Brefeldin A effects on the trans-Golgi network and Golgi bodies inBotryococcus braunii are not uniform during the cell cycle

Summary Using cryo-fixation and freeze-substitution electron microscopy, the effects of brefeldin A (BFA) on the structure of the trans-Golgi network (TGN), the endoplasmic reticulum (ER), and Golgi bodies in the unicellular green algaBotryococcus braunii were examined at various stages of the cell cycle. In the presence of BFA, all the TGNs of interphase and dividing cells aggregated to form a single tubular mass. In contrast, the TGNs decomposed just after cell division and disappeared during cell wall formation. Throughout the cell cycle, the TGN produced at least six kinds of vesicles, of which two were not formed in the presence of BFA: vesicles with a diameter of 200 nm and fibrillar substances, which formed in interphase cells; and vesicles with a diameter of 180–240 nm, which may participate in septum formation. In addition, the number of clathrin-coated vesicles attaching to the TGN decreased. In interphase cells, BFA induced the disassembly of Golgi bodies and an increase in the smooth-ER cisternae at the cis-side of Golgi bodies. This result may suggest the existence of retrograde transport from the Golgi bodies to the ER in the presence of BFA. These drastic structural changes in the Golgi bodies and the ER of interphase cells were not observed in BFA-treated dividing cells.Abbreviations BFA brefeldin A - ER endoplasmic reticulum - TGN trans-Golgi network     

Effects of brefeldin A on the Golgi apparatus, the nuclear envelope, and the endoplasmic reticulum in a green alga,Scenedesmus acutus

Summary The effects of brefeldin A (BFA) on the structure of the Golgi apparatus, the nuclear envelope, and the endoplasmic reticulum (ER), and on the thiamine pyrophosphatase (TPPase) activity in these organelles were examined in a green alga,Scenedesmus acutus, to obtain evidence for the existence of a retrograde transport from the Golgi apparatus to the ER via the nuclear envelope. InScenedesmus, Golgi bodies are situated close to the nuclear envelope throughout the cell cycle and receive the transition vesicles not directly from the ER, but from the nuclear envelope. BFA induced the disassembly of Golgi bodies and an increase in the ER cisternae at the trans-side of decomposed Golgi bodies in interphase cells and multinuclear cells before septum formation. The accumulated ER cisternae connected to the nuclear envelope at one part. TPPase activity was detected in all cisternae of Golgi bodies, but not in the nuclear envelope or the ER in nontreated cells. On the contrary, in BFA-treated cells, TPPase activity was detected in the nuclear envelope and the ER in addition to the decomposed Golgi bodies. When septum-forming cells were treated with BFA, the disassembly of Golgi bodies was less than that in interphase cells, and TPPase activity was detected in the Golgi cisternae but not in the nuclear envelope or the ER. These results suggest mat BFA blocks the anterograde transport from the nuclear envelope to the Golgi bodies but does not block the retrograde transport from the Golgi bodies to the nuclear envelope in interphase and multinuclear cells.Abbreviations BFA brefeldin A - ER endoplasmic reticulum - TPPase thiamine pyrophosphatase     

Disruption of the Golgi apparatus with brefeldin A does not destabilize the associated detyrosinated microtubule network.

Stable subsets of microtubules (MTs) are often enriched in detyrosinated alpha-tubulin. Recently it has been found that the Golgi apparatus is associated with a subset of relatively stable MTs and that detyrosinated MTs colocalize spatially and temporally with the Golgi apparatus in several cell lines. To determine whether the Golgi apparatus actively stabilizes associated MTs and thus allows their time-dependent detyrosination, we have used the drug brefeldin A (BFA) to disrupt the Golgi apparatus and have monitored changes in the Golgi apparatus and MT populations using simultaneous immunofluorescence and fluorescent lectin microscopy. We found that although BFA caused the Golgi apparatus to completely redistribute to the endoplasmic reticulum (ER), the detyrosinated MTs were not disrupted and remained in a juxtanuclear region. By Western blot analysis we found that even after 6 h of continuous exposure of cells to BFA, there was no detectable reduction in the level of detyrosinated alpha-tubulin. Simultaneous treatment with nocodazole and BFA led to a complete disruption of all MTs and normal Golgi structure/organization. Upon removal of nocodazole in the continued presence of BFA, we found that the detyrosinated MTs reformed in a compact juxtanuclear location in the absence of an intact Golgi complex. Finally, we found that the detyrosinated MTs colocalized precisely with a BFA-resistant structure that binds to the lectin, wheat germ agglutinin. We conclude that the juxtanuclear detyrosinated MTs are not actively stabilized by association with BFA-sensitive Golgi membranes. However, another closely associated structure which binds wheat germ agglutinin may serve to stabilize the juxtanuclear MTs. Alternatively, the MT organizing center (MTOC) and/or MT-associated proteins (MAPs) may organize and stabilize the juxtanuclear detyrosinated MTs.     

Microtubule-dependent retrograde transport of proteins into the ER in the presence of brefeldin A suggests an ER recycling pathway

Characteristics of brefeldin A (BFA)-induced redistribution of Golgi proteins into the endoplasmic reticulum (ER) and its relationship to an ER retrieval pathway were investigated. Retrograde movement of Golgi proteins into the ER occurred via long, tubulovesicular processes extending out of the Golgi along microtubules. Microtubule-disrupting agents (i.e., nocodazole), energy poisons, and reduced temperatures inhibited this pathway. In BFA-treated cells Golgi proteins appeared to cycle between the ER and an intermediate compartment marked by a 53 kd protein. Addition of nocodazole disrupted this dynamic cycle by preferentially inhibiting retrograde movement, causing Golgi proteins to accumulate in the intermediate compartment. In the absence of BFA, such an ER cycling pathway appeared to be followed normally by the 53 kd protein but not by Golgi proteins, as revealed by temperature shift experiments. We propose that BFA induces the interaction of the Golgi with an intermediate "recycling" compartment that utilizes a microtubule-dependent pathway into the ER.     

Brefeldin A induces a microtubule-dependent fusion of galactosyltransferase-containing vesicles with the rough endoplasmic reticulum

The fungal drug brefeldin A (BFA) has recently been found to induce a redistribution of medial- and cis-Golgi components to the endoplasmic reticulum (ER), raising the possibility of the existence of a retrograde pathway from the Golgi complex to the ER. Here, we demonstrate a BFA-induced reversible rearrangement of the trans-Golgi membrane protein galactosyltransferase (Gal-T) to the ER in HeLa cells. With immunofluorescence microscopy we have shown that BFA first caused a rapid change of Gal-T immunolabelling from a normal Golgi complex pattern to long and slender structures emanating from the cell centre and co-localizing with tubulin. Then immunofluorescence became ER-like. This effect was not dependent on ongoing protein synthesis and was reversed to normal within 120 min after removal of the drug. Restoration of the Golgi complex after removal of brefeldin A was energy-dependent but not mediated by microtubules nor dependent on protein synthesis. BFA-induced backflow of Gal-T was inhibited by nocodazole, a microtubule-disrupting agent. Immunoelectron microscopy showed that BFA treatment resulted in the fusion of Gal-T-containing vesicles with the ER. Furthermore, sucrose gradient centrifugation showed a significant shift in density of mature Gal-T polypeptides upon BFA treatment: about 40% of the enzyme migrated from its original density (1.13 g/ml) to the density of rough ER (1.19 g/ml). Thus, BFA caused microtubule-dependent vesicular backflow from a trans-Golgi component to the ER followed by fusion of the Golgi-derived vesicles with the ER.     

Brefeldin A induces reversible dissociation of the Golgi apparatus in the green algaMicrasterias

Summary The fungal metabolite brefeldin A (BFA) causes inhibition of cell growth inMicrasterias denticulata after 2 h incubation, combined with slight malformation of the cell shape. The BFA effects on cell development are accompanied by a gradual decrease in the number of Golgi cisternae and severe structural and morphological changes of the dictyosomes which are already visible after only 10 min exposure. When the treatment is prolonged the number of dictyosomes is markedly reduced, leading to almost complete loss of Golgi bodies, particularly in the young semicell. Groups of primary wall material-containing vesicles accumulated in areas of former dictyosomes, and previously unknown vesicular bodies are found. Restitution of almost normal dictyosomes occurs within 5 h when the cells are allowed to recover from BFA treatment.Micrasterias cells incubated in BFA at concentrations below 15 M maintain their ability to divide over several generations. Our results indicate that, of the various inhibitors of the secretory pathway tested against growingMicrasterias cells, BFA is the only drug which induces complete and reversible dissociation of dictyosomes in the growing semicell. This allows deductions about the function of the processes targeted by BFA during cell development inMicrasterias.Abbreviations BFA brefeldin A - CPA cyclopiazonic acid - ER endoplasmic reticulum - TM tunicamycin     

Brefeldin A affects growth, endoplasmic reticulum, Golgi bodies, tubular vacuole system, and secretory pathway in Pisolithus tinctorius

Brefeldin A (BFA) reduced radial growth in Pisolithus tinctorius at a concentration as low as 2 microM. Use of endoplasmic reticulum (ER)-Tracker dye, unconjugated BFA, and fluorescent BFA (BODIPY-BFA) allowed comparison of the effects of BFA on the endomembrane system of P. tinctorius at the light and electron microscope levels. Both ER-Tracker dye and BODIPY-BFA have been shown previously to label the ER. Unconjugated BFA and BODIPY-BFA modified the ER network and disrupted the tubular vacuole system in the tip region. The ultrastructure in freeze-substituted hyphae showed that BFA treatment resulted in (i) disruption of the Spitzenk?rper, (ii) reduction in number of apical vesicles, (iii) redistribution and mild dilation of ER, and (iv) persistence and increased size and complexity of Golgi bodies. The effects of BFA on the ER were only partially reversible in the time period examined. We conclude that in P. tinctorius, BFA as the free metabolite or BODIPY-BFA affects the tubular vacuole system as well as anterograde membrane flow between the ER and the Golgi bodies and post-Golgi transport.     

Golgi proteins persist in the tubulovesicular remnants found in brefeldin A-treated pancreatic acinar cells.

Brefeldin A (BFA) blocks protein export from the endoplasmic reticulum (ER) and causes dismantling of the Golgi cisternae with relocation of resident Golgi proteins to the ER in many cultured cell lines. We examined the effects of BFA on Golgi organization and the distribution of Golgi markers in the rat exocrine pancreas. Immediately after BFA addition, Golgi stacks began to disorganize and Golgi cisternae to vesiculate, and by 15 min no intact Golgi cisternae remained. However, even after prolonged BFA incubation, clusters of small vesicles surrounded by transitional elements of the ER persisted both in the Golgi region and dispersed throughout the apical cytoplasm. These vesicles were morphologically heterogeneous in the density of their content and in the presence of cytoplasmic coats. Immunogold labeling demonstrated that some vesicles within the clusters contained gp58, a cis Golgi marker, and some contained alpha-mannosidase II, a middle/trans Golgi marker in this cell type. Neither marker was detected in the rough ER by immunogold or immunofluorescence labeling. When AlF4- was added during BFA treatment some of the vesicles in the clusters appeared coated. When microsomes were subfractionated into Golgi (light) and rough ER (heavy) fractions on sucrose density gradients, greater than 65% of alpha-mannosidase II and galactosyltransferase activities were found in light fractions (1.14-1.16 g/ml) in both control and BFA-treated lobules. In both cases equally low enzyme activity was recovered in heavier fractions (1.2-1.23 g/ml) containing RNA and alpha-glucosidase activity. However, 5 to 8% of the total recovered RNA consistently codistributed with the Golgi enzyme peak. These results indicate that BFA rapidly inhibits secretion and causes dismantling of the Golgi stacks in pancreatic acinar cells, but clusters of vesicles consisting of bona fide Golgi remnants persist even with prolonged exposure to BFA. Many of the vesicles contain Golgi markers by immunolabeling. By cell fractionation Golgi membrane enzyme activities are recovered in equal amounts in light (Golgi) fractions in both controls and BFA-treated specimens. These findings indicate that in the exocrine pancreas there is a dissociation of BFA's effects on the exocytic pathway: there is a block in transport and Golgi organization is disrupted, but remnant Golgi vesicles and tubules persist and retain Golgi membrane antigens and enzyme activities.     

Influenza virus hemagglutinin trimers and monomers maintain distinct biochemical modifications and intracellular distribution in brefeldin A-treated cells.

Brefeldin A (BFA) induces the retrograde transport of proteins from the Golgi complex (GC) to the endoplasmic reticulum (ER). It is uncertain, however, whether the drug completely merges the ER with post-ER compartments, or whether some of their elements remain physically and functionally distinct. We investigated this question by the use of monoclonal antibodies specific for monomers and trimers of the influenza virus hemagglutinin (HA). In untreated influenza virus-infected cells, monomers and trimers almost exclusively partition into the ER and GC, respectively. In BFA-treated cells, both monomers and trimers are detected in the ER by immunofluorescence. Cell fractionation experiments indicate, however, that whereas HA monomers synthesized in the presence of BFA reside predominantly in vesicles with a characteristic density of the ER, HA trimers are primarily located in lighter vesicles characteristic of post-ER compartments. Biochemical experiments confirm that in BFA-treated cells, trimers are more extensively modified than monomers by GC-associated enzymes. Additional immunofluorescence experiments reveal that in BFA-treated cells, HA monomers can exist in an ER subcompartment less accessible to trimers and, conversely, that trimers are present in a vesicular compartment less accessible to monomers. These findings favor the existence of a post-ER compartment for which communication with the ER is maintained in the presence of BFA and suggest that trimers cycle between this compartment and the ER, but have access to only a portion of the ER.     

Effects of methyl benzimidazoIe-2-yl carbamate on microtubule and actin cytoskeleton inAspergillus nidulans

Summary The effects of methyl benzimidazole-2-yl carbamate (MBC) on microtubule and actin cytoskeleton were analyzed by indirect immunofluorescence and transmission electron microscopy in a wild-type strain and a benomyl-resistant mutant (benA ₁₀) ofAspergillus nidulans. The treatment of the wild-type strain with sublethal doses of MBC not only caused depolymerization of cytoplasmic microtubules (MTs), but also changed the pattern of actin at the hyphal tips. In the MBC-treated hyphae, the actin fluorescence was concentrated at the very tip region of the hypha, whereas in the control hyphae, the actin fluorescence was weak at the very tip and strong below the tip. The dose of MBC used for the wild-type strain did not depolymerize the MTs or modify the actin organization at the apex in the mutant strain, which confirmed that the change in actin distribution in the wild-type strain was due to the disruption of MTs. In the mutant strain, a seven times higher concentration of MBC than in the wild-type strain was required to depolymerize MTs and to alter the actin organization at the apex. The ultrastructural study of the MBC-treated hyphae revealed that the area containing apical vesicles was larger and the number of microvesicles was higher than in control hyphae. These changes probably resulted from the disassembly of MTs and the reorientation of actin cytoskeleton in MBC-treated apexes and suggested that MTs would organize the actin at the apex, which in turn would restrict the vesicle fusion to a narrow area at the hyphal tip. In treated hyphae of both strains without cytoplasmic MTs, mitotic spindles were detected although in lower number and with slightly modified morphology.Abbreviations DAPI 4,6-diamidino-2-phenylindole - DMSO dimethyl sulfoxide - EM electron microscopy - ER endoplasmic reticulum - IIP indirect immunofluorescence - MBC methyl benzimidazole-2-yl carbamate - MTs microtubules     

New cellulose synthesizing complexes (terminal complexes) involved in animal cellulose biosynthesis in the tunicateMetandrocarpa uedai

Summary Subcellular compartments comprising the endomembrane system in filamentous fungi are poorly characterized with most showing significant morphological differences from eukaryotic cells. For example, many filamentous fungi lack stacked Golgi-body cisternae, but contain Golgi equivalents — single cisternae or tubules which appear to serve the same functions. To help identify fungal endomembrane compartments and interrelationships between them we used a pharmacological agent, brefeldin A, known to affect specific endomembrane organelles in other organisms, most prominently the Golgi apparatus. At 10 g/ml brefeldin A, radial hyphal growth of the rice blast pathogenMagnaporthe grisea on solid agar medium was reduced by 96% over an initial 48 h, but recovered and was reduced by only 20% over a subsequent 72 h exposure. Light microscopic examination of individual living hyphae showed that apical elongation generally halted within 1 min after exposure to brefeldin A. Acute effects of 14 g/ml brefeldin A were characterized ultrasiructurally in cells prepared by freeze substitution. These included the appearance of two types of cisternae with unusual morphology, associated with ca. 45 nm diameter vesicles, as well as the unexpected persistence and increase in complexity of the Golgi equivalents. Also observed were (1) reduced numbers of apicale vesicles and disruption of Spitzenkörper organization, (2) apical clusters of 30–35 nm diameter microvesicles and associated tubular arrays, (3) dilation of rough endoplasmic reticulum, (4) packets of membrane-bounded electron-opaque cell wall inclusions, and (5) altered morphology of some vacuolar compartments. The distribution of concanavalin A binding sites, previously mapped to particular endomembrane compartments, was documented to aid the interpretation of these results. We conclude that brefeldin A effects on cells ofM. grisea differ from those reported with plant and animal cells, perhaps reflecting underlying differences in the endomembrane systems among these eukaryotes.Abbreviations BFA brefeldin A - ConA concanavalin A - ER endoplasmic reticulum - PDA potato dextrose agar - RER rough endoplasmic reticulum     

Membranes in the spindle of Iris pollen mother cells during the second division of meiosis

Summary Spindle membrane in the second division of meiosis inIris comprises endoplasmic reticulum (ER) and small spherical vesicles (40–80 nm diameter). The vesicles are in all respects similar to those of dictyosomes and it is proposed that they originate in the Golgi system. ER on the other hand is generated from the nuclear envelope (NE). Invaginations and evaginations of the inner and outer membranes of the NE are present at interphase and they enlarge during prophase II and break to form spindle ER elements at the beginning of prometaphase II. The area enclosed by this ER continues to increase until the entire spindle is filled with irregular profiles at mid-prometaphase II. During late-prometaphase II the quantity of ER decreases and, concurrent with an increase in the number of microtubules (MTs), the cisternae align alongside MT bundles and accumulate at the poles. It is suggested that ER increases in quantity by growth of NE membranes. Furthermore, the alignment of ER along the interpolar axis and its transport polewardsprior to anaphase suggest the action of a force on each ER element.     

Cortical ultrastructure of freeze-substituted protonemata of the mossFunaria hygrometrica

Summary The ultrastructural organization of the cortical cytoplasm has been examined in caulonemata, branches and buds of the mossFunaria hygrometrica, which were prepared by rapid freeze-fixation and freeze-substitution (FS). The same structural components occur in the cortex of all three cell types: microtubules (MTs), endoplasmic reticulum (ER), coated and uncoated vesicles, coated pits, and dictyosomes. However, the configuration and density of the cortical ER varies between the three. Caulonemata have an open, polygonal network of ER associated with long MTs oriented mostly parallel to the length of the cell. Lamellar ER, covered with polysomes, is interspersed in the network. Branches have a more tightly arranged ER network, at places occurring in a thick layer, and occasional polysome-decorated lamellae. MTs, which extend to the tip of the branch, are oriented mainly parallel to the cell's long axis and are associated with the cortical ER. Buds have the tightest ER network, which is frequently arranged in a thick layer. Tubules in the polygonal ER of buds are densely covered with ribosomes, whereas tubules in the ER network of caulonemata and branches range from nearly smooth to moderately rough. Closely-spaced ER lamellae, with many polysomes, occur in some buds. The MTs of buds extend into the apical dome and are associated with the cortical ER, but are more randomly oriented than in caulonemata or branches. Close appositions between the ER and PM are observed in all three cells, but are more frequent in buds.Abbreviations DiOC₆(3) 3,3-dihexyloxacarbocyanine iodide - ER endoplasmic reticulum - FS freeze-substitution - MT microtubule - MF microfilament - PM plasma membrane     

Brefeldin A arrests the maturation and egress of herpes simplex virus particles during infection.

Herpes simplex virus (HSV) requires the host cell secretory apparatus for transport and processing of membrane glycoproteins during the course of virus assembly. Brefeldin A (BFA) has been reported to induce retrograde movement of molecules from the Golgi to the endoplasmic reticulum and to cause disassembly of the Golgi complex. We examined the effects of BFA on propagation of HSV type 1. Release of virions into the extracellular medium was blocked by as little as 0.3 microgram of BFA per ml when present from 2 h postinfection. Characterization of infected cells revealed that BFA inhibited infectious viral particle formation without affecting nucleocapsid formation. Electron microscopic analyses of BFA-treated and untreated cells (as in control cells) demonstrated that viral particles were enveloped at the inner nuclear membrane in BFA-treated cells and accumulated aberrantly in this region. Most of the progeny virus particles observed in the cytoplasm of control cells, but not that of BFA-treated cells, were enveloped and contained within membrane vesicles, whereas many unenveloped nucleocapsids were detected in the cytoplasm of BFA-treated cells. This suggests that BFA prevents the transport of enveloped particles from the perinuclear space to the cytoplasmic vesicles. These findings indicate that BFA-induced retrograde movement of molecules from the Golgi complex to the endoplasmic reticulum early in infection arrests the ability of host cells to support maturation and egress of enveloped viral particles. Furthermore, we demonstrate that the effects of BFA on HSV propagation are not fully reversible, indicating that maturation and egress of HSV type 1 particles relies on a series of events which cannot be easily reconstituted after the block to secretion is relieved.     

Disruption of endoplasmic reticulum to Golgi transport leads to the accumulation of large aggregates containing beta-COP in pancreatic acinar cells.

When transport between the rough endoplasmic reticulum (ER) and Golgi complex is blocked by Brefeldin A (BFA) treatment or ATP depletion, the Golgi apparatus and associated transport vesicles undergo a dramatic reorganization. Because recent studies suggest that coat proteins such as beta-COP play an important role in the maintenance of the Golgi complex, we have used immunocytochemistry to determine the distribution of beta-COP in pancreatic acinar cells (PAC) in which ER to Golgi transport was blocked by BFA treatment or ATP depletion. In controls, beta-COP was associated with Golgi cisternae and transport vesicles as expected. Upon BFA treatment, PAC Golgi cisternae are dismantled and replaced by clusters of remnant vesicles surrounded by typical ER transitional elements that are generally assumed to represent the exit site of vesicular carriers for ER to Golgi transport. In BFA-treated PAC, beta-COP was concentrated in large (0.5-1.0 micron) aggregates closely associated with remnant Golgi membranes. In addition to typical ER transitional elements, we detected a new type of transitional element that consists of specialized regions of rough ER (RER) with ribosome-free ends that touched or extended into the beta-COP containing aggregates. In ATP-depleted PAC, beta-COP was not detected on Golgi membranes but was concentrated in similar large aggregates found on the cis side of the Golgi stacks. The data indicate that upon arrest of ER to Golgi transport by either BFA treatment or energy depletion, beta-COP dissociates from PAC Golgi membranes and accumulates as large aggregates closely associated with specialized ER elements. The latter may correspond to either the site of entry or exit for vesicles recycling between the Golgi and the RER.     

Occurence of microtubules and microfilaments,and origin of septa in dikaryotic hyphae ofSchizophyllum commune

Summary In an electron microscopic study on the dikaryotic hyphae ofSchizophyllum commune, microtubules were observed during the nuclear division, and close to the non-dividing nuclei of apical cells and older cells. Microtubules of the spindle were connected with semicircular bodies at nuclear poles. Microfilaments were detected in the distal part of the apical cells. Vesicles similar to those in the tips of the hyphae occured also at the sites of septa formation. The occurrence of microtubules and the structure of semicircular bodies are compared with those in other basidiomycetes. It is suggested that vesicles are involved in the primary growth of the septal cross wall.     

Localized accumulation of cell wall mannoproteins and endomembranes induced by brefeldin A in hyphal cells of the dimorphic yeastCandida albicans

Summary Candida albicans, a dimorphic yeast, has the abililty to switch its growth form between budding growth and hyphal growth. Since fungal growth involves secretory processes, spatial control of secretion should play a crucial role in such a morphogenetic transition. Brefeldin A (BFA), an inhibitor of the membrane trafficking system of eukaryotes, increases the occurrence of Golgi-like cisternae in the yeast. In the present study, BFA was used to obtain further insights into the spatial organization of secretory processes in hyphal growth ofC. albicans. BFA completely inhibited the formation and growth of germ tubes at a concentration of 35 M or higher. Electron microscopy of BFA-untreated germinated cells revealed many vesicles in the apical region and Golgi-like cisternae in the cytoplasm. In cells treated with 35 M BFA, the vesicles disappeared from the apical region, and, instead, stacked membrane cisternae and membrane-enclosed spherical dense bodies accumulated in the subapical region. These accumulated structures were positive for both polysaccharide staining and immunocytochemical staining with antibodies raised against cell surface antigens ofC. albicans, as were Golgi cisternae in BFA-untreated cells. In cells treated with a higher concentration of BFA (140 M), the structures that appeared in cells treated with 35 M BFA were no longer observed and the endoplasmic reticulum was extended and positive for polysaccharide staining. These results suggested that BFA affects different steps of membrane trafficking in a concentration-dependent manner. The accumulated structures induced by 35 M BFA seemed to be the altered forms of Golgi cisternae. Their accumulation in the subapical region of the germ tube might indicate that the step(s) in membrane trafficking that are associated with the Golgi pathway are vectorially organized in hyphal growth ofC. albicans.Abbrevations BFA brefeldin A - BSA bovine serum albumin - CBB Coomassie brilliant blue - Con A concanavalin A - HRP horseradish peroxidase     

Inhibition of membrane tubule formation and trafficking by isotetrandrine, an antagonist of G-protein-regulated phospholipase A2 enzymes

Previous studies have established a role for cytoplasmic phospholipase A(2) (PLA(2)) activity in tubule-mediated retrograde trafficking between the Golgi complex and the endoplasmic reticulum (ER). However, little else is known about how membrane tubule formation is regulated. This study demonstrates that isotetrandrine (ITD), a biscoclaurine alkaloid known to inhibit PLA(2) enzyme activation by heterotrimeric G-proteins, effectively prevented brefeldin A (BFA)-induced tubule formation from the Golgi complex and retrograde trafficking to the ER. In addition, ITD inhibited BFA-stimulated tubule formation from the trans-Golgi network and endosomes. ITD inhibition of the BFA response was potent (IC(50) approximately 10-20 microM) and rapid (complete inhibition with a 10-15-min preincubation). ITD also inhibited normal retrograde trafficking as revealed by the formation of nocodazole-induced Golgi mini-stacks at ER exit sites. Treatment of cells with ITD alone caused the normally interconnected Golgi ribbons to become fragmented and dilated, but cisternae were still stacked and located in a juxtanuclear position. These results suggest that a G-protein-binding PLA(2) enzyme plays a pivotal role in tubule mediated trafficking between the Golgi and the ER, the maintenance of the interconnected ribbons of Golgi stacks, and tubule formation from endosomes.     

Brefeldin A induces callose formation in onion inner epidermal cells

Summary The antibiotic fungal toxin brefeldin A (BFA) causes synthesis of additional cell wall material in adult differentiated onion inner epidermal cells at concentrations of 5–30 g/ml. This tertiary wall contains callose and is layered on the secondary cellulosic wall in a time- and dose-dependent manner. Initially, callose is found in pit fields in the form of small vesicular patches. With time and dose, depositions grow in size and form large plugs invaginating into the cell, where the adjacent cytoplasm forms bulky accumulations and contains many organelles including endomembranes. Within the cytoplasm, BFA exerts the characteristic morphological effects on the secretory system including changes of the Golgi stacks, formation of large vesicles, and proliferation of dilated cisternae of the endoplasmic reticulum. Higher concentrations of BFA (60 g/ml) lead to disintegration of the Golgi apparatus; they have no effects on the cell wall, no callose synthesis occurs. We conclude from these observations that BFA has two independent targets in onion cells. BFA acts on the plasma membrane, hence operating as an elicitor of plant defense reactions and thus activates callose synthesis. BFA acts also on the membranes of the secretory system and influences budding and fusion of vesicles at the endoplasmic reticulum and at the dictyosomes. These two mechanisms occur in parallel, suggesting that the secretory system still can play its presumed role in callose synthesis. Only when dictyosomes are completely disintegrated, no more callose is formed.Abbreviations BFA Brefeldin A - PM plasma membrane - GA Golgi apparatus - ER endoplasmic reticulum - GS glucan synthetase Dedicated to Professor Walter Gustav Url on the occasion of his 70th birthday     

Peroxisomal membrane ascorbate peroxidase is sorted to a membranous network that resembles a subdomain of the endoplasmic reticulum

The peroxisomal isoform of ascorbate peroxidase (APX) is a novel membrane isoform that functions in the regeneration of NAD(+) and protection against toxic reactive oxygen species. The intracellular localization and sorting of peroxisomal APX were examined both in vivo and in vitro. Epitope-tagged peroxisomal APX, which was expressed transiently in tobacco BY-2 cells, localized to a reticular/circular network that resembled endoplasmic reticulum (ER; 3,3'-dihexyloxacarbocyanine iodide-stained membranes) and to peroxisomes. The reticular network did not colocalize with other organelle marker proteins, including three ER reticuloplasmins. However, in vitro, peroxisomal APX inserted post-translationally into the ER but not into other purified organelle membranes (including peroxisomal membranes). Insertion into the ER depended on the presence of molecular chaperones and ATP. These results suggest that regions of the ER serve as a possible intermediate in the sorting pathway of peroxisomal APX. Insight into this hypothesis was obtained from in vivo experiments with brefeldin A (BFA), a toxin that blocks vesicle-mediated protein export from ER. A transiently expressed chloramphenicol acetyltransferase-peroxisomal APX (CAT-pAPX) fusion protein accumulated only in the reticular/circular network in BFA-treated cells; after subsequent removal of BFA from these cells, the CAT-pAPX was distributed to preexisting peroxisomes. Thus, plant peroxisomal APX, a representative enzymatic peroxisomal membrane protein, is sorted to peroxisomes through an indirect pathway involving a preperoxisomal compartment with characteristics of a distinct subdomain of the ER, possibly a peroxisomal ER subdomain.