RESUMO
The transmembrane emp24 domain-containing (TMED) proteins, also called p24 proteins, are members of a family of sorting receptors present in all representatives of the Eukarya and abundantly present in all subcompartments of the early secretory pathway, namely the endoplasmic reticulum (ER), the Golgi, and the intermediate compartment. Although essential during the bidirectional transport between the ER and the Golgi, there is still a lack of information regarding the TMED's structure across different subfamilies. Besides, although the presence of a TMED homo-oligomerization was suggested previously based on crystallographic contacts observed for the isolated Golgi Dynamics (GOLD) domain, no further analyses of its presence in solution were done. Here, we describe the first high-resolution structure of a TMED1 GOLD representative and its biophysical characterization in solution. The crystal structure showed a dimer formation that is also present in solution in a salt-dependent manner, suggesting that the GOLD domain can form homodimers in solution even in the absence of the TMED1 coiled-coil region. A molecular dynamics description of the dimer stabilization, with a phylogenetic analysis of the residues important for the oligomerization and a model for the orientation towards the lipid membrane, are also presented.
Assuntos
Complexo de Golgi/química , Simulação de Acoplamento Molecular , Filogenia , Proteínas de Transporte Vesicular/química , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Humanos , Domínios Proteicos , Termodinâmica , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
The cell surface and extracellular matrix polysaccharide, heparan sulfate (HS) conveys chemical information to control crucial biological processes. HS chains are synthesized in a non-template driven process mainly in the Golgi apparatus, involving a large number of enzymes capable of subtly modifying its substitution pattern, hence, its interactions and biological effects. Changes in the localization of HS-modifying enzymes throughout the Golgi were found to correlate with changes in the structure of HS, rather than protein expression levels. Following BFA treatment, the HS-modifying enzymes localized preferentially in COPII vesicles and at the trans-Golgi. Shortly after heparin treatment, the HS-modifying enzyme moved from cis to trans-Golgi, which coincided with increased HS sulfation. Finally, it was shown that COPI subunits and Sec24 gene expression changed. Collectively, these findings demonstrate that knowledge of the ER-Golgi dynamics of HS-modifying enzymes via vesicular trafficking is a critical prerequisite for the complete delineation of HS biosynthesis.
Assuntos
Vesículas Revestidas pelo Complexo de Proteína do Envoltório/enzimologia , Retículo Endoplasmático/enzimologia , Complexo de Golgi/enzimologia , Heparitina Sulfato/biossíntese , Transporte Biológico/efeitos dos fármacos , Brefeldina A/farmacologia , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/genética , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Retículo Endoplasmático/química , Retículo Endoplasmático/efeitos dos fármacos , Regulação da Expressão Gênica , Complexo de Golgi/química , Complexo de Golgi/efeitos dos fármacos , Heparina/farmacologia , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Plasmídeos/química , Plasmídeos/metabolismo , Cultura Primária de Células , Transfecção , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
Golgi Reassembly and Stacking Proteins (GRASPs) were firstly described as crucial elements in determining the structure of the Golgi complex. However, data have been accumulating over the years showing GRASPs can participate in various cell processes beyond the Golgi maintenance, including cell adhesion and migration, autophagy and unconventional secretion of proteins. A comprehensive understanding of the GRASP functions requires deep mechanistic knowledge of its structure and dynamics, especially because of the unique structural plasticity observed for many members of this family coupled with their high promiscuity in mediating protein-protein interactions. Here, we critically review data regarding the structural biophysics of GRASPs in the quest for understanding the structural determinants of different functionalities. We dissect GRASP structure starting with the full-length protein down to its separate domains (PDZ1, PDZ2 and SPR) and outline some structural features common to all members of the GRASP family (such as the presence of many intrinsically disordered regions). Although the impact of those exquisite properties in vivo will still require further studies, it is possible, from our review, to pinpoint factors that must be considered in future interpretation of data regarding GRASP functions, thus bringing somewhat new perspectives to the field.
Assuntos
Biofísica , Complexo de Golgi/ultraestrutura , Proteínas da Matriz do Complexo de Golgi/ultraestrutura , Conformação Proteica , Cristalografia por Raios X , Complexo de Golgi/química , Proteínas da Matriz do Complexo de Golgi/química , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/ultraestruturaRESUMO
Rab proteins are present in all eukaryotic lineages and regulate vesicular trafficking. Entamoeba histolytica has approximately 100 genes encoding Rab proteins, among which 16 have homology with human Rab proteins. Human Rab21 participates in integrin recycling, and thus amoebic Rab21 was believed to regulate the mobilization of Ehß1FNR (integrin-like fibronectin receptor related with human integrin ß1). We analyzed the distribution of EhRab21 using a polyclonal antibody produced with a specific peptide against the amoebic Rab protein, using confocal microscopy and specific probes for different organelles. EhRab21 was not associated with Ehß1FNR in fibronectin-stimulated trophozoites. However, EhRab21 was relocalized to lysosomes in erythrophagocytosis assays and was also found in Golgi-positive structures and the nuclear periphery. These results suggest that EhRab21, unlike its human homologue, is not present in the recycling pathway. However, according to the results, EhRab21 may regulate the trafficking between lysosomes and the Golgi apparatus.
Assuntos
Entamoeba histolytica/química , Entamoeba histolytica/fisiologia , Eritrócitos/metabolismo , Fagocitose , Proteínas rab de Ligação ao GTP/análise , Núcleo Celular/química , Complexo de Golgi/química , Lisossomos/químicaRESUMO
Given their obligate intracellular lifestyle, Chlamydia trachomatis ensure that they have access to multiple host sources of essential lipids by interfering with vesicular transport. These bacteria hijack Rab6-, Rab11- and Rab14-controlled trafficking pathways to acquire sphingomyelin from the Golgi complex. Another important source of sphingolipids, phospholipids and cholesterol are multivesicular bodies (MVBs). Despite their participation in chlamydial inclusion development and bacterial replication, the molecular mechanisms mediating the interaction between MVBs and chlamydial inclusions remain unknown. In the present study, we demonstrate that Rab39a labels a subset of late endocytic vesicles - mainly MVBs - that move along microtubules. Moreover, Rab39a is actively recruited to chlamydial inclusions throughout the pathogen life cycle by a bacterial-driven process that depends on the Rab39a GTP- or GDP-binding state. Interestingly, Rab39a participates in the delivery of MVBs and host sphingolipids to maturing chlamydial inclusions, thereby promoting inclusion growth and bacterial development. Taken together, our findings indicate that Rab39a favours chlamydial replication and infectivity. This is the first report showing that a late endocytic Rab GTPase is involved in chlamydial infection development.
Assuntos
Chlamydia trachomatis/metabolismo , Corpos Multivesiculares/metabolismo , Esfingolipídeos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Chlamydia trachomatis/patogenicidade , Colesterol/metabolismo , Complexo de Golgi/química , Células HeLa , Interações Hospedeiro-Patógeno/genética , Humanos , Corpos Multivesiculares/microbiologia , Fosfolipídeos/metabolismo , Esfingomielinas/metabolismo , Vesículas Transportadoras/metabolismoRESUMO
Tritrichomonas foetus is a protist that causes bovine trichomoniasis and presents a well-developed Golgi. There are very few studies concerning the Golgi in trichomonads. In this work, monoclonal antibodies were raised against Golgi of T. foetus and used as a tool on morphologic and biochemical studies of this organelle. Among the antibodies produced, one was named mAb anti-Golgi 20.3, which recognized specifically the Golgi complex by fluorescence and electron microscopy. By immunoblotting this antibody recognized two proteins with 60 and 66 kDa that were identified as putative beta-tubulin and adenosine triphosphatase, respectively. The mAb 20.3 also recognized the Golgi complex of the Trichomonas vaginalis, a human parasite. In addition, the nucleotide coding sequences of these proteins were identified and included in the T. foetus database, and the 3D structure of the proteins was predicted. In conclusion, this study indicated: (1) adenosine triphosphatase is present in the Golgi, (2) ATPase is conserved between T. foetus and T. vaginalis, (3) there is new information concerning the nucleic acid sequences and protein structures of adenosine triphosphatase and beta-tubulin from T. foetus and (4) the mAb anti-Golgi 20.3 is a good Golgi marker and can be used in future studies.
Assuntos
Adenosina Trifosfatases/metabolismo , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Complexo de Golgi/ultraestrutura , Infecções Protozoárias em Animais/parasitologia , Tritrichomonas foetus/ultraestrutura , Adenosina Trifosfatases/química , Adenosina Trifosfatases/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Sequência de Bases , Bovinos , Feminino , Complexo de Golgi/química , Complexo de Golgi/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão/veterinária , Microscopia de Fluorescência/veterinária , Modelos Moleculares , Proteínas de Protozoários/química , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Alinhamento de Sequência/veterinária , Análise de Sequência de DNA/veterinária , Trichomonas vaginalis/enzimologia , Trichomonas vaginalis/imunologia , Tritrichomonas foetus/enzimologia , Tritrichomonas foetus/genética , Tritrichomonas foetus/imunologiaRESUMO
Brucella abortus elicits a vigorous Th1 immune response which activates cytotoxic T lymphocytes. However, B. abortus persists in its hosts in the presence of CD8(+) T cells, establishing a chronic infection. Here, we report that B. abortus infection of human monocytes/macrophages inhibited the IFN-γ-induced MHC-I cell surface expression. This phenomenon was dependent on metabolically active viable bacteria. MHC-I down-modulation correlated with the development of diminished CD8(+) cytotoxic T cell response as evidenced by the reduced expression of the activation marker CD107a on CD8(+) T lymphocytes and a diminished percentage of IFN-γ-producing CD8(+) T cells. Inhibition of MHC-I expression was not due to changes in protein synthesis. Rather, we observed that upon B. abortus infection MHC-I molecules were retained within the Golgi apparatus. Overall, these results describe a novel mechanism based on the intracellular sequestration of MHC-I molecules whereby B. abortus would avoid CD8(+) cytotoxic T cell responses, evading their immunological surveillance.
Assuntos
Brucella abortus/imunologia , Brucella abortus/fisiologia , Linfócitos T CD8-Positivos/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Evasão da Resposta Imune , Macrófagos/imunologia , Macrófagos/microbiologia , Células Cultivadas , Complexo de Golgi/química , Humanos , Interferon gama/metabolismo , Transporte ProteicoRESUMO
Proteoglycans encompass a heterogeneous group of glycoconjugates where proteins are substituted with linear, highly negatively charged glycosaminoglycan chains. Sulphated glycosaminoglycans are ubiquitous to the animal kingdom of the Eukarya domain. Information on the distribution and characterisation of proteoglycans in invertebrate tissues is limited and restricted to a few species. By the use of multidimensional protein identification technology and immunohistochemistry, this study shows for the first time the presence and tissue localisation of different proteoglycans, such as perlecan, aggrecan, and heparan sulphate proteoglycan, amongst others, in organs of the gastropoda Achatina fulica. Through a proteomic analysis of Golgi proteins and immunohistochemistry of tissue sections, we detected the machinery involved in glycosaminoglycan biosynthesis, related to polymer formation (polymerases), as well as secondary modifications (sulphation and uronic acid epimerization). Therefore, this work not only identifies both the proteoglycan core proteins and glycosaminoglycan biosynthetic enzymes in invertebrates but also provides a novel method for the study of glycosaminoglycan and proteoglycan evolution.
Assuntos
Enzimas/análise , Proteoglicanas/biossíntese , Proteoglicanas/química , Proteômica/métodos , Caramujos/metabolismo , Animais , Enzimas/química , Enzimas/metabolismo , Glicosaminoglicanos/química , Glicosaminoglicanos/metabolismo , Complexo de Golgi/química , Complexo de Golgi/metabolismo , Modelos Animais , Proteoglicanas/análise , Proteoma/análise , Caramujos/química , Caramujos/genética , Caramujos/ultraestrutura , Distribuição Tecidual , Vertebrados/metabolismoRESUMO
Although the molecular and cellular basis of particular events that lead to the biogenesis of membranes in eukaryotic cells has been described in detail, understanding of the intrinsic complexity of the pleiotropic response by which a cell adjusts the overall activity of its endomembrane system to accomplish these requirements is limited. Here we carried out an immunocytochemical and biochemical examination of the content and quality of the endoplasmic reticulum (ER) and Golgi apparatus membranes in two in vivo situations characterized by a phase of active cell proliferation followed by a phase of declination in proliferation (rat brain tissue at early and late developmental stages) or by permanent active proliferation (gliomas and their most malignant manifestation, glioblastomas multiforme). It was found that, in highly proliferative phases of brain development (early embryo brain cells), the content of ER and Golgi apparatus membranes, measured as total lipid phosphorous content, is higher than in adult brain cells. In addition, the concentration of protein markers of ER and Golgi is also higher in early embryo brain cells and in human glioblastoma multiforme cells than in adult rat brain or in nonpathological human brain cells. Results suggest that the amount of endomembranes and the concentration of constituent functional proteins diminish as cells decline in their proliferative activity.
Assuntos
Encéfalo/citologia , Proliferação de Células , Retículo Endoplasmático/química , Complexo de Golgi/química , Membranas Intracelulares/química , Animais , Western Blotting , Encéfalo/crescimento & desenvolvimento , Encéfalo/fisiologia , Encéfalo/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Feminino , Glioblastoma/química , Glioblastoma/metabolismo , Glioblastoma/patologia , Complexo de Golgi/ultraestrutura , Humanos , Imuno-Histoquímica , Membranas Intracelulares/ultraestrutura , Lipídeos de Membrana/análise , Proteínas de Membrana/análise , Fósforo/análise , Ratos , Ratos WistarRESUMO
Reactionary dentine and reparative dentine are two strategies used by the dentine-pulp complex to respond to injury. The reactionary dentine is secreted by original odontoblasts, while the reparative dentine is formed by odontoblast-like cells. Osteopontin (OPN) is a non-collagenous protein usually present in the repair of mineralized tissues. It is likely to be present in newly formed dentine but there are no studies attempting to detect it in reactionary and reparative dentine. The aim of the present study was to examine the ultrastructural characteristics, as well as the presence and distribution of OPN in reactionary and reparative dentine by provoking extrusion of the rat incisor. The right upper incisors of 3-month-old male rats were extruded 3 mm and then repositioned into their original sockets. At 3, 7, 10, 15, 20, 30 and 60 days after surgery, the incisors were fixed in glutaraldehyde-formaldehyde and then processed for scanning and transmission electron microscopy and for immunocytochemistry for OPN. After extrusive trauma, the dentine-pulp interface showed the presence of reactionary and reparative dentine, which varied in aspect, thickness and related cells. OPN was not detected in the physiological and reactionary dentine, while it was strongly immunoreactive in the matrix that surrounded the entrapped cells of reparative dentine. In addition, original odontoblasts subjacent to the physiological dentine contained OPN in their Golgi region. The present findings showed that reparative dentine shares some structural characteristics with primary bone, especially in relation to its OPN content. The odontoblast-like cells resemble osteoblasts rather than odontoblasts.
Assuntos
Dentina/lesões , Dentinogênese , Incisivo/lesões , Osteopontina/análise , Cicatrização , Animais , Dentina/química , Dentina/ultraestrutura , Complexo de Golgi/química , Complexo de Golgi/ultraestrutura , Imuno-Histoquímica , Masculino , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Osteoblastos/química , Osteoblastos/ultraestrutura , Distribuição Aleatória , Ratos , Ratos WistarRESUMO
GEM (glycosphingolipid-enriched microdomains) are specialized detergent-resistant domains of the plasma membrane in which some gangliosides concentrate. Although genesis of GEM is considered to occur in the Golgi complex, where the synthesis of gangliosides also occurs, the issue concerning the incorporation of ganglioside species into GEM is still poorly understood. In this work, using Chinese hamster ovary K1 cell clones with different glycolipid compositions, we compared the behaviour with cold Triton X-100 solubilization of plasma membrane ganglioside species with the same species newly synthesized in Golgi membranes. We also investigated whether three ganglioside glycosyltransferases (a sialyl-, a N-acetylgalactosaminyl- and a galactosyl-transferase) are included or excluded from GEM in Golgi membranes. Our data show that an important fraction of plasma membrane G(M3), and most G(D3) and G(T3), reside in GEM. Immunocytochemical examination of G(D3)-expressing cells showed G(D3) to be distributed as cold-detergent-resistant patches in the plasma membrane. These patches did not co-localize with a glycosylphosphatidylinositol-anchored protein used as GEM marker, indicating a heterogeneous composition of plasma membrane GEM. In Golgi membranes we were unable to find evidence for GEM localization of either ganglioside glycosyltransferases or newly synthesized gangliosides. Since the same ganglioside species appear in plasma membrane GEM, it was concluded that in vivo nascent G(D3), G(T3) and G(M3) segregate from their synthesizing transferases and then enter GEM. This latter event could have taken place shortly after synthesis in the Golgi cisternae, along the secretory pathway and/or at the cell surface.
Assuntos
Detergentes/química , Gangliosídeos/biossíntese , Gangliosídeos/metabolismo , Glicosiltransferases/metabolismo , Complexo de Golgi/química , Membranas Intracelulares/química , Animais , Células CHO/química , Células CHO/enzimologia , Células CHO/metabolismo , Extratos Celulares/química , Linhagem Celular , Membrana Celular/química , Cricetinae , Complexo de Golgi/enzimologia , Humanos , Membranas Intracelulares/enzimologia , Microdomínios da Membrana/química , Octoxinol/metabolismo , Sialiltransferases/biossínteseRESUMO
We present observations on the fine structure and the division process of the Golgi complex in the protists Trichomonas vaginalis and Tritrichomonas foetus, parasites of the urogenital tract of humans and cattle, respectively. The Golgi in trichomonads is a prominent structure, associated with striated parabasal filaments to which this organelle seems to be connected. We followed by immunofluorescence and electron microscopy the Golgi in interphasic and mitotic cells. Ultrastructural studies were performed using fast-freezing fixation, immunocytochemistry using antisera to the known adhesins AP65 and AP51, cytochemistry (acid phosphatase, Ca++-ATPase, zinc iodide-osmium tetroxide technique (ZIO), for analysis of distribution of the endoplasmic reticulum and Golgi complex, and Thiéry's techniques), routine and serial thin-sections. Three-dimensional reconstruction, NBD-ceramide, fluorescent lectin (WGA) and nocodazole treatments were also used. We demonstrate that: (1) the Golgi in trichomonads is a single-copy organelle; (2) presents a fenestrated structure; (3) is formed by 8-12 saccules; (4) is connected to the parabasal filaments by thin filamentous bridges; (5) by cytochemistry, presents a positive reaction for the lectin WGA, Ca++-ATPase, acid phosphatase, ZIO and Thiéry's techniques; (6) does not appear to break down at any point of the cell cycle; (7) elongates during the cell cycle by lateral growth; (8) is labeled by anti-glutamylated tubulin antibodies, but it is not fragmented by nocodazole treatment; (9) before mitosis, the already elongated Golgi ribbon undergoes progressive medial fission, cisternae by cisternae, starting at the cisternae adjacent to the cell surface and ending with the cis-most cisternae; (10) the Golgikinesis originates two small Golgi ribbons; (11) the Golgi is intensely labeled with the antisera to the AP65 and AP51 adhesins in T. vaginalis, thus seeming to be a key station in the production of adhesins.
Assuntos
Complexo de Golgi/ultraestrutura , Trichomonas vaginalis/ultraestrutura , Tritrichomonas foetus/ultraestrutura , Fosfatase Ácida/metabolismo , Animais , ATPases Transportadoras de Cálcio/metabolismo , Bovinos , Ciclo Celular/fisiologia , Simulação por Computador , Técnica de Fratura por Congelamento , Substituição ao Congelamento , Complexo de Golgi/química , Complexo de Golgi/metabolismo , Humanos , Trichomonas vaginalis/metabolismo , Tritrichomonas foetus/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
The plant alkaloid ryanodine (Ry) is a high-affinity modulator of ryanodine receptor (RyR) Ca(2+) release channels. Although these channels are present in a variety of cell types, their functional role in nerve cells is still puzzling. Here, a monosubstituted fluorescent Ry analogue, B-FL-X Ry, was used to reveal the distribution of RyRs in cultured rat sympathetic neurons. B-FL-X Ry competitively inhibited the binding of [3H]Ry to rabbit skeletal muscle SR membranes, with an IC(50) of 150 nM, compared to 7 nM of unlabeled Ry. Binding of B-FL-X Ry to the cytoplasm of sympathetic neurons is saturable, reversible and of high affinity. The pharmacology of B-FL-X Ry showed marked differences with unlabeled Ry, which are partially explained by its lower affinity: (1) use-dependent reversible inhibition of caffeine-induced intracellular Ca(2+) release; (2) diminished voltage-gated Ca(2+) influx, due to a positive shift in the activation of voltage gated Ca(2+) currents. B-FL-X Ry-stained sympathetic neurons, viewed under confocal microscopy, showed conspicuous labeling of crescent-shaped structures pertaining to the Golgi complex, a conclusion supported by experiments showing co-localization with Golgi-specific fluorescent probes and the breaking up of crescent-shaped staining after treatment with drugs that disassemble Golgi complex. The presence of RyRs to the Golgi could be confirmed with specific anti-RyR(2) antibodies, but evidence of caffeine-induced Ca(2+) release from this organelle could not be obtained using fast confocal microscopy. Rather, an apparent decrease of the cytosolic Ca(2+) signal was detected close to this organelle. In spite of that, short-term incubation with brefeldin A (BFA) suppressed the fast component of caffeine-induced Ca(2+) release, and the Ca(2+) release process lasted longer and appeared less organized. These observations, which suggest a possible role of the Golgi complex in Ca(2+) homeostasis and signaling in nerve cells, could be relevant to reports involving derangement of the Golgi complex as a probable cause of some forms of progressive neuronal degeneration, such as Alzheimer's disease and amyotrophic lateral sclerosis.
Assuntos
Sinalização do Cálcio/fisiologia , Complexo de Golgi/metabolismo , Neurônios/fisiologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Rianodina/análogos & derivados , Rianodina/metabolismo , Animais , Antibacterianos/farmacologia , Especificidade de Anticorpos , Compostos de Boro/metabolismo , Brefeldina A/farmacologia , Cafeína/farmacologia , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Corantes Fluorescentes , Complexo de Golgi/química , Macrolídeos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Neurônios/química , Neurônios/ultraestrutura , Inibidores de Fosfodiesterase/farmacologia , Ratos , Canal de Liberação de Cálcio do Receptor de Rianodina/análise , Canal de Liberação de Cálcio do Receptor de Rianodina/imunologia , Gânglio Cervical Superior/citologiaRESUMO
The temporal subcellular localization of the structural proteins VP2 and VP3 of infectious pancreatic necrosis virus was analyzed with monoclonal antibodies conjugated with fluorochromes. Early in the infection both proteins were colocalized in the cytosol, at later times, VP2 was visualized as inclusion bodies around the nuclei of the cells and, sometimes, it was found in elongated tubular structures that might correspond to the type I tubules seen in cells infected with another Birnavirus. As VP2 is a glycosylated protein we determined its subcellular localization compared with that of ER and Golgi probes. These results suggest that VP2 is glycosylated freely in the cytoplasm.
Assuntos
Capsídeo/análise , Vírus da Necrose Pancreática Infecciosa/química , Animais , Proteínas do Capsídeo , Células Cultivadas , Retículo Endoplasmático/química , Glicosilação , Complexo de Golgi/química , Salmão , Replicação ViralRESUMO
Both mitotic and apoptotic cells display hypercondensation of the chromatin and loss of the nuclear envelope (Lazebnik et al., 1993). Herein, we describe a third similarity between the two processes. We have observed, initially in apoptotic cells of the PC-12 lineage clusters of 40-60 (approximately 50) nm vesicles adjoined by a minor contingent of tubule vesicular elements of 100-200 nm which are indistinguishable from their vesicular counterparts in mitotic PC-12 cells. The clusters of approximately 50 nm vesicles were subsequently observed in all studied rat tissue cells in apoptosis (plasma cells and macrophages, secretory epithelial cells from pancreatic acini, ventral lobe of prostate and mammary gland). Clusters of approximately 50 nm vesicles comparable to those of the PC-12 cells were found in HeLa cells treated with human alfa TNF, in WEHI-3 cells exposed to VM 26 (a teneposide) (Sesso et al., 1997) and in HL-60 cells treated with thapsigargin. PC-12 and HeLa cells affixed to coverslips were double labelled and examined with the fluorescence microscope to reveal simultaneously the disposition of the chromatin with Hoechst stain and the distribution of the fluorescence of Golgi or of Golgi-associated proteins. A common pattern of fluorescence was observed in a minor proportion of apoptotic cells using three different antibodies used. The label frequently appeared as finely dispersed granules in the cytoplasm. In some apoptotic cells, relatively coarse granules were observed. This pattern of label distribution is compatible with the disposition of vesicular clusters we have encountered in apoptotic PC-12 cells sectioned serially or semi serially. In such sections of both mitotic and apoptotic PC-12 cells, we noticed that the conglomerates of 50 nm vesicles were frequently associated with cisternae of the rough ER. Vesicles of similar size were also noted pinching off from the extremities of Golgi cisternae reduced in size. These cisternae diminish in length and width when they are in the process of disassembling at the very beginning of mitosis and in apoptosis.
Assuntos
Apoptose/fisiologia , Mitose/fisiologia , Animais , Cromatina/ultraestrutura , Citoplasma/ultraestrutura , Retículo Endoplasmático Rugoso/ultraestrutura , Feminino , Imunofluorescência , Complexo de Golgi/química , Complexo de Golgi/ultraestrutura , Células HL-60 , Células HeLa/ultraestrutura , Humanos , Masculino , Microscopia Eletrônica de Transmissão e Varredura , Células PC12/ultraestrutura , RatosRESUMO
Euglena gracilis, a unicellular flagellated alga, can display numerous shape changes. These changes are most probably caused by a pellicle and an internal cytoskeleton. In this paper we studied the distribution of the cytoskeletal proteins actin, alpha-actinin, tropomyosin and tubulin in dark-adapted Euglena, using immunofluorescence microscopy. We found that F-actin, alpha-actinin, tropomyosin and tubulin have a distribution that is coincident in the plasma membrane and, in addition, alpha-actinin and tropomyosin are seen in small patches in the cytoplasm, and tubulin in the flagella. We have also studied the distribution of the endoplasmic reticulum, nucleus, and Golgi apparatus of these cells, using fluorescent probes. Both the endoplasmic reticulum and the Golgi apparatus have a meshwork pattern distributed throughout the cytoplasm, and the nucleus has a chromatin evenly distributed in the nucleoplasm.
Assuntos
Actinina/análise , Actinas/análise , Euglena gracilis/química , Tropomiosina/análise , Tubulina (Proteína)/análise , Animais , Clorofila/análise , Citoesqueleto/química , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Complexo de Golgi/química , Microscopia de FluorescênciaRESUMO
Using cell-fractionation techniques (differential and discontinuous gradient centrifugation), we obtained a highly enriched fraction containing the Golgi complex of Tritrichomonas foetus. This fraction was further subfractionated by sodium carbonate (150 mM) treatment at pH 11.5, leading to the isolation of the Golgi content and membrane subfractions. Both fractions were characterized by electron microscopy. The protein content of membrane and luminal subfractions was about 40% and 60% of the total Golgi protein, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed an enrichment of 15 protein bands in the Golgi fraction, with molecular masses varying between 15 and 116 kDa. Alkaline treatment released some proteins into the medium, but most of them were associated with the Golgi membrane.
Assuntos
Complexo de Golgi/ultraestrutura , Proteínas de Protozoários/análise , Tritrichomonas foetus/ultraestrutura , Animais , Fracionamento Celular/métodos , Centrifugação Zonal/métodos , Eletroforese em Gel de Poliacrilamida/métodos , Complexo de Golgi/química , Tritrichomonas foetus/químicaRESUMO
Caltrins, the small, basic proteins of the seminal vesicle secretion that inhibit calcium transport into epididymal spermatozoa, and consequently the onset of the acrosome reaction and the hyperactivated motility, were localized in the epithelial cells and the lumen of the seminal vesicles of the guinea pig by an immunocytochemical procedure and electron microscopy. Rabbit antisera against each protein (caltrin I or II), and goat anti-rabbit IgG antiserum labeled with colloidal gold were used to detect the caltrin immunoreaction. The subcellular distribution of the gold labeling was occasionally localized in the rough endoplasmic reticulum but mainly within big secretory vacuoles containing low electron-dense material, which are components of the Golgi complex known as condensing vacuoles. These are involved in the intracellular transport, storage, and discharge of secretory proteins. Gold-labeled material released to the lumen was also detected. There was no clear evidence that the discharge was mediated by an exocytotic process. Immunoreaction was observed neither in the electron-dense core nor in the electron-lucent halo of the typical secretory granules of the epithelial cells of the seminal vesicles. Using light microscope immunocytochemistry, intense positive immunoreactivity was detected in the material secreted to the lumen but not on the epithelial cell layer. Only those cells undergoing a degenerative process and showing a picnotic nucleus and condensed cytoplasmic matrix exhibited detectable immunoreaction when gold label and silver intensification were applied. The same distribution of the immunoprobes was obtained by electron or light microscopy when antiserum to either I or II was used. It would appear that the two caltrin proteins of the guinea pig are synthesized in the rough endoplasmic reticulum of the epithelial cells and transported quickly to the Golgi complex where the secretory vacuoles (condensing vacuoles) are formed. The proteins are transported by the secretory vacuoles to the apical ends of the cells to be discharged into the lumen.
Assuntos
Proteínas Secretadas pela Próstata , Proteínas/análise , Glândulas Seminais/química , Animais , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/ultraestrutura , Retículo Endoplasmático Rugoso/química , Retículo Endoplasmático Rugoso/ultraestrutura , Epitélio/química , Epitélio/metabolismo , Epitélio/ultraestrutura , Complexo de Golgi/química , Complexo de Golgi/ultraestrutura , Cobaias , Imuno-Histoquímica , Masculino , Microscopia Imunoeletrônica , Proteínas/metabolismo , Proteínas de Plasma Seminal , Glândulas Seminais/metabolismo , Glândulas Seminais/ultraestruturaRESUMO
Rabbits aged 1, 4, 10, 15, and 20 days, and 4 months were anesthetized and perfused with 4% formaldehyde. One eye of each rabbit was processed for paraffin embedding, while the other eye was embedded intact in methacrylate. Rabbits aged 1 and 15 days and 4 months were perfused with 2.5% glutaraldehyde, and the eyes were processed for Epon embedding. The paraffin sections were immunostained to allow detection of a high molecular weight cartilage matrix glycoprotein (CMGP), which is synthesized by the ciliary body and found in the vitreous in adult animals, using a specific mouse monoclonal antibody. CMGP was identified in the vitreous and in the inner layer of the ciliary epithelium only after the fifteenth day of life in amounts comparable to those detected in adult rabbits. Before this time immunostaining with the monoclonal antibody was seen only in the apical region of the inner ciliary epithelial cells. However, electron microscopic observations revealed that the cytoplasmic organelles responsible for the secretion of glycoproteins, i.e. the rough endoplasmic reticulum, Golgi apparatus, and vesicles, were present in the inner layer of ciliary epithelial cells as early as the first day of life. Anteroposterior sections of whole eyes embedded in methacrylate revealed a relatively dense meshwork of vitreous fibrils on the first day of life. The blood vessels were concentrated at the posterior region of the lens, and isolated cells were visible. The blood vessels were not seen after the age of 15 days, and the fiber meshwork and cells were inconspicuous by then.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Proteínas da Matriz Extracelular , Corpo Vítreo/ultraestrutura , Animais , Vasos Sanguíneos/ultraestrutura , Corpo Ciliar/ultraestrutura , Retículo Endoplasmático/química , Glicoproteínas/análise , Complexo de Golgi/química , Proteínas Matrilinas , Microscopia Eletrônica , Coelhos , Fatores de Tempo , Corpo Vítreo/química , Corpo Vítreo/crescimento & desenvolvimentoRESUMO
Heparan sulphate proteoglycan is the predominant proteoglycan synthesized by the parenchymal cells of the rat submandibular gland. A polyclonal antibody was used to localize this proteoglycan in the adult rat submandibular gland. Localization was accomplished by indirect immunoperoxidase cytochemistry at the light and electron microscopic levels. Heparan sulphate proteoglycan was localized in a continuous, linear pattern in the lamina densa of the basement membrane surrounding all of the epithelial components of the gland as well as the basement membrane of the capillaries and small arterioles in the glandular stroma. In addition, heparan sulphate proteoglycan was seen in vesicles and pits along the acinar cell basal plasmalemma adjacent to the basement membrane and in the endoplasmic reticulum and Golgi apparatus of the acinar cells.