Golgi-disturbing Agent Research Articles

Golgi tethering factor golgin-97 suppresses breast cancer cell invasiveness by modulating NF-\u03baB activity

BackgroundGolgin-97 is a tethering factor in the trans-Golgi network (TGN) and is crucial for vesicular trafficking and maintaining cell polarity. However, the significance of golgin-97 in human diseases such as cancer remains unclear.MethodsWe searched for a potential role of golgin-97 in cancers using Kaplan-Meier Plotter (http://kmplot.com) and Oncomine (www.oncomine.org) datasets. Specific functions of golgin-97 in migration and invasion were examined in golgin-97-knockdown and golgin-97-overexpressing cells. cDNA microarray, pathway analysis and qPCR were used to identify gene profiles regulated by golgin-97. The role of golgin-97 in NF-κB signaling pathway was examined by using subcellular fractionation, luciferase reporter assay, western blot analysis and immunofluorescence assay (IFA).ResultsWe found that low expression of golgin-97 correlated with poor overall survival of cancer patients and was associated with invasiveness in breast cancer cells. Golgin-97 knockdown promoted cell migration and invasion, whereas re-expression of golgin-97 restored the above phenotypes in breast cancer cells. Microarray and pathway analyses revealed that golgin-97 knockdown induced the expression of several invasion-promoting genes that were transcriptionally regulated by NF-κB p65. Mechanistically, golgin-97 knockdown significantly reduced IκBα protein levels and activated NF-κB, whereas neither IκBα levels nor NF-κB activity was changed in TGN46- or GCC185-knockdown cells. Conversely, golgin-97 overexpression suppressed NF-κB activity and restored the levels of IκBα in golgin-97-knockdown cells. Interestingly, the results of Golgi-disturbing agent treatment revealed that the loss of Golgi integrity was not involved in the NF-κB activation induced by golgin-97 knockdown. Moreover, both TGN-bound and cytosolic golgin-97 inhibited NF-κB activation, indicating that golgin-97 functions as an NF-κB suppressor regardless of its subcellular localization.ConclusionOur results collectively demonstrate a novel and suppressive role of golgin-97 in cancer invasiveness. We also provide a new avenue for exploring the relationship between the TGN, golgin-97 and NF-κB signaling in tumor progression.

Chondroitin sulfate N-acetylgalactosaminyltransferase-2 contributes to the replication of infectious bursal disease virus via interaction with the capsid protein VP2.

Infectious bursal disease virus (IBDV) is a birnavirus that causes a highly contagious immunosuppressive disease in young chickens. The capsid protein VP2 of IBDV plays multiple roles in its life cycle. To more comprehensively understand the functions of VP2 involved in the communication between virus and host, we used yeast two-hybrid screening to identify the cellular factors that interact with this protein. We found that chondroitin sulfate N-acetylgalactosaminyltransferase-2 (CSGalNAcT2), a typical type II transmembrane protein located in Golgi apparatus, could interact with VP2, and we confirmed this interaction by co-immunoprecipitation and confocal laser scanning microscopy assays. Additionally, up-regulation of CSGalNAcT2 during IBDV infection was observed. Overexpression and siRNA-mediated knockdown of CSGalNAcT2 assays suggested that CSGalNAcT2 promoted IBDV replication. Moreover, this enhancing effect of CSGalNAcT2 could be inhibited by Brefeldin A, which is a Golgi-disturbing agent. This indicated that the integrity of Golgi apparatus structure was involved in the function of CSGalNAcT2. Taken together, we concluded that CSGalNAcT2, located in the Golgi apparatus, contributed to the replication of IBDV via interaction with VP2.

Golgin-84-associated Golgi fragmentation triggers tau hyperphosphorylation by activation of cyclin-dependent kinase-5 and extracellular signal-regulated kinase

Tau hyperphosphorylation is a critical event in Alzheimer's disease, in which the neuronal Golgi fragmentation occurs earlier than tau hyperphosphorylation. However, the intrinsic link between Golgi impairment and tau pathology is missing. By electron microscopy and western blotting, we observed in the present study that the neuronal Golgi fragmentation was increased age-dependently with a correlated tau hyperphosphorylation in the brains of C57BL/6 mice aged from 4 to 16 months. Simultaneously, golgin-84 and Golgi reassembly stacking protein 65, 2 important Golgi matrix proteins, were decreased in the brains of elder mice. Further studies in HEK293/tau cells showed that Golgi-disturbing agents, brefeldin A and nocodazole induced tau hyperphosphorylation. Knockdown of golgin-84, not Golgi reassembly stacking protein 65, by small interfering RNA was sufficient to induce tau hyperphosphorylation, while over-expressing golgin-84 arrested the brefeldin A-induced Golgi fragmentation and tau hyperphosphorylation. Finally, we demonstrated that cyclin-dependent kinase-5 and extracellular signal-regulated kinase were activated after golgin-84 knockdown, and simultaneous inhibition of these kinases abolished the golgin-84 deficit-induced tau hyperphosphorylation. These data suggest Golgi fragmentation could be an upstream event triggering tau hyperphosphorylation through golgin-84 deficit-induced activation of cyclin-dependent kinase-5 and extracellular signal-regulated kinase.

A CREB3–ARF4 signalling pathway mediates the response to Golgi stress and susceptibility to pathogens

Treatment of cells with brefeldin A (BFA) blocks secretory vesicle transport and causes a collapse of the Golgi apparatus. To gain more insight into the cellular mechanisms mediating BFA toxicity, we conducted a genome-wide haploid genetic screen that led to the identification of the small G protein ADP-ribosylation factor 4 (ARF4). ARF4 depletion preserves viability, Golgi integrity and cargo trafficking in the presence of BFA, and these effects depend on the guanine nucleotide exchange factor GBF1 and other ARF isoforms including ARF1 and ARF5. ARF4 knockdown cells show increased resistance to several human pathogens including Chlamydia trachomatis and Shigella flexneri. Furthermore, ARF4 expression is induced when cells are exposed to several Golgi-disturbing agents and requires the CREB3 (also known as Luman or LZIP) transcription factor, whose downregulation mimics ARF4 loss. Thus, we have uncovered a CREB3-ARF4 signalling cascade that may be part of a Golgi stress response set in motion by stimuli compromising Golgi capacity.

Modulation of Matrix Metalloprotease-2 Levels by Mechanical Loading of Three-Dimensional Mesenchymal Stem Cell Constructs: Impact onIn VitroTube Formation

Angiogenesis is essential to tissue reconstitution, is sensitive to mechanical stresses, and currently represents one of the major challenges in tissue engineering. The pro-angiogenic matrix metalloprotease-2 (MMP-2) is upregulated in mechanically loaded mesenchymal stem cells (MSCs). Therefore, MMP-2 may provide a regulating link between angiogenesis and the surrounding mechanical conditions. This study aimed to modulate MMP-2 levels by mechanical loading of MSCs embedded in a three-dimensional matrix as well as to investigate the mechanism of MMP-2 regulation along with its contribution to angiogenesis stimulation. MMP-2-inducing conditions (30% compression, 1 Hz, 72 h) were defined after varying loading parameters. Addition of the Golgi-disturbing agent Brefeldin A suppressed this mechanical upregulation of MMP-2. Analysis of enzymatic activities demonstrated an enhancement of pro-MMP-2, mature MMP-2, and tissue inhibitor of metalloproteases-2. Further, mechano-regulation of MMP-14 and mature MMP-2 was dependent upon the activity of furin, a proprotein processing endoprotease. Angiogenesis was stimulated by conditioned media from MSCs loaded at inducing conditions. This augmentation of angiogenesis was hindered by inhibition of pro-MMP-2 and mature MMP-2. In conclusion, mechanical stimulation of MSCs in a three-dimensional matrix induces pro-MMP-2 secretion and MMP-2 activation, potentially via the activation complex consisting of MMP-2/-14/tissue inhibitor of metalloproteases-2. Mechano-regulated pro-MMP-2 and mature MMP-2 seem to contribute to angiogenesis stimulation. Thus, an application of these loading parameters could augment vascularization of tissue-engineered constructs based on the described MMP-2-dependent mechanism.

Differential palmitoylation of the endosomal SNAREs syntaxin 7 and syntaxin 8

Palmitoylation is a posttranslational modification that regulates protein trafficking and stability. In this study we investigated whether the endosomal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins syntaxin 7 and syntaxin 8 are modified with palmitate. Using metabolic labeling and site-directed mutagenesis, we show that human syntaxins 7 and 8 are modified with palmitate through a thioester linkage. Palmitoylation is dependent upon cysteine 239 of human syntaxin 7 and cysteine 214 of syntaxin 8, residues that are located on the cytoplasmic face of the transmembrane domain (TMD). Palmitoylation of syntaxin 8 is minimally affected by the Golgi-disturbing agent brefeldin A (BFA), whereas BFA dramatically inhibits palmitoylation of syntaxin7. The differential effect of BFA suggests that palmitoylation of syntaxins 7 and 8 occurs in distinct subcellular compartments. Palmitoylation does not affect the rate of protein turnover of syntaxins 7 and 8 nor does it influence the steady-state localization of syntaxin 8 in late endosomes. Syntaxin 7 actively cycles between endosomes and the plasma membrane. Palmitoylation-defective syntaxin 7 is selectively retained on the plasma membrane, suggesting that palmitoylation is important for intercompartmental transport of syntaxin 7.

Golgi-Disturbing Agents Lead to the Elimination of Intracellular Toxoplasma gondii

The Golgi apparatus is responsible for the genesis of secretory organelles of Toxoplasma gondii and lipid traffic to the vacuole. This study used anti-Golgi agents to demonstrate the importance of Golgi in Toxoplasma development. Monensin, Brefeldin A, Retinoic Acid and Okadaic Acid reduced the infection, leading to parasite elimination. Mon, BFA and RA affected secretory organelles and the Golgi Complex of the parasites, with faster parasite elimination in the presence of Monensin; in addition, the vesicular transit of host cell C6-NBD-ceramide metabolites was interrupted, but the GC of host cells was preserved. Our results suggest that several targets in the secretory pathway are affected in the intracellular Toxoplasma rather than in the host cells, resulting in interruption of parasite development and its elimination from the intracellular medium.

Inhibition of Autotaxin by Lysophosphatidic Acid and Sphingosine 1-Phosphate

Autotaxin (ATX) or nucleotide pyrophosphatase/phosphodiesterase 2 (NPP2) is an NPP family member that promotes tumor cell motility, experimental metastasis, and angiogenesis. ATX primarily functions as a lysophospholipase D, generating the lipid mediator lysophosphatidic acid (LPA) from lysophosphatidylcholine. ATX uses a single catalytic site for the hydrolysis of both lipid and non-lipid phosphodiesters, but its regulation is not well understood. Using a new fluorescence resonance energy transfer-based phosphodiesterase sensor that reports ATX activity with high sensitivity, we show here that ATX is potently and specifically inhibited by LPA and sphingosine 1-phosphate (S1P) in a mixed-type manner (Ki approximately 10(-7) M). The homologous ecto-phosphodiesterase NPP1, which lacks lysophospholipase D activity, is insensitive to LPA and S1P. Our results suggest that, by repressing ATX activity, LPA can regulate its own biosynthesis in the extracellular environment, and they reveal a novel role for S1P as an inhibitor of ATX, in addition to its well established role as a receptor ligand.

Mu-opioid receptor trafficking on inhibitory synapses in the rat brainstem.

Whole-cell recordings were made from identified gastric-projecting rat dorsal motor nucleus of the vagus (DMV) neurons. The amplitude of evoked IPSCs (eIPSCs) was unaffected by perfusion with met-enkephalin (ME) or by mu-, delta-, or kappa-opioid receptor selective agonists, namely D-Ala2-N-Me-Phe4-Glycol5-enkephalin (DAMGO), cyclic [D-Pen2-D-Pen5]-enkephalin, or trans-3,4-dichloro-N-methyl-N-[2-(1-pyrolytinil)-cyclohexyl]-benzeneacetamide methane sulfonate (U50,488), respectively. Brief incubation with the adenylate cyclase activator forskolin or the nonhydrolysable cAMP analog 8-bromo-cAMP, thyrotropin releasing hormone, or cholecystokinin revealed the ability of ME and DAMGO to inhibit IPSC amplitude; this inhibition was prevented by pretreatment with the mu-opioid receptor (MOR1) selective antagonist D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2. Conversely, incubation with the adenylate cyclase inhibitor dideoxyadenosine, with the protein kinase A (PKA) inhibitor N-[2-(p-Bromocinnamyl-amino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H89), or with the Golgi-disturbing agent brefeldin A, blocked the ability of forskolin to facilitate the inhibitory actions of ME. Immunocytochemical experiments revealed that under control conditions, MOR1 immunoreactivity (MOR1-IR) was colocalized with glutamic acid decarboxylase (GAD)-IR in profiles apposing DMV neurons only after stimulation of the cAMP-PKA pathway. Pretreatment with H89 or brefeldin A or incubation at 4 degrees C prevented the forskolin-mediated insertion of MOR1 on GAD-IR-positive profiles. These results suggest that the cAMP-PKA pathway regulates trafficking of mu-opioid receptors into the cell surface of GABAergic nerve terminals. By consequence, the inhibitory actions of opioid peptides in the dorsal vagal complex may depend on the state of activation of brainstem vagal circuits.

Brefeldin A enhancesHelicobacter pylorivacuolating cytotoxin-induced vacuolation of epithelial cells

Intracellular VacA localises to the vacuolar (late endosome/lysosome) membrane, but little is known about the trafficking of the toxin beyond this region. We show that the Golgi-disturbing agent brefeldin A (BFA) enhances VacA-induced vacuolation of epithelial cells by Helicobacter pylori co-culture and, importantly, BFA treatment induces vacuolation by less toxic forms of VacA. The effect is BFA dose-dependent and occurs within 2.5 h. These data suggest that VacA may be routed deeper within the cell than the vacuole, and that vacuolation is minimised when this occurs efficiently. This may explain why some forms of VacA do not cause vacuolation and why vacuolation is minimal at the low bacteria:cell ratios observed in vivo.

Regulation of matrix metalloproteinase MT1-MMP/MMP-2 in cardiac fibroblasts by TGF-beta1 involves furin-convertase.

Heart failure is characterized by an imbalance of matrix synthesis/turnover, finally resulting in fibrosis. Cardiac myocytes and fibroblasts play a pivotal role in the remodeling process. Cardiac remodeling involves the expression of TGF-beta1 and matrix metalloproteinases (MMPs) in cardiac fibroblasts (CFBs). Furin, a subtilisin/kexin-like proprotein convertase (PC), activates TGF-beta1 and membrane-bound MT1-MMP, which facilitates pro-gelatinase A (MMP-2) activation. Even though several reports identified TGF-beta1 as a pro-fibrotic cytokine in the heart, it increases MMP-activity and cell migration/invasion in several cell types. The present study was done to investigate the contribution of TGF-beta1 and furin to CFBs MMP-activity and motility. Stimulation of CFBs from adult Sprague-Dawley rats with TGF-beta1 (20 ng/ml) induced furin, but had no effect on the closely related PC5. Inhibition of furin inhibited angiotensin II-induced TGF-beta1 activation, indicating that TGF-beta1 amplifies its activating convertase in CFBs. Pretreatment of CFBs with TGF-beta1 (20 ng/ml, 24 h) increased their migration by about two-fold (p<0.05), which was accompanied by an enhanced expression and activity of MT1-MMP and MMP-2. Brefeldin A (BFA), a Golgi-disturbing agent, inhibited MT1-MMP activation, indicating that it occurs in the trans-Golgi network (TGN), where furin is concentrated and colocalized with MT1-MMP. Inhibition of furin significantly inhibited TGF-beta1-induced MT1-MMP/MMP-2 activation. Furthermore, inhibition of furin attenuated TGF-beta1-enhanced migration on gelatin-coated membranes (p<0.05). This was comparable to the effects of the MMP-inhibitor GM6001, pointing out that MMPs are major mediators of TGF-beta1-enhanced CFB motility. We demonstrate that TGF-beta1 induces MMP-activity in CFBs, thereby facilitating CFBs motility. Furthermore, TGF-beta1 amplifies its activating convertase furin, which is also required for MT1-MMP/MMP-2 activation in CFBs. Thus, furin is central for TGF-beta1 and MT1-MMP activation and might be a novel target in cardiac remodeling.

Brefeldin A enhances Helicobacter pylori vacuolating cytotoxin-induced vacuolation of epithelial cells

Intracellular VacA localises to the vacuolar (late endosome/lysosome) membrane, but little is known about the trafficking of the toxin beyond this region. We show that the Golgi-disturbing agent brefeldin A (BFA) enhances VacA-induced vacuolation of epithelial cells by Helicobacter pylori co-culture and, importantly, BFA treatment induces vacuolation by less toxic forms of VacA. The effect is BFA dose-dependent and occurs within 2.5 h. These data suggest that VacA may be routed deeper within the cell than the vacuole, and that vacuolation is minimised when this occurs efficiently. This may explain why some forms of VacA do not cause vacuolation and why vacuolation is minimal at the low bacteria:cell ratios observed in vivo.

Rescuing the traffic-deficient mutants of rat mu-opioid receptors with hydrophobic ligands.

Deletion of a sequence near the fifth transmembrane domain (258RLSKV262, i3-1 mutant) and a motif residing at the proximal carboxyl tail (344KFCTR348, C-2 mutant) resulted in mu-opioid receptor mutants that were poorly expressed on the surface of transfected human embryonic kidney 293 cells. Treatment with the opioid antagonist naloxone, the agonist etorphine, and other hydrophobic ligands enhanced cell surface expression of i3-1 and C-2 mutants. The observed enhancement was time- and concentration-dependent, required the ligands to be membrane permeable, and was not the result of the reversal of the constitutive activities of the mutant receptors. The binding of the ligands resulted in the trafficking of the mutant receptors retained in the endoplasmic reticulum to the cell surface. The cell surface-expressed mutant C-2, but not i3-1, fully retained ability to mediate inhibition of adenylyl cyclase activity. Furthermore, the Golgi-disturbing agents brefeldin A and monensin completely blocked naloxone-enhanced expression of i3-1 and C-2 mutants. Results of these studies suggest that intracellular interactions of agonist and antagonist with mutant receptors can serve as chaperones in the trafficking of the mutants to the cell surface.

Coordinated regulation and colocalization of alphav integrin and its activating enzyme proprotein convertase PC5 in vivo.

Integrin alphav is involved in intracellular-extracellular signaling important for cytoskeleton alterations and control of cell movement. In vitro experiments indicate that the integrin alphav-subunit undergoes post-translational endoproteolytic cleavage. This type of activation requires the presence of suitable kexin/subtilisin-like proprotein convertases. In vitro experiments have demonstrated that, among several proprotein convertases, PC5A, and to a threefold lesser extent furin, can activate alphav integrin. The biological significance of these in vitro data would be further supported by a coexpression and coordinated regulation of the gene expression of alphav integrin and its activating enzyme PC5 in vivo. In the present study we investigated the regulation of alphav integrin and PC5 following balloon injury in vivo. Comparative immunocytochemistry revealed a coordinated regulation of alphav integrin and PC5 during vascular remodeling in rodents. Integrin alphav was found to be upregulated in PCNA-positive, proliferating vascular smooth muscle cells. Northern blots revealed no significant regulation of furin mRNA, whereas PC5A mRNA increased during vascular remodeling, suggesting that PC5 is the major convertase during neointima formation in vivo. Incubation of vascular smooth muscle cells with the Golgi-disturbing agent brefeldin A inhibited alphav integrin maturation, indicating that endoproteolytic cleavage occurs in the trans-Golgi network, were PC5 is localized. Thus, the present study further supports the concept that activation of alphav integrin occurs in the trans-Golgi network in vascular smooth muscle cells and involves PC5.

Sub-cellular trafficking of phytochemicals explored using auto-fluorescent compounds in maize cells

BackgroundLittle is known regarding the trafficking mechanisms of small molecules within plant cells. It remains to be established whether phytochemicals are transported by pathways similar to those used by proteins, or whether the expansion of metabolic pathways in plants was associated with the evolution of novel trafficking pathways. In this paper, we exploited the induction of green and yellow auto-fluorescent compounds in maize cultured cells by the P1 transcription factor to investigate their targeting to the cell wall and vacuole, respectively.ResultsWe investigated the accumulation and sub-cellular localization of the green and yellow auto-fluorescent compounds in maize BMS cells expressing the P1 transcription factor from an estradiol inducible promoter. We established that the yellow fluorescent compounds accumulate inside the vacuole in YFBs that resemble AVIs. The green fluorescent compounds accumulate initially in the cytoplasm in large spherical GFBs. Cells accumulating GFBs also contain electron-dense structures that accumulate initially in the ER and which later appear to fuse with the plasma membrane. Structures resembling the GFBs were also observed in the periplasmic space of plasmolized cells. Ultimately, the green fluorescence accumulates in the cell wall, in a process that is insensitive to the Golgi-disturbing agents BFA and monensin.ConclusionsOur results suggest the presence of at least two distinct trafficking pathways, one to the cell wall and the other to the vacuole, for different auto-fluorescent compounds induced by the same transcription factor in maize BMS cells. These compartments represent two of the major sites of accumulation of phenolic compounds characteristic of maize cells. The secretion of the green auto-fluorescent compounds occurs by a pathway that does not involve the TGN, suggesting that it is different from the secretion of most proteins, polysaccharides or epicuticular waxes. The yellow auto-fluorescent compounds accumulate in a vacuolar compartment, in structures that resemble the AVIs present in many cells accumulating anthocyanins. Together, our studies suggest that the accumulation of auto-fluorescent compounds can provide a powerful tool to dissect the trafficking of phytochemicals, knowledge necessary for the efficient engineering of plant metabolism.

Differential Effect of Brefeldin A on the Palmitoylation of Surfactant Protein C Proprotein Mutants

The surfactant protein C precursor (proSP-C) is palmitoylated on two cysteines adjacent to its transmembrane domain. We showed previously that palmitoylation of proSP-C occurs in a postendoplasmic reticulum compartment and is not affected by the Golgi-disturbing agent brefeldin A (BFA). In contrast, the investigations presented here showed that BFA almost completely abolished palmitoylation of proSP-C mutants that contained alterations in the region between the palmitoylated cysteines and the transmembrane domain, including a Pro 30 to Leu mutant associated with interstitial lung disease. This differential effect of BFA was not caused by differences in the palmitoylation kinetics between wild-type proSP-C and the mutants and was not mimicked by nocodazole and monensin. However, differences between the mutants and wild-type proSP-C in the relative degree of processing suggest that BFA may unmask a difference in routing. This would imply that the amino acids just N-terminal of the transmembrane domain may be important for a proper sorting of proSP-C.

Molecular and cellular mechanisms of adipose secretion: Comparison of leptin and angiotensinogen

Besides their function of lipid storage, the adipose cells secrete a number of proteins of physiopathological importance. To get further insights into this function, which remains poorly characterized, we sought to compare the mechanisms and regulation of secretion of two individual proteins in the same cells. Leptin and angiotensinogen were chosen and assessed by radioimmunoassay and quantitative immunoblotting, respectively, in primary culture of epididymal adipose cells from young obese Zucker rats. Leptin was secreted at a steady rate of 4 ng/10(6) cells/h over 2-6 h. Despite secretion, leptin cellular content remained stable at 3 ng/10(6) cells. In contrast, the rate of angiotensinogen secretion decreased regularly from 45 arbitrary units/10(6) cells/h at 2 h, to half this value at 6 h, although cell content remained constant at 100 arbitrary units/10(6) cells. Inhibition of protein synthesis by cycloheximide depleted the cells from leptin, but not from angiotensinogen for up to 6 h. Insulin increased leptin secretion (+75%) and cell content (+70 %), without affecting angiotensinogen. Secretion of both proteins was inhibited by Golgi-disturbing agents, brefeldin A and monensin. The presence of brefeldin A led to a specific rise in leptin cell content, an effect inhibited by cycloheximide and enhanced by insulin (+80%). These data show that leptin and angiotensinogen are both secreted through Golgi-dependent pathways and that their respective intracellular pool exhibit distinct turn-over rate and insulin sensitivity. These characteristics might account for the differential response of these adipose proteins to variations in the systemic environment.

Golgi-disturbing agents.

Pharmacological agents have proven useful for gaining fundamental insights into the biology of the Golgi apparatus. This review summarizes pertinent and recent work on the effects on this organelle of monensin, brefeldin A, bafilomycin, ilimaquinone, okadaic acid, retinoic acid, and nocodazole. The molecular targets of monensin, brefeldin A, ilimaquinone, and retinoic acid remain to be elucidated whereas those for bafilomycin (vacuolar H+-ATPase), okadaic acid (serine/threonine phosphatases types 1, 2a, and 2b), and nocodazole (microtubules) are reasonably well understood. The molecular target of brefeldin has not been defined, but has been suggested to involve guanine nucleotide exchange proteins acting on ADP-ribosylation factor 1. Whether a defined molecular target can be found for monensin must be questioned since its main action consists in exchanging protons for Na+ which leads to osmotic swelling of post-Golgi endosomal structures and Golgi subcompartments by virtue of its membrane-associated effect as a cationophore. Brefeldin A was one of the most thoroughly investigated Golgi-disturbing agents and proved instrumental in unraveling retrograde flow mechanisms in the secretory pathways. Okadaic acid attracted interest for its properties mimicking mitotic fragmentation of the Golgi apparatus. Nocodazole was instrumental in establishing the cytoskeletal anchoring of the Golgi apparatus close to the microtubular organizing center.

Double immunofluorescent staining of α2,6 sialyltransferase and β1,4 galactosyltransferase in monensin‐treated cells: Evidence for different Golgi compartments?

Beta 1,4 galactosyl- and alpha 2,6 sialyltransferase (gal-T EC 2.4.1.22 and sialyl-T EC 2.4.99.1) sequentially elongate and terminate complex N-glycan chains of glycoproteins. Both enzymes reside in trans Golgi cisternae; their ultrastructural relationship, however, is unknown. To delineate their respective Golgi compartment(s) we conducted a double label immunofluorescent study by conventional and confocal laser scanning microscopy in HepG2, HeLa, and other cells in presence of Golgi-disturbing agents. Polyclonal, peptide-specific antibodies to human sialyl-T expressed as a beta-galactosidase-sialyl-T fusion protein in E. coli were developed and applied together with mABs to human milk gal-T. In untreated HepG2 and HeLa cells Golgi morphology identified by immunofluorescent labeling of sialyl-T and gal-T, respectively, was nearly identical. Treatment of cells with brefeldin A (BFA) led to rapid and coordinated disappearance of immunostaining of both enzymes; after BFA washout, vesicular structures reappeared which first stained for gal-T followed by sialyl-T; in the reassembled Golgi apparatus sialyl-T and gal-T were co-localized again. In contrast, monensin treatment produced a reversible swelling and scattering of gal-T positive Golgi elements while sialyl-T positive structures showed little change. Treatment with nocodazole led to dispersal of Golgi elements in which gal-T and sialyl-T remained co-localized. Treatment with chloroquine affected Golgi structures less than monensin and led to condensation of gal-T positive and to slight enlargement of sialyl-T positive structures. Sequential recovery from BFA of gal-T and sialyl-T and their segregation by monensin suggest that these enzymes are targeted to different Golgi subcompartments.