tool for biological tissue analysis as it allows direct chemical distribution imaging, even at low element concentrations. A microcomputer based digital imaging system achieving acquisition at low light level is presented. It includes a high sensitivity video camera connected to a specialized image processor subsystem. Acquired images consist of 512 x 512 pixels with 8 bits accuracy. Real-time image processing software has been implemented so that image processing may be performed on-line. Image processing software allows off-line image manipulation and correlation for biological interpretation of elemental mapping images. System capabilities are illustrated by a study of stable and radio iodine mapping in rat thyroid tissue.
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Processamento de Imagem Assistida por Computador , Microcomputadores , Software , Glândula Tireoide/anatomia & histologia , Algoritmos , Animais , Espectrometria de Massas , RatosRESUMO
Secondary ion mass spectrometry (SIMS) microscopy provides a direct mapping of 127I in thyroid follicles. Thyroid tissue should be fixed and embedded in methacrylate resin, then cut in 3 microns-thick sections which are placed on a gold coverslip. The histological structure of the tissue is determined by the phosphorus ion (31P) which is present in large amounts in nuclei and phosphorylated molecules in the cytoplasm. An image processing system is used to superimpose images of 127I and 31P: this system allows measurement of local concentration of 127I in the follicular cell and follicular lumen compartments. A study in 8 subjects with normal thyroid glands showed that the level of 127I within follicular cells (430 +/- 250 micrograms/g; m +/- SE) was 6 to 7 times lower than the level in the follicular lumen (2.780 +/- 230 micrograms/g). In simple goiter (9 patients with macrofollicular adenomas), follicular lumen (346 +/- 17 micrograms/g) and cellular (68 +/- 6 micrograms/g) concentrations of 127I were decreased fivefold but the ratio of concentrations remained similar to that seen in normal tissue. In hyperfunctioning nodules (2 microfollicular and 3 macrofollicular adenomas), follicular cell and follicular lumen 127I levels varied widely and showed considerable overlap (2 to 16,000 micrograms/g and 1-21,000 micrograms/g, respectively). In benign cold nodules (1 microfollicular adenoma and 2 macrofollicular adenomas), follicular cell and follicular lumen 127I levels were barely detectable (3 +/- 0.6 micrograms/g and 6 +/- 0.7 micrograms/g respectively). In the 9 malignant nodules studied, no difference in 127I level profile was found with benign nodules.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Bócio Nodular/patologia , Espectrometria de Massas/métodos , Glândula Tireoide/ultraestrutura , Nódulo da Glândula Tireoide/ultraestrutura , Humanos , Técnicas In Vitro , Radioisótopos do Iodo/análise , Radioisótopos de Fósforo , Valores de Referência , Nódulo da Glândula Tireoide/fisiopatologiaRESUMO
Previous studies using overexpressed polymeric immunoglobulin receptor in cultured neurons have suggested that these cells may use a dendroaxonal transcytotic pathway (Ikonen et al., 1993; de Hoop et al., 1995). By using a combination of semiquantitative light microscopy, video microscopy, and a biochemical assay, we show that this pathway is used by the endogenous ligand transferrin (Tf) and its receptor. Labeled Tf added to fully mature hippocampal neurons changes the intracellular distribution of its receptor from preferentially dendritic shortly after addition to dendritic and axonal at longer times. Incubation of living neurons with (caged)FITC-Tf followed by uncaging in the dendrites results in the later appearance of fluorescence in the axon of the same cell. In "chambered" sympathetic neurons in culture, 125I-Tf or iron as 55Fe-Tf added to the cell body/dendrite chamber is recovered in the axonal chamber, showing that internalized ligand from the cell body-dendrite area is released at the axonal end. Finally, we show that excitatory neurotransmitters increase Tf receptor transcytosis, whereas inhibitory neurotransmitters reduce it. The dendritic uptake, transcytotic transport, and axonal release of physiologically active Tf demonstrated here could be envisioned for other trophic factors and therefore have important consequences for neuronal anterograde target maturation. Moreover, the changes in transcytosis after neurotransmitter addition may be important in the cellular responses that follow electrical activation.
Assuntos
Transporte Axonal/fisiologia , Axônios/metabolismo , Dendritos/metabolismo , Endocitose/fisiologia , Hipocampo/citologia , Receptores da Transferrina/metabolismo , Gânglio Cervical Superior/citologia , Transferrina/metabolismo , Animais , Transporte Axonal/efeitos dos fármacos , Axônios/ultraestrutura , Transporte Biológico/efeitos dos fármacos , Células Cultivadas , Endocitose/efeitos dos fármacos , Corantes Fluorescentes , Ácido Glutâmico/farmacologia , Hipocampo/metabolismo , Processamento de Imagem Assistida por Computador , Ferro/metabolismo , Microscopia de Fluorescência , Microscopia de Vídeo , N-Metilaspartato/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Ratos , Rodaminas , Gânglio Cervical Superior/metabolismo , Transmissão Sináptica , Ácido gama-Aminobutírico/farmacologiaRESUMO
We have shown previously that intracellular phagosome movement requires microtubules. Here we provide evidence that within cells phagosomes display two different kinds of microtubule-based movements in approximately equal proportions. The first type occurs predominantly in the cell periphery, often shortly after the phagosome is formed, and at speeds below 0.1 microm/second. The second is faster (0.2-1.5 micron/second) and occurs mainly after phagosomes have reached the cell interior. Treating cells with nanomolar concentrations of taxol or nocodazole alters microtubule dynamics without affecting either total polymer mass or microtubule organisation. Such treatments slow the accumulation of phagosomes in the perinuclear region and reduce the number of slow movements by up to 50% without affecting the frequency of fast movements. This suggests that a proportion of slow movements are mediated by microtubule dynamics while fast movements are powered by microtubule motors. In macrophages, interphase microtubules radiate from the microtubule organising centre with their plus-end towards the cell periphery. To understand the behaviour of 'early' phagosomes at the cell periphery we investigated their ability to bind microtubule plus-ends in vitro. We show that early phagosomes have a strong preference for microtubule plus-ends, whereas 'late' phagosomes do not, and that plus-end affinity requires the presence of microtubule-associated proteins within cytosol. We suggest that phagosomes can bind to the plus-ends of dynamic microtubules and move by following their shrinkage or growth.
Assuntos
Microtúbulos/fisiologia , Fagossomos/fisiologia , Actinas/fisiologia , Animais , Linhagem Celular , Camundongos , Proteínas Associadas aos Microtúbulos/fisiologia , Movimento , RatosRESUMO
The mechanisms and carriers responsible for exocytic protein trafficking between the trans-Golgi network (TGN) and the plasma membrane remain unclear. To investigate the dynamics of TGN-to-plasma membrane traffic and role of the cytoskeleton in these processes we transfected cells with a GFP-fusion protein, vesicular stomatitis virus G protein tagged with GFP (VSVG3-GFP). After using temperature shifts to block VSVG3-GFP in the endoplasmic reticulum and subsequently accumulate it in the TGN, dynamics of TGN-to-plasma membrane transport were visualized in real time by confocal and video microscopy. Both small vesicles (<250 nm) and larger vesicular-tubular structures (>1.5 microm long) are used as transport containers (TCs). These TCs rapidly moved out of the Golgi along curvilinear paths with average speeds of approximately 0.7 micrometer/second. Automatic computer tracking objectively determined the dynamics of different carriers. Fission and fusion of TCs were observed, suggesting that these late exocytic processes are highly interactive. To directly determine the role of microtubules in post-Golgi traffic, rhodamine-tubulin was microinjected and both labeled cargo and microtubules were simultaneously visualized in living cells. These studies demonstrated that exocytic cargo moves along microtubule tracks and reveals that carriers are capable of switching between tracks.
Assuntos
Membrana Celular/metabolismo , Complexo de Golgi/metabolismo , Glicoproteínas de Membrana , Microtúbulos/metabolismo , Animais , Transporte Biológico/fisiologia , Linhagem Celular , Citoesqueleto/fisiologia , Exocitose/fisiologia , Imunofluorescência , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Macropodidae , Microinjeções , Microscopia de Vídeo , Microtúbulos/fisiologia , Organelas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
This study provides a detailed description of the anatomical defects in the Hoxa-1-/- mutant mice previously generated in our laboratory (T. Lufkin, A. Dierich, M. LeMeur, M. Mark and P. Chambon, 1991; Cell 66, 1105-1119). Three-dimensional reconstructions of the Hoxa-1-/- rhombencephalon reveals that it bears only five rhombomeric structures (ie. morphological segments) instead of the normal seven. The first three of these rhombomeres appear normal as judged from the distribution pattern of CRABPI transcripts in the neurectoderm and from the histological analysis of the cranial nerve components derived from these structures. In contrast, the neural-crest-cell-free region normally located opposite rhombomere 5 is lacking in Hoxa-1-/- embryos, and motor neurons of the facial and abducens nerves, which normally differentiate within rhombomeres 4, 5 and 6, are missing in Hoxa-1-/- fetuses. These morphological data, combined with the determination of the molecular positional identities of the rhombomeres 4 and 5 (P. Dollé, T. Lufkin, R. Krumlauf, M. Mark, D. Duboule and P. Chambon, 1993; Proc. Natl. Acad. Sci. USA, in press), suggest that rhombomere 4 is markedly reduced, whereas rhombomere 5 is almost absent. Thus, the remnants of rhombomeres 4 and 5 appear to be fused caudally with rhombomere 6 to form a single fourth rhombomeric structure. Moreover, the migration of neural crest cells contributing to the glossopharyngeal and vagus nerves occurs in a more rostral position, resulting in abnormalities of these cranial nerves, which were visualized by whole-mount anti-neurofilament immunostaining. The mutual relationship along the rostrocaudal axis between the otic pit and the neuroepithelial site of int-2 protein secretion (a putative otogenic cue) is not significantly changed in Hoxa-1-/- embryos. However, the abnormal relationship between the rhombencephalon and the epithelial inner ear may account for the aplasia and faulty differentiation of the membranous labyrinth, the disruption of the cartilaginous otic capsule and the disorganisation of some middle ear structures. This phenotype is compared with that of the Hoxa-1-/- mutants generated by O. Chisaka, T. S. Musci and M. R. Capecchi, 1992 (Nature 335, 516-520) and with that of the mice homozygous for the kreisler mutation.
Assuntos
Orelha/embriologia , Camundongos Mutantes/embriologia , Rombencéfalo/embriologia , Animais , Nervos Cranianos/embriologia , Processamento de Imagem Assistida por Computador , Técnicas Imunoenzimáticas , Camundongos , Camundongos Mutantes/genética , Morfogênese/fisiologia , Crista Neural/fisiologia , FenótipoRESUMO
O-glycosylation of proteins is initiated by a family of UDP-N-acetylgalactosamine:polypeptide N-acetylgalactos-aminyltransferases (GalNAc-T). In this study, we have localized endogenous and epitope-tagged human GalNAc-T1, -T2 and -T3 to the Golgi apparatus in HeLa cells by subcellular fractionation, immunofluorescence and immunoelectron microscopy. We show that all three GalNAc-transferases are concentrated about tenfold in Golgi stacks over Golgi associated tubular-vesicular membrane structures. Surprisingly, we find that GalNAc-T1, -T2 and -T3 are present throughout the Golgi stack suggesting that initiation of O-glycosylation may not be restricted to the cis Golgi, but occur at multiple sites within the Golgi apparatus. GalNAc-T1 distributes evenly across the Golgi stack whereas GalNAc-T2 and -T3 reside preferentially on the trans side and in the medial part of the Golgi stack, respectively. Moreover, we have investigated the possibility of O-glycan initiation in pre-Golgi compartments such as the ER. We could not detect endogenous polypeptide GalNAc-transferase activity in the ER of HeLa cells, neither by subcellular fractionation nor by situ glycosylation of an ER-retained form of CD8 (CD8/E19). However, upon relocation of chimeric GalNAc-T1 or -T2 to the ER, CD8/E19 is glycosylated with different efficiencies indicating that all components required for initiation of O-glycosylation are present in the ER except for polypeptide GalNAc-transferases.