HT-2 toxin impairs porcine oocyte in vitro maturation through disruption of endomembrane system
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Brefeldin A
Nocodazole
Endomembrane system
COPI
Summary— The effects of the drug Brefeldin A, shown to block the translocation of proteins between the endoplasmic reticulum and the Golgi apparatus in animal cells, were studied on different plant cell systems. In suspension culture cells and root tissues, the Golgi aparatus was affected by Brefeldin A treatments resulting in distortion and dissociation of the Golgi stacks, coupled with appearance of numerous vesicles in the cytoplasm. This process was reversible. Therefore, Brefeldin A provides a powerful tool with which to study Golgi dynamics and function in plant as well as in animal cells.
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N ‐Ethylmaleimide‐sensitive factor (NSF) is required for multiple pathways of vesicle‐mediated protein transport. Microinjection of a monoclonal anti‐NSF antibody almost completely blocked brefeldin A‐promoted Golgi disassembly without affecting the rapid release of β‐COP, a subunit of the Golgi coat proteins (COPI), from the Golgi apparatus. Similar results were obtained using a dominant‐negative NSF which is known to compete with endogenous NSF. The present results suggest that an NSF‐mediated step is present in the brefeldin A‐promoted disassembly of the Golgi apparatus.
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Brefeldin A (BFA) has a profound effect on the structure of the Golgi apparatus, causing Golgi proteins to redistribute into the ER minutes after drug treatment. Here we describe the dissociation of a 110-kD cytoplasmically oriented peripheral membrane protein (Allan, V. J., and T. E. Kreis. 1986. J. Cell Biol. 103:2229-2239) from the Golgi apparatus as an early event in BFA action, preceding other morphologic changes. In contrast, other peripheral membrane proteins of the Golgi apparatus were not released but followed Golgi membrane into the ER during BFA treatment. The 110-kD protein remained widely dispersed throughout the cytoplasm during drug treatment, but upon removal of BFA it reassociated with membranes during reformation of the Golgi apparatus. Although a 30-s exposure to the drug was sufficient to cause the redistribution of the 110-kD protein, removal of the drug after this short exposure resulted in the reassociation of the 110-kD protein and no change in Golgi structure. If cells were exposed to BFA for 1 min or more, however, a portion of the Golgi membrane was committed to move into and out of the ER after removal of the drug. ATP depletion also caused the reversible release of the 110-kD protein, but without Golgi membrane redistribution into the ER. These findings suggest that the interaction between the 110-kD protein and the Golgi apparatus is dynamic and can be perturbed by metabolic changes or the drug BFA.
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ABSTRACT The fungal fatty acid derivative Brefeldin A (BFA), has been used to study the reversible distribution of a Golgi glycoprotein, the JIM 84 epitope, into the cytosol of higher plant cells. Treatment of both maize and onion root tip cells resulted in a rearrangement of the Golgi stacks into either circular formations or a perinuclear distribution. The Golgi cisternae became curved and vesiculated and in cells where the Golgi apparatus was totally dispersed the JIM 84 epitope was associated with large areas in the cytosol which were also vesiculated. On removal of the BFA the Golgi apparatus reformed and the JIM 84 epitope was again located in the cisternal stacks. This mode of BFA action is compared with that so far described for animal cells.
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Brefeldin A
Nocodazole
COPII
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Golgi apparatus changes start at 90 to 120 seconds following to the onset of Brefeldin A (BFA)-administration and after coated regions had already disappeared at 30 to 60 seconds. The Golgi apparatus stacks disassemble and long tubules grow out of the Golgi region. After 3 to 5 min, a Golgi apparatus is missing in the cells and instead, the cytoplasm is occupied by a network of membrane tubules being interwoven with membranes of the endoplasmic reticulum. At places, glomerular membrane figures form conspicuous "organelles" in BFA-treated cells (cf. Figs. 39 and 40).
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Evidence has been presented both for and against obligate retrograde movement of resident Golgi proteins through the endoplasmic reticulum (ER) during nocodazole-induced Golgi ministack formation. Here, we studied the nocodazole-induced formation of ministacks using phospholipase A 2 (PLA 2 ) antagonists, which have been shown previously to inhibit brefeldin A–stimulated Golgi-to-ER retrograde transport. Examination of clone 9 rat hepatocytes by immunofluorescence and immunoelectron microscopy revealed that a subset of PLA 2 antagonists prevented nocodazole-induced ministack formation by inhibiting two different trafficking pathways for resident Golgi enzymes; at 25 μM, retrograde Golgi-to-ER transport was inhibited, whereas at 5 μM, Golgi-to-ER trafficking was permitted, but resident Golgi enzymes accumulated in the ER. Moreover, resident Golgi enzymes gradually redistributed from the juxtanuclear Golgi or Golgi ministacks to the ER in cells treated with these PLA 2 antagonists alone. Not only was ER-to-Golgi transport of resident Golgi enzymes inhibited in cells treated with these PLA 2 antagonists, but transport of the vesicular stomatitis virus G protein out of the ER was also prevented. These results support a model of obligate retrograde recycling of Golgi resident enzymes during nocodazole-induced ministack formation and provide additional evidence that resident Golgi enzymes slowly and constitutively cycle between the Golgi and ER.
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Human autoantibodies offer unique tools for the study of cellular constituents since they usually recognize highly conserved components, the most difficult to detect due to their low immunogenicity. The serum from a patient with Sjögren's syndrome (RM serum) showing a very high reactivity to the Golgi complex has been shown to immunoprecipitate and to immunodetect by Western blotting experiments a protein mol wt 210,000 (p210) that was shown to be peripheral and cytoplasmically disposed. A close examination of the p210 labeling revealed some differences with Golgi markers: RM serum staining was slightly more extensive than several Golgi markers and showed a discontinuous or granular appearance. Nocodazole induced a specific and early segregation of many p210-associated vesicles or tubules from Golgi apparatus. Upon brefeldin A treatment, p210 did not redistribute in the ER as did other Golgi proteins. In contrast, it exhibited a vesicular pattern reminiscent to that displayed by proteins residing in the intermediate compartment. Double staining immunofluorescence using the RM serum and the marker of the intermediate compartment, p58, revealed segregation of both proteins in control conditions but colocalization in BFA-treated cells. We have further demonstrated by combining different drug treatments that p210-containing elements in brefeldin A-treated cells belong indeed to the intermediate compartment. Experiments on brefeldin A recovery suggested that these p210 elements might play a role in reformation and repositioning of the Golgi apparatus. Ultrastructural localization performed by immunoperoxidase staining allowed us to establish that p210 interacted with the external side of an abundant tubulo-vesicular system on the cis side of the Golgi complex which extended to connecting structures and vesicles between saccules or stacks of cisternae, p210 appears to be a novel protein residing in the cis-Golgi network that may cycle between the Golgi apparatus and the intermediate compartment.
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Nocodazole
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Cellular compartment
Vesicular Transport Proteins
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Immunoprecipitation
Golgi membrane
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The Golgi apparatus forms stacks of cisternae in many eukaryotic cells. However, little is known about how such a stacked structure is formed and maintained. To address this question, plant cells provide a system suitable for live-imaging approaches because individual Golgi stacks are well separated in the cytoplasm. We established tobacco BY-2 cell lines expressing multiple Golgi markers tagged by different fluorescent proteins and observed their responses to brefeldin A (BFA) treatment and BFA removal. BFA treatment disrupted cis, medial, and trans cisternae but caused distinct relocalization patterns depending on the proteins examined. Medial- and trans-Golgi proteins, as well as one cis-Golgi protein, were absorbed into the endoplasmic reticulum (ER), but two other cis-Golgi proteins formed small punctate structures. After BFA removal, these puncta coalesced first, and then the Golgi stacks regenerated from them in the cis-to-trans order. We suggest that these structures have a property similar to the ER-Golgi intermediate compartment and function as the scaffold of Golgi regeneration.
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