RESUMO
Magnetotactic bacteria biomineralize intracellular magnetic nanocrystals surrounded by a lipid bilayer called magnetosomes. Due to their unique characteristics, magnetite magnetosomes are promising tools in Biomedicine. However, the uptake, persistence, and accumulation of magnetosomes within mammalian cells have not been well studied. Here, the endocytic pathway of magnetite magnetosomes and their effects on human cervix epithelial (HeLa) cells were studied by electron microscopy and high spatial resolution nano-analysis techniques. Transmission electron microscopy of HeLa cells after incubation with purified magnetosomes showed the presence of magnetic nanoparticles inside or outside endosomes within the cell, which suggests different modes of internalization, and that these structures persisted beyond 120 h after internalization. High-resolution transmission electron microscopy and electron energy loss spectra of internalized magnetosome crystals showed no structural or chemical changes in these structures. Although crystal morphology was preserved, iron oxide crystalline particles of approximately 5 nm near internalized magnetosomes suggests that minor degradation of the original mineral structures might occur. Cytotoxicity and microscopy analysis showed that magnetosomes did not result in any apparent effect on HeLa cells viability or morphology. Based on our results, magnetosomes have significant biocompatibility with mammalian cells and thus have great potential in medical, biotechnological applications.
Assuntos
Endocitose , Óxido Ferroso-Férrico/metabolismo , Magnetossomos/metabolismo , Biotecnologia/métodos , Sobrevivência Celular , Endossomos/metabolismo , Endossomos/ultraestrutura , Células HeLa , Humanos , Teste de Materiais , Microscopia Eletrônica de Transmissão , Testes de ToxicidadeRESUMO
Axonal transport is required for neuronal development and survival. Transport from the axon to the soma is driven by the molecular motor cytoplasmic dynein, yet it remains unclear how dynein is spatially and temporally regulated. We find that the dynein effector Hook1 mediates transport of TrkB-BDNF-signaling endosomes in primary hippocampal neurons. Hook1 comigrates with a subpopulation of Rab5 endosomes positive for TrkB and BDNF, which exhibit processive retrograde motility with faster velocities than the overall Rab5 population. Knockdown of Hook1 significantly reduced the motility of BDNF-signaling endosomes without affecting the motility of other organelles. In microfluidic chambers, Hook1 depletion resulted in a significant decrease in the flux and processivity of BDNF-Qdots along the mid-axon, an effect specific for Hook1 but not Hook3. Hook1 depletion inhibited BDNF trafficking to the soma and blocked downstream BDNF- and TrkB-dependent signaling to the nucleus. Together, these studies support a model in which differential association with cargo-specific effectors efficiently regulates dynein in neurons.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Dineínas do Citoplasma/metabolismo , Endossomos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/metabolismo , Receptor trkB/metabolismo , Animais , Transporte Axonal , Sítios de Ligação , Fator Neurotrófico Derivado do Encéfalo/genética , Núcleo Celular/metabolismo , Dineínas do Citoplasma/química , Dineínas do Citoplasma/genética , Endossomos/ultraestrutura , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/metabolismo , Hipocampo/ultraestrutura , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Modelos Moleculares , Neurônios/ultraestrutura , Cultura Primária de Células , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Secundária de Proteína , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Receptor trkB/genética , Transdução de Sinais , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismo , Proteína Vermelha FluorescenteRESUMO
Dendritic arborization of neurons is regulated by brain-derived neurotrophic factor (BDNF) together with its receptor, TrkB. Endocytosis is required for dendritic branching and regulates TrkB signaling, but how postendocytic trafficking determines the neuronal response to BDNF is not well understood. The monomeric GTPase Rab11 regulates the dynamics of recycling endosomes and local delivery of receptors to specific dendritic compartments. We investigated whether Rab11-dependent trafficking of TrkB in dendrites regulates BDNF-induced dendritic branching in rat hippocampal neurons. We report that TrkB in dendrites is a cargo for Rab11 endosomes and that both Rab11 and its effector, MyoVb, are required for BDNF/TrkB-induced dendritic branching. In addition, BDNF induces the accumulation of Rab11-positive endosomes and GTP-bound Rab11 in dendrites and the expression of a constitutively active mutant of Rab11 is sufficient to increase dendritic branching by increasing TrkB localization in dendrites and enhancing sensitization to endogenous BDNF. We propose that Rab11-dependent dendritic recycling provides a mechanism to retain TrkB in dendrites and to increase local signaling to regulate arborization.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/farmacologia , Dendritos/efeitos dos fármacos , Endossomos/efeitos dos fármacos , Proteínas de Ligação ao GTP/metabolismo , Neurônios/citologia , Análise de Variância , Animais , Anticorpos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Carbazóis/farmacologia , Células Cultivadas , Dendritos/fisiologia , Dendritos/ultraestrutura , Embrião de Mamíferos , Endocitose/efeitos dos fármacos , Endossomos/ultraestrutura , Inibidores Enzimáticos/farmacologia , Feminino , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Guanosina Trifosfato/metabolismo , Hipocampo/citologia , Alcaloides Indólicos/farmacologia , Masculino , Microscopia Confocal , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação/genética , Miosinas/metabolismo , Neurônios/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia , Ratos , Receptor trkB/metabolismo , Tiazolidinas/farmacologia , TransfecçãoRESUMO
Oropouche virus (ORO), family Bunyaviridae, is the second most frequent cause of arboviral febrile illness in Brazil. Studies were conducted to understand ORO entry in HeLa cells. Chlorpromazine inhibited early steps of ORO replication cycle, consistent with entry/uncoating. The data indicate that ORO enters HeLa cells by clathrin-coated vesicles, by a mechanism susceptible to endosomal acidification inhibitors. Transmission electron microscopy and immunofluorescence indicated that ORO associates with clathrin-coated pits and can be found in association with late endosomes in a time shorter than 1h.
Assuntos
Ácidos/metabolismo , Infecções por Bunyaviridae/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Endossomos/metabolismo , Orthobunyavirus/fisiologia , Internalização do Vírus , Brasil , Infecções por Bunyaviridae/virologia , Vesículas Revestidas por Clatrina/ultraestrutura , Vesículas Revestidas por Clatrina/virologia , Endossomos/ultraestrutura , Endossomos/virologia , Células HeLa , Humanos , Orthobunyavirus/ultraestruturaRESUMO
Reservosomes are late endosomes present only in members of the Schizotrypanum subgenus of the Trypanosoma genus and are defined as the site of storage of endocytosed macromolecules and lysosomal enzymes. They have been extensively described in Trypanosoma cruzi epimastigote: are bounded by a membrane unit, present an electron-dense protein matrix with electron-lucent lipid inclusions, being devoid of inner membranes. Here we performed a detailed ultrastructural analysis of these organelles using a variety of electron microscopy techniques, including ultrathin sectioning, uranyl acetate stained preparations, and freeze fracture, either in intact epimastigotes or in isolated reservosomes. New informations were obtained. First, both isolated and in situ reservosomes presented small profiles of inner membranes that are morphologically similar to the membrane surrounding the organelle. In uranyl acetate stained preparations, internal membrane profiles turned out to be longer than they appeared in ultrathin section images and traversed the organelle diameter. Internal vesicles were also found. Second, endocytosed cargo are not associated with internal vesicles and reach reservosomes on board of vesicles that fuse with the boundary membrane, delivering cargo directly into reservosome lumen. Third, electron-lucent bodies with saturated lipid core surrounded by a membrane monolayer and with unusual rectangular shape were also observed. Fourth, it was possible to demonstrate the presence of intramembranous particles on the E face of both internal vesicles and the surrounding membrane. Collectively, these results indicate that reservosomes have a complex internal structure, which may correlate with their multiple functions.
Assuntos
Endossomos/ultraestrutura , Trypanosoma cruzi/ultraestrutura , Animais , Microscopia Crioeletrônica , Membranas Intracelulares/ultraestrutura , Microscopia Eletrônica de Transmissão , Microtomia , Compostos Organometálicos , Coloração e RotulagemRESUMO
Canova is a homeopathic medication with immunomodulatory properties, recommended for diseases where the immune system is depressed. Our research aims to study the activation of mice peritoneal macrophages when submitted to in vivo and in vitro Canova treatment. Morphological parameters and acid phosphatase activity were analyzed using light and transmission electron microscopy. Differential interference contrast microscopy, including serial time acquisition in living cells, was also performed. The results demonstrated a greater spreading ability in Canova treated macrophages, a higher phagocytic activity of non-infective microorganisms (Saccharomyces cerevisiae and Tripanosoma cruzi epimastigotes) and a tendency to lower the phagocytic activity of the infective microorganisms T. cruzi trypomastigotes and Leishmania amazonensis, when compared with control cells. Acid phosphatase activity was analyzed and showed that Canova treatment stimulates an increase of the endosomal/lysosomal system. Treated macrophages that do or do not interact with yeast present a higher number of acid phosphatase marked vesicles compared to control cells. In contrast, the activity of tartrate resistant acid phosphatase (TRAP), is lower in Canova treated macrophages. The net results demonstrate that Canova medication is an effective stimulator of macrophage activity.
Assuntos
Fatores Imunológicos/farmacologia , Macrófagos Peritoneais/efeitos dos fármacos , Materia Medica/farmacologia , Extratos Vegetais/farmacologia , Fosfatase Ácida/metabolismo , Animais , Células Cultivadas , Endossomos/ultraestrutura , Humanos , Fatores Imunológicos/administração & dosagem , Injeções Subcutâneas , Leishmania/imunologia , Lisossomos/ultraestrutura , Macrófagos Peritoneais/enzimologia , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/ultraestrutura , Masculino , Materia Medica/administração & dosagem , Microscopia Confocal , Microscopia de Interferência , Fagocitose , Extratos Vegetais/administração & dosagem , Saccharomyces cerevisiae/imunologia , Trypanosoma cruzi/imunologiaRESUMO
Hepatopancreal tissue of the crab Ucides cordatus was investigated by light and electron microscopy. The observed epithelial cells were: E-cells (embryonic), located in the distal portion of the hepatopancreal tubules, R-cells (resorptive) F-cells (fibrillar) and B-cells (blister or secretory), found in its intermediate and proximal regions. Two types of electron-dense granules (EDGs) were found frequently in the cells of the proximal portion of the hepatopancreal tubule. Both types of EDGs presented alternating concentric electron-dense and electron-lucent layers. In order to better characterize these granules, energy dispersive X-ray analysis (EDXA) and glucose-6-phosphatase (G6Pase) cytochemistry were performed. One type of spherical granule was seen inside vacuoles surrounded by an association of myelin-like membranes as well as some small membrane-bound vesicles. This type of granule neither presented detectable Ca and P on EDXA spectra nor G6Pase cytochemical reaction products. The second type of granule had O, P and Ca characteristic peaks. G6Pase cytochemical products were observed inside these structures and showed that this mineralized type was surrounded by endoplasmic reticulum membranes. This result suggests that in U. cordatus the endoplasmic reticulum is associated with the genesis of mineralized EDGs. While amorphous mineral granules may be associated with a storage of Ca and P for the new carapace synthesis, EDGs covered by the non-mineralized spherical multi-layered membranes may be associated with late endosomes. No specific secretory pathway however was determined for the EDGs at the epithelial proximal portion.
Assuntos
Braquiúros/ultraestrutura , Células Epiteliais/ultraestrutura , Fígado/ultraestrutura , Pâncreas/ultraestrutura , Animais , Braquiúros/metabolismo , Calcificação Fisiológica/fisiologia , Cálcio/metabolismo , Grânulos Citoplasmáticos/metabolismo , Grânulos Citoplasmáticos/ultraestrutura , Microanálise por Sonda Eletrônica , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Endossomos/metabolismo , Endossomos/ultraestrutura , Células Epiteliais/metabolismo , Glucose-6-Fosfatase/metabolismo , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Fígado/metabolismo , Masculino , Microscopia Eletrônica , Pâncreas/metabolismo , Fosfatos/metabolismo , Vesículas Secretórias/metabolismo , Vesículas Secretórias/ultraestruturaRESUMO
Reservosomes are acidic compartments present at the posterior region of epimastigote forms of Trypanosoma cruzi that store proteins and lipids. During metacyclogenesis, they consume their contents and disappear. Reservosomes are rich in cruzipain, the main proteolytic enzyme of this parasite. By centrifugation in a sucrose gradient, we have obtained a highly purified subcellular fraction containing reservosomes from 5-day-old Y strain epimastigotes. Transmission electron microscopy showed that the fraction contained well-preserved organelles. The protein profile of the organelle analyzed by SDS-PAGE depicted a wide range of protein bands, predominating those corresponding to a triplet of 60-51 kDa and a doublet of 25-23 kDa. Protease activity in substrate-containing gels, in the presence or absence of protease inhibitors, showed that cysteine proteinase is enriched and very active in the purified fraction. Enzymatic assays demonstrated the absence of pyrophosphatase, an acidocalcisome marker, and succinate cytochrome c reductase, a mitochondrial marker, although these enzymes were active in other regions of the purification sucrose gradient. Thin layer chromatographic neutral lipid analysis of purified reservosomes demonstrated that the organelle stores large amounts of ergosterol and esterified cholesterol. Phospholipid analysis indicated phosphatidylcholine and phosphatidylethanolamine as the major constituents of reservosome membranes.
Assuntos
Endossomos , Frações Subcelulares , Trypanosoma cruzi/ultraestrutura , Animais , Centrifugação com Gradiente de Concentração/métodos , Endossomos/química , Endossomos/ultraestrutura , Lipídeos/análise , Microscopia Eletrônica , Proteínas/análise , Frações Subcelulares/química , Frações Subcelulares/ultraestrutura , Trypanosoma cruzi/crescimento & desenvolvimentoRESUMO
The localization of the glucocorticoid-inducible protein annexin 1 (ANX-1) in leukocytes during the process of experimental inflammation has been studied using immunocytochemistry. ANX- 1 immunoreactivity was detected in extravasated neutrophils and eosinophils as well as in resident tissue mast cells. Following injection of carrageenin, the mesenteric tissue was highly inflamed with large presence of leukocytes (predominantly neutrophils with a small percentage of eosinophils) adherent to post-capillary venules and extravasated in the perivascular tissue. ANX-1 immunoreactivity was detected in the cytosol of neutrophils and eosinophils mainly associated with granules and/or vesicles. A good degree of localization in the endosomes was observed in the neutrophils. In both cell types, some ANX-1 immunoreactivity in the nucleus and in the plasma membrane was also detected. Resident mast cells were also activated. Mast cells were positive for ANX-1, without apparent changes in protein content in relation to their activation status. Degranulated mast cells still presented ANX-1 associated with the granule matrix. In conclusion, this study demonstrated the presence of ANX-1 in leukocytes that play a central role in the host inflammatory response. These are the extravasating polymorphonuclear cells, or the resident mast cells. These data provide morphological support to the notion that endogenous and exogenous ANX-1 are able to modulate the reactivity of these cell types, and more generally, of the experimental inflammatory reaction.
Assuntos
Anexina A1/metabolismo , Compartimento Celular/fisiologia , Quimiotaxia de Leucócito/fisiologia , Inflamação/metabolismo , Membranas Intracelulares/metabolismo , Leucócitos/metabolismo , Organelas/metabolismo , Animais , Carragenina/farmacologia , Endossomos/imunologia , Endossomos/metabolismo , Endossomos/ultraestrutura , Imuno-Histoquímica , Inflamação/imunologia , Inflamação/patologia , Membranas Intracelulares/imunologia , Membranas Intracelulares/ultraestrutura , Leucócitos/imunologia , Leucócitos/ultraestrutura , Masculino , Microcirculação/imunologia , Microcirculação/metabolismo , Microcirculação/ultraestrutura , Microscopia Eletrônica , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/ultraestrutura , Organelas/imunologia , Organelas/ultraestrutura , Peritonite/imunologia , Peritonite/metabolismo , Peritonite/patologia , Ratos , Ratos Sprague-DawleyRESUMO
Giardia lamblia, a primitive eukaryotic cell, lacks organelles such as mitochondria, peroxisomes, and a typical Golgi complex and presents a system of vesicles located below the plasma membrane. We used fluorescence and electron microscopy to better characterize the peripheral vesicles. Incubation of living cells with acridine orange showed that the peripheral vesicles correspond to an acidic compartment. Incubation with lucifer yellow, and with horseradish peroxidase, showed labeling of the peripheral vesicles even after several hours. Acid phosphatase was localized in the endoplasmic reticulum and in most of the peripheral vesicles. On the other hand, glucose 6-phosphatase, an endoplasmic reticulum marker, was observed in the endoplasmic reticulum cisternae and in some peripheral vesicles. A similar labeling pattern was observed using the zinc iodide technique, which reveals SH-containing proteins. Three-dimensional reconstruction and electron microscopy tomography of cells stained for acid phosphatase and glucose-6-phosphatase revealed the connection between some vesicles and profiles of the endoplasmic reticulum. Taken together, our observations suggest that trophozoites of G. lamblia present an endosomal-lysosomal system concentrated in a single system, the peripheral vesicles, which may represent an ancient organellar system that later on subdivided into compartments such as early and late endosomes and lysosomes.