-
Archives of Histology and Cytology Aug 2002One hundred years have passed since the discovery of "the internal reticular apparatus" by Camillo GOLGI. Investigations into the structure and function of the "Golgi... (Review)
Review
One hundred years have passed since the discovery of "the internal reticular apparatus" by Camillo GOLGI. Investigations into the structure and function of the "Golgi apparatus" have raised more and more challenging issues for cell biologists. After long debate, many new findings have accumulated in the last 10 years as a result of the availability of elegant new genetic, biochemical and morphological tools. This, in turn, has raised many new questions to be solved. In addition, numerous new findings have led to some confusion on the understanding of the Golgi apparatus. This review article deals with several modern aspects of vesicular transport versus cisternal maturation. Disruption of the stacked structure in mitotic and drug-induced conditions is also discussed to demonstrate the importance of structural integrity in the Golgi apparatus.
Topics: Animals; Brefeldin A; Endoplasmic Reticulum; Golgi Apparatus; Membrane Proteins; Mitosis; Models, Biological; Models, Structural; Okadaic Acid; Transport Vesicles
PubMed: 12389660
DOI: 10.1679/aohc.65.209 -
Cellular and Molecular Life Sciences :... Sep 2008Cholesterol, certain lipids, membrane-bound and soluble proteins, as well as viruses that are synthesized in the endoplasmic reticulum (ER), reach the plasma membrane... (Review)
Review
Cholesterol, certain lipids, membrane-bound and soluble proteins, as well as viruses that are synthesized in the endoplasmic reticulum (ER), reach the plasma membrane (PM) via non-classical pathway(s) that remain poorly understood. Typical for this transport is (i) its insensitivity to brefeldin A (BFA), which dissociates selected coat complexes from membranes, resulting in the disassembly of the Golgi apparatus; (ii) its rapid kinetics as compared to the classical secretory pathway; and (iii) its role in the trafficking of lipid raft components. Based on results showing that the intermediate compartment (IC) at the ER-Golgi boundary constitutes a stable tubular network that maintains its dynamics in the presence of BFA, we propose that two bidirectional Golgi-bypass pathways to the PM exist, a direct route from early IC elements, and another, reminiscent of the yeast secretory pathway, from late IC elements via the endosomal system. These pathways have implications for the organization of the secretory processes in different cell types.
Topics: Biological Transport; Brefeldin A; Cell Membrane; Golgi Apparatus; Humans; Membrane Proteins; Protein Synthesis Inhibitors; Protein Transport; Vacuoles
PubMed: 18726174
DOI: 10.1007/s00018-008-8355-0 -
FEMS Immunology and Medical Microbiology Sep 2002Paecilomyces sp. and Aspergillus clavatus, which were isolated from Taxus mairei and Torreya grandis from southeast China, produced toxic metabolites when grown in...
Paecilomyces sp. and Aspergillus clavatus, which were isolated from Taxus mairei and Torreya grandis from southeast China, produced toxic metabolites when grown in liquid culture. Nuclear magnetic resonance techniques, infrared spectrometry, electrospray ionization mass spectroscopy and X-ray analysis identified brefeldin A, a bioactive metabolite produced by a number of fungal species belonging to the genera Alternaria, Ascochyta, Penicillium, Curvularia, Cercospora and Phyllosticta. This is the first report of the isolation of the cytotoxin from Paecilomyces sp. and A. clavatus. The relevance of brefeldin A to the association between these fungi and their host plants is discussed.
Topics: Aspergillus; Brefeldin A; Cell Line; China; Cytotoxins; Ecosystem; Humans; Microscopy, Electron, Scanning; Molecular Structure; Mycotoxins; Paecilomyces; Plants, Medicinal; Taxus; Tracheophyta
PubMed: 12208606
DOI: 10.1111/j.1574-695X.2002.tb00602.x -
STAR Protocols Mar 2021Characterizing cytokine production is important for properly understanding immunologic responses. Cytokine reporter mice are limited by the need to cross markers into...
Characterizing cytokine production is important for properly understanding immunologic responses. Cytokine reporter mice are limited by the need to cross markers into various knockout backgrounds and by availability of reporters of interest. To overcome this, we utilize injection of brefeldin A into mice to enable flow cytometric analysis of cytokine production during a bacterial infection. While we evaluate IFN-γ production during infection, this protocol can be applied to other cytokines and other mouse models. For complete details on the use and execution of this protocol, please refer to Kovacs et al. (2020) and Liu and Whitton (2005).
Topics: Animals; Brefeldin A; Burkholderia; Burkholderia Infections; Flow Cytometry; Interferon-gamma; Mice
PubMed: 33458706
DOI: 10.1016/j.xpro.2020.100244 -
Genes Sep 2022Interleukin 17F (IL17F) has been found to be involved in various inflammatory pathologies and has recently become a target for therapeutic purposes. In contrast to IL17F...
Interleukin 17F (IL17F) has been found to be involved in various inflammatory pathologies and has recently become a target for therapeutic purposes. In contrast to IL17F secreted by immune cells, the focus of this study is to describe the triggers of IL17F release in non-immune cells with a particular focus on IL17F-induced fibrosis. IL17F induction was examined in human lung epithelial (BEAS-2B) and myeloid cell lines as well as in peripheral blood mononuclear cells after in vitro exposure to aqueous cigarette smoke extract (CSE), inorganic mercury, cadmium or the apoptosis inducer brefeldin A. Fibrosis was examined in vitro, evaluating the transition of human primary dermal fibroblasts to myofibroblasts. We observed that all stressors were able to induce IL17F gene expression regardless of cell type. Interestingly, its induction was associated with cytotoxic/apoptotic signs. Inhibiting oxidative stress by N-acetylcysteine abrogated CSE-induced cytotoxic and -inducing effects. The induction of was accompanied by IL17F protein expression. The transition of fibroblasts into myofibroblasts was not influenced by either recombinant IL17F or supernatants of CSE-exposed BEAS-2B. In addition to IL17F secretion by specialized or activated immune cells, we underscored the cell type-independent induction of IL17F by mechanisms of inhibitable oxidative stress-induced cytotoxicity. However, IL17F was not involved in dermal fibrosis under the conditions used in this study.
Topics: Humans; Acetylcysteine; Interleukin-17; Leukocytes, Mononuclear; Brefeldin A; Cadmium; Apoptosis; Oxidative Stress; Nicotiana; Fibrosis; Mercury
PubMed: 36292624
DOI: 10.3390/genes13101739 -
American Journal of Physiology.... Sep 2018Manganese (Mn) toxicity arises from nutritional problems, community and occupational exposures, and genetic risks. Mn blood levels are controlled by hepatobiliary...
Manganese (Mn) toxicity arises from nutritional problems, community and occupational exposures, and genetic risks. Mn blood levels are controlled by hepatobiliary clearance. The goals of this study were to determine the cellular distribution of Mn transporters in polarized hepatocytes, to establish an in vitro assay for hepatocyte Mn efflux, and to examine possible roles the Mn transporters would play in metal import and export. For these experiments, hepatocytoma WIF-B cells were grown for 12-14 days to achieve maximal polarity. Immunoblots showed that Mn transporters ZIP8, ZnT10, ferroportin (Fpn), and ZIP14 were present. Indirect immunofluorescence microscopy localized Fpn and ZIP14 to WIF-B cell basolateral domains whereas ZnT10 and ZIP8 associated with intracellular vesicular compartments. ZIP8-positive structures were distributed uniformly throughout the cytoplasm, but ZnT10-positive vesicles were adjacent to apical bile compartments. WIF-B cells were sensitive to Mn toxicity, showing decreased viability after 16 h exposure to >250 μM MnCl. However, the hepatocytes were resistant to 4-h exposures of up to 500 μM MnCl despite 50-fold increased Mn content. Washout experiments showed time-dependent efflux with 80% Mn released after a 4 h chase period. Hepcidin reduced levels of Fpn in WIF-B cells, clearing Fpn from the cell surface, but Mn efflux was unaffected. The secretory inhibitor, brefeldin A, did block release of Mn from WIF-B cells, suggesting vesicle fusion may be involved in export. These results point to a possible role of ZnT10 to import Mn into vesicles that subsequently fuse with the apical membrane and empty their contents into bile. NEW & NOTEWORTHY Polarized WIF-B hepatocytes express manganese (Mn) transporters ZIP8, ZnT10, ferroportin (Fpn), and ZIP14. Fpn and ZIP14 localize to basolateral domains. ZnT10-positive vesicles were adjacent to apical bile compartments, and ZIP8-positive vesicles were distributed uniformly throughout the cytoplasm. WIF-B hepatocyte Mn export was resistant to hepcidin but inhibited by brefeldin A, pointing to an efflux mechanism involving ZnT10-mediated uptake of Mn into vesicles that subsequently fuse with and empty their contents across the apical bile canalicular membrane.
Topics: Animals; Biological Transport; Brefeldin A; Cation Transport Proteins; Cell Line; Cell Membrane; Cell Polarity; Cytoplasmic Vesicles; Hepatocytes; Hepcidins; Humans; Manganese; Protein Synthesis Inhibitors
PubMed: 29792530
DOI: 10.1152/ajpgi.00103.2018 -
Virology Jul 2007Brefeldin A is a macrolide compound that interferes with the secretory pathway and also affects protein synthesis in mammalian cells. As a result, this antibiotic...
Brefeldin A is a macrolide compound that interferes with the secretory pathway and also affects protein synthesis in mammalian cells. As a result, this antibiotic impedes the maturation of viral glycoproteins of enveloped viruses and viral genome replication in several virus species. In the present work, we show that translation of subgenomic mRNA from Sindbis virus, which in contrast to cellular translation is resistant to brefeldin A after prolonged treatment. The phosphorylation of eIF2alpha as a result of brefeldin A treatment correlates with the inhibition of cellular translation, while late viral protein synthesis is resistant to this phosphorylation. The effect of brefeldin A on Sindbis virus replication was also examined using a Sindbis virus replicon. Although brefeldin A delayed viral RNA synthesis, translation by non-replicative viral RNAs was not affected, reinforcing the idea that brefeldin A delays viral RNA replication, but does not directly affect Sindbis virus protein synthesis.
Topics: Alphavirus Infections; Animals; Brefeldin A; Cell Line; Eukaryotic Initiation Factor-2; Phosphorylation; Protein Biosynthesis; Protein Synthesis Inhibitors; RNA, Messenger; RNA, Viral; Replicon; Sindbis Virus; Viral Proteins; Virus Replication
PubMed: 17360015
DOI: 10.1016/j.virol.2007.02.001 -
Proceedings of the National Academy of... Feb 2005Guanine nucleotide-exchange proteins activate ADP-ribosylation factors by accelerating the replacement of bound GDP with GTP. Mammalian brefeldin A-inhibited guanine...
Guanine nucleotide-exchange proteins activate ADP-ribosylation factors by accelerating the replacement of bound GDP with GTP. Mammalian brefeldin A-inhibited guanine nucleotide-exchange proteins, BIG1 and BIG2, are important activators of ADP-ribosylation factors for vesicular trafficking. To identify proteins that interact with BIG2, we used cDNA constructs encoding BIG2 sequences in a yeast two-hybrid screen of a human heart library. Clone p2-5-3, encoding a form of human exocyst protein Exo70, interacted with BIG2 amino acids 1-643 and 1-832, but not 644-832, which was confirmed by coimmunoprecipitation of in vitro-translated BIG2 N-terminal segments and 2-5-3. By immunofluorescence microscopy, endogenous BIG2 and Exo70 in HepG2 cells were visualized at Golgi membranes and apparently at the microtubule-organizing center (MTOC). Both were identified in purified centrosomes. Immunoreactive Exo70 and BIG2 partially or completely overlapped with gamma-tubulin at the MTOC in cells inspected by confocal microscopy. In cells incubated with brefeldin A, most of the BIG2, Exo70, and trans-Golgi protein p230 were widely dispersed from their perinuclear concentrations, but small amounts always remained, apparently at the MTOC. After disruption of microtubules with nocodazole, BIG2 and Exo70 were widely distributed in cells and remained only partially colocalized with p230, BIG2 more so than Exo70. We conclude that in HepG2 cells BIG2 and Exo70 interact in trans-Golgi network and centrosomes, as well as in exocyst structures or complexes that move along microtubules to the plasma membrane, consistent with a functional association in both early and late stages of vesicular trafficking.
Topics: Amino Acid Sequence; Brefeldin A; Cell Line; Exocytosis; Guanine Nucleotide Exchange Factors; Humans; Microtubule-Organizing Center; Molecular Sequence Data; Nocodazole; Two-Hybrid System Techniques; trans-Golgi Network
PubMed: 15705715
DOI: 10.1073/pnas.0409871102 -
Journal of Cell Science Apr 2018Although the Golgi complex has a conserved morphology of flattened stacked cisternae in most eukaryotes, it has lost the stacked organisation in several lineages,...
Although the Golgi complex has a conserved morphology of flattened stacked cisternae in most eukaryotes, it has lost the stacked organisation in several lineages, raising the question of what range of morphologies is possible for the Golgi. In order to understand this diversity, it is necessary to characterise the Golgi in many different lineages. Here, we identify the Golgi complex in , one of the first descriptions of an unstacked Golgi organelle in a non-parasitic eukaryote, other than fungi. We provide a comprehensive list of Golgi-associated membrane trafficking genes encoded in two species of and show that nearly all are expressed in mouse-passaged cells. We then study distribution of the Golgi marker ()CopB by fluorescence in , identifying membranous structures that are disrupted by Brefeldin A treatment, consistent with Golgi localisation. Confocal and immunoelectron microscopy reveals that COPB localises to tubular membranous structures. Our data identify the Golgi organelle for the first time in this major eukaryotic lineage, and provide the rare example of a tubular morphology, representing an important sampling point for the comparative understanding of Golgi organellar diversity.This article has an associated First Person interview with the first author of the paper.
Topics: Adenosine Triphosphatases; Animals; Brefeldin A; Eukaryotic Cells; Golgi Apparatus; Humans; Membrane Transport Proteins; Mice; Naegleria; Phylogeny; Protein Transport
PubMed: 29535209
DOI: 10.1242/jcs.213306 -
Proceedings of the National Academy of... Mar 2000Two brefeldin A (BFA)-inhibited guanine nucleotide-exchange proteins for ADP-ribosylation factors, 200-kDa BIG1 and 190-kDa BIG2, were copurified from bovine brain...
Two brefeldin A (BFA)-inhibited guanine nucleotide-exchange proteins for ADP-ribosylation factors, 200-kDa BIG1 and 190-kDa BIG2, were copurified from bovine brain cytosol associated with >670-kDa macromolecular complexes. When observed by immunofluorescence in HeLa S3 and HepG2 cells, endogenous BIG1 and coexpressed BIG2 were distributed in a punctate pattern throughout the cytosol, and also concentrated in the perinuclear region, where endogenous BIG1 and BIG2 each partially colocalized with Golgi-specific 58K protein and gamma-adaptin. On Western blot analysis, both BIG1 and BIG2 were clearly more abundant in the cytosol than in the microsomal fractions. After density gradient centrifugation of a microsomal fraction, BIG1 and BIG2 were recovered in the same fraction as beta-COP, a marker for Golgi membranes. When cytosol from HeLa S3 cells was subjected to gel filtration and fractions were analyzed by Western blotting, the largest percentages of both BIG1 and BIG2 were detected in fractions containing proteins with a molecular mass of >670 kDa. Western blotting using anti-peptide antibodies specific for BIG1 or BIG2 demonstrated that approximately 70% of BIG2 was immunoprecipitated along with 100% of BIG1 by the anti-BIG1 IgG, and approximately 75% of BIG1 was coprecipitated with 100% of BIG2 by the anti-BIG2 IgG. All observations were consistent with the conclusion that significant fractions of BIG1 and BIG2 exist as components of the same macromolecular complexes in bovine brain cytosol and are similarly localized in cultured cells.
Topics: ADP-Ribosylation Factors; Blotting, Western; Brefeldin A; Cytosol; Dose-Response Relationship, Drug; GTP-Binding Proteins; Golgi Apparatus; Guanine Nucleotide Exchange Factors; HeLa Cells; Humans; Microscopy, Fluorescence; Microsomes; Peptides; Precipitin Tests; Protein Synthesis Inhibitors; Time Factors; Transfection; Tumor Cells, Cultured
PubMed: 10716990
DOI: 10.1073/pnas.97.6.2567