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PubMed1985

Kinetics of monensin-induced swelling of Golgi apparatus cisternae of H-2 hepatoma cells.

In H-2 hepatoma cells grown in culture, a pattern of response to the sodium-selective ionophore, monensin, was observed in which cisternal swelling commenced at one pole of the Golgi apparatus and was accompanied by the accumulation of swollen vacuoles in the cytoplasm in the absence of a decrease in the number of cisternae per stack. The results suggest that the swollen vacuoles are formed from Golgi apparatus cisternae that are replaced by formation of new cisternae. Depending on monensin concentration, between 2 and 4 such vacuoles accumulated per Golgi apparatus stack within 12.5 min after monensin addition. The rate of vacuole formation was greatest at 10(-6) M monensin and least at 10(-8) M monensin over the range 10(-5) to 10(-8) M monensin. The general pattern of early response to the ionophore is that formation of normal secretory vesicles is blocked, after which the Golgi apparatus continues to function for a time to release membrane as swollen cisternal units that accumulate in the cytoplasm at or near the trans Golgi apparatus face. These phenomena all precede the general swelling response normally observed in glutaraldehyde-fixed animal material and illustrate that the early dynamic responses to monensin are not confined to plant cells.

Animals↗
PubMed2005

Subcompartmentalizing the Golgi apparatus.

The subcompartmentalized structure of the Golgi apparatus contributes to efficient glycosylation in the secretory pathway. Subcompartmentalization driven by maturation relies primarily on constant and accurate vesicle-mediated local recycling of Golgi residents. The precision of this vesicle transport is dependent on the interplay between the key factors that mediate vesicle budding and fusion--the coat proteins and the SNARE fusion machinery. These alone, however, may not be sufficient to ensure establishment of compartments de novo, and additional regulatory mechanisms operate to modify their activity.

Biological Transport↗
PubMed1991

A part of glucosylceramide formed from exogenous lactosylceramide is not degraded to ceramide but re-cycled and glycosylated in the Golgi apparatus.

The subcellular fate of glucosylceramide (GlcCer) formed from exogenous lactosylceramide (LacCer) in rat liver is investigated. LacCer radiolabeled on different positions of the molecule was intravenously administered to rats as a liposomal dispersion. A Golgi apparatus fraction 140-fold enriched in specific markers and constituted by intact cisternal stacks, as well as the lysosomal and plasma membrane fractions concurrently prepared from the same homogenate, were then studied in order to determine the time course of radioactive glycosphingolipids. LacCer quickly decreased with time in the plasma membrane, whereas in the lysosomes it increased up to 4 h and decreased thereafter. In both fractions results were regardless of the labeling position. In the Golgi apparatus, LacCer increased up to 12 h and then decreased. In this fraction, the radioactivity values of [Glc-3H]LacCer were over twice those of [Gal-3H]LacCer. GlcCer was found only after [Glc-3H]LacCer administration. In the lysosomes, its time course provided a peak similar in shape but delayed in timing with respect to that of LacCer. Conversely, in the Golgi apparatus GlcCer was earlier formed, but earlier consumed, than LacCer. Gangliosides increased in the Golgi apparatus until 4 h and then decreased after 12 h, whereas in the plasma membrane they were progressively accumulated. In both fractions the amount of [Glc-3H]gangliosides was over twice that of [Gal-3H]gangliosides was over twice that of [Gal-3H]gangliosides. Since we demonstrated that the sugars released in the course of LacCer degradation (LacCer----galactose + GlcCer----glucose + ceramide) are not incorporated into glycoconjugates, we conclude that a part of GlcCer formed during the lysosomal degradation of LacCer actually reaches the Golgi apparatus where it undergoes successive glycosylation.

Animals↗
PubMed1988

Cytochemical study of the Golgi apparatus and related organelles of the secretory ameloblasts of rat molar tooth germs cultured with and without colchicine.

The Golgi apparatus and Golgi-associated endoplasmic reticulum lysosome (GERL) were examined in the ameloblasts with a cytochemical marker, osmium impregnation, and two enzyme markers, thiamine pyrophosphatase (TPPase) and acid phosphatase (ACPase). In control cultured germs, osmium deposit appeared in one to two immature side cisternae of Golgi stacks; TPPase activity was restricted in a few mature side cisternae and condensing vacuoles. ACPase activity existed in the GERL and, sometimes, in the mature side-cisternae and condensing vacuoles. These findings show that Golgi stacks of ameloblasts consist of several distinct compartments. In colchicine-treated tooth germs, there were morphological and cytochemical changes in both Golgi stacks and GERL. The Golgi apparatus was fragmented and its stacks were scattered throughout the supranuclear region. In some stacks, the number of osmium-positive cisternae was greater than normal; in others they were absent. TPPase and ACPase activity was absent or diminished. These findings suggest the importance of microtubules in the organization of Golgi complex and GERL in the secretory ameloblast.

Acid Phosphatase↗
PubMed1993

Cholesterol deprivation affects the fluorescence properties of a ceramide analog at the Golgi apparatus of living cells.

Previous studies have established that a fluorescent analog of ceramide, N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)] -6-aminohexanoyl-D-erythro-sphingosine (C6-NBD-Cer), is a vital stain for the Golgi apparatus and a useful tool for studying the sorting and transport of sphingolipids along the secretory pathway in animal cells. Here, we examine the effects of various culture conditions on labeling of the Golgi apparatus of human skin fibroblasts by C6-NBD-Cer and demonstrate that cholesterol deprivation affects the fluorescence properties of the probe at this organelle. Labeling of the Golgi apparatus by C6-NBD-Cer was dramatically reduced in cells grown in medium containing lipoprotein-deficient serum compared to cells grown in medium containing normal serum. Quantitative fluorescence microscopy showed that this apparent reduction in labeling resulted from accelerated photo-bleaching of the fluorescent analog. C6-NBD-Cer labeling of the Golgi apparatus was restored in cholesterol-deprived cells by stimulating endogenous cholesterol biosynthesis with mevalonic acid or by adding exogenous nonlipoprotein cholesterol or low density lipoprotein to the culture medium. In addition, when cells grown in medium containing normal serum were perforated and treated with cholesterol oxidase, an apparent reduction in labeling resulted, further implicating an intracellular pool of cholesterol in the potentiation of C6-NBD-Cer fluorescence. These results demonstrate that cytological studies using C6-NBD-Cer are affected by cholesterol deprivation and suggest that this fluorescent lipid may be used to monitor cholesterol at the Golgi apparatus of living cells.

4-Chloro-7-nitrobenzofurazan↗
PubMed2001

Direct targeting of cis-Golgi matrix proteins to the Golgi apparatus.

The targeting route of newly synthesized GM130 and GRASP65 to the Golgi apparatus was investigated by three different approaches. First, localization of pulse labeled GM130 and GRASP65 in normal rat kidney (NRK) cells was traced by subcellular fractionation followed by immunoprecipitation. Immediately after the pulse labeling, GM130 and GRASP65 were found in the Golgi but not in the endoplasmic reticulum (ER) membrane fractions, whereas a control Golgi membrane protein was still found in the ER membrane fractions. Second, epitope tagged GM130 and GRASP65 were expressed in NRK cells by plasmid microinjection into the nuclei and their localization was analyzed by immunofluorescence. When ER to Golgi transport was inhibited by prior microinjection of a GTP-restricted mutant of Sar1 protein into the cytosol, the expressed GM130 and GRASP65 showed clear Golgi localization. Last, binding of GM130 and GRASP65 to the membranes was analyzed in vitro. In vitro synthesized GM130 and GRASP65 specifically bound to purified Golgi membranes but not to microsomal membranes. The bound GM130 and GRASP65 were found to form a complex with pre-existing counterparts on the Golgi membrane. These results strongly suggested that GM130 and GRASP65 are directly targeted to the Golgi membrane without initial assembly on the ER and subsequent vesicular transport to the Golgi apparatus.

Animals↗
PubMed1992

A monoclonal antibody recognizing Golgi apparatus produced using affinity purified material from a patient with connective tissue disease.

Serum antibodies recognizing the Golgi apparatus have been reported in patients with connective tissue diseases, but little is known of their significance. Serum from a systemic lupus erythematosus patient with polymyositis was found to have high titers of anti-Golgi apparatus antibody. This serum recognized a 64 kD polypeptide in immunoblotting with HEp-2 cells. To verify that the 64 kD polypeptide was associated with the Golgi apparatus and to characterize which Golgi component was recognized, a monoclonal antibody was produced. IgG, isolated from this serum, was used in affinity chromatography to produce purified material which was used to generate a mouse monoclonal antibody. The monoclonal antibody had an indirect immunofluorescent pattern identical to that produced by the patient's serum, and similarly recognized a 64kD polypeptide in immunoblotting. A 59 kD polypeptide was also recognized by the monoclonal antibody, suggesting that the antigens recognized by the monoclonal and serum antibodies may be only partially identical. The antigen appears to be a glycoprotein and an integral component of the Golgi cisternae membranes.

Aged↗
PubMed1996

The Golgi apparatus of spinal cord motor neurons in transgenic mice expressing mutant Cu,Zn superoxide dismutase becomes fragmented in early, preclinical stages of the disease.

Dominant mutations of the SOD1 gene encoding Cu,Zn superoxide dismutase have been found in members of certain families with familial amyotrophic lateral sclerosis (ALS). To better understand the contribution of SOD1 mutations in the pathogenesis of familial ALS, we developed transgenic mice expressing one of the mutations found in familial ALS. These animals display clinical and pathological features closely resembling human ALS. Early changes observed in these animals were intra-axonal and dendritic vacuoles due to dilatation of the endoplasmic reticulum and vacuolar degeneration of mitochondria. We have reported that the Golgi apparatus of spinal cord motor neurons in patients with sporadic ALS is fragmented and atrophic. In this study we show that spinal cord motor neurons of transgenic mice for an SOD1 mutation display a lesion of the Golgi apparatus identical to that found in humans with sporadic ALS. In these mice, the stacks of the cisternae of the fragmented Golgi apparatus are shorter than in the normal organelle, and there is a reduction in Golgi-associated vesicles and adjacent cisternae of the rough endoplasmic reticulum. Furthermore, the fragmentation of the Golgi apparatus occurs in an early, presymptomatic stage and usually precedes the development of the vacuolar changes. Transgenic mice overexpressing the wild-type human superoxide dismutase are normal. In familial ALS, an early lesion of the Golgi apparatus of motor neurons may have adverse functional effects, because newly synthesized proteins destined for fast axoplasmic transport pass through the Golgi apparatus.

Aging↗
PubMed2000

Implication of sphingolipid metabolism in the stability of the Golgi apparatus.

We examined the effects of short chain and long chain ceramides on the stability of the Golgi apparatus. Short chain ceramides, C(2)- and C(6)-ceramides, blocked brefeldin A-induced Golgi disassembly without affecting the rapid release of Golgi coat proteins, whereas they did not inhibit brefeldin A-induced tubulation of endosomes. Both short chain ceramides also retarded Golgi disassembly induced by nordihydroguaiaretic acid and nocodazole, suggesting that they stabilize the Golgi apparatus. In contrast to short chain ceramides, natural long chain ceramides, when incorporated into cells or formed within cells upon treatment with sphingomyelinase or metabolic inhibitors, enhanced brefeldin A-induced Golgi disassembly. These results suggest that sphingolipid metabolism is implicated in the stability of the Golgi apparatus.

Animals↗
PubMed1994

Disruption of the Golgi apparatus by brefeldin A blocks cell polarization and inhibits directed cell migration.

The role of the Golgi apparatus in the motile activity of fibroblasts was examined with brefeldin A (BFA), which disrupts the Golgi apparatus in a variety of cells. Upon incubation with BFA, Swiss mouse 3T3 fibroblasts lost their typical polarized morphology, in which the leading edge is characterized by intensive lamellipodia formation. BFA affected cell asymmetry as demonstrated by a decrease in the morphometric indices, dispersion, and elongation. After BFA treatment, cells showed little protrusional activity and did not form a dense actin network at the leading edge, and consequently the rate of cell migration into an experimental wound was significantly reduced. In addition, BFA prevented an increase in pseudopodial activity and prevented the formation of long processes induced by phorbol 12-myristate 13-acetate. The effects of BFA on cell shape and protrusional activity were quantitatively similar to those observed with the microtubule-disrupting agent nocodazole, although BFA had no effect on microtubule integrity. These results suggest that the integrity of both the Golgi apparatus and microtubules is necessary for the generation and maintenance of fibroblast asymmetry, which is a prerequisite for directed cell migration.

3T3 Cells↗
PubMed1997

Functional protein prenylation is required for the brefeldin A-dependent retrograde transport from the Golgi apparatus to the endoplasmic reticulum.

In cells exposed to brefeldin A (BFA), enzymes of the Golgi apparatus are redistributed to the endoplasmic reticulum (ER) by retrograde membrane flow, where they may cause modifications on resident ER proteins. We have used a truncated form of the rough ER-specific type I transmembrane glycoprotein ribophorin I as a probe to detect Golgi glycosyltransferases relocated to the ER in a BFA-dependent fashion. This polypeptide (RI332) comprises the 332 amino-terminal amino acids of ribophorin I and behaves like a luminal ER protein when expressed in HeLa cells. Upon treatment of the cells with BFA, RI332 becomes quantitatively O-glycosylated by Golgi glycosyltransferases that are transported back to the ER. Here we demonstrate that pretreatment of the cells with lovastatin, an inhibitor of HMG-CoA reductase, abrogates this modification and that mevalonate, the product formed in the step inhibited by the drug, is able to counteract the effect of lovastatin. We also show by immunofluorescence using mannosidase II as a Golgi marker that the BFA-induced retrograde transport of Golgi enzymes is blocked by lovastatin, although electron microscopy indicates that BFA causes disassembly of the Golgi apparatus into swollen vesicles and tubules. Our observations support the role of a prenylated protein, such as the geranylgeranylated small G protein Rab6, in the retrograde transport from the Golgi apparatus to the ER, since lovastatin acts by inhibiting its prenylation.

Animals↗
PubMed1981

The golgi apparatus: two organelles in tandem.

The Golgi apparatus consists of distinct cis and trans compartments that may act sequentially to refine the protein export of the endoplasmic reticulum by removing escaped endoplasmic reticulum proteins. Refinement may be a multistage process akin to fractional distillation; the stack of cisternae comprising the cis Golgi may be the plates in this distillation tower. The trans Golgi, consisting of the last one or two cisternae, may be the receiver that collects from the cis Golgi only its most refined fraction for later distribution to specific locations throughout the cell.

Animals↗
PubMed1972

Lipoprotein particles from the Golgi apparatus of guinea-pig liver.

1. A cell fraction has been isolated from guinea-pig liver and shown to be rich in Golgi apparatus by electron microscopy. The activity of UDP-d-galactose-N-acetylglucosamine galactosyltransferase was over 100-fold greater in this cell fraction than in the liver homogenate. These data support the conclusion that the fraction was enriched in Golgi apparatus. 2. The Golgi cisternae and secretory vesicles contained electron-dense particles of 10-80nm diameter. Disruption of the Golgi apparatus cell fraction released these particles, which were separated into VLD (very-low-density) and LD (low-density) species on the basis of their density. 3. The Golgi VLD particles possessed morphological, flotational, chemical and immunochemical properties which closely resembled those of the serum VLD lipoproteins from the same animals. 4. The Golgi LD particles were rich in phospholipid, containing 48.1% by weight. The chemical composition of these particles was quite distinct from that of the serum LD lipoproteins, but did, however, show some similarity to that of the serum VLD lipoproteins. A marked resemblance was noted in the chemical characteristics of the Golgi LD and VLD particles (with the exception of triglyceride content). In addition, these two species of Golgi particles possessed the same antigenic determinant. 5. The results suggest that the Golgi VLD particles are the precursors of the serum VLD lipoproteins. On the basis of similarities in gross chemical composition and in the antigenic determinant of the Golgi LD and VLD particles, we conclude that the LD particles are probably the precursors of the VLD particles. In view of the marked differences in gross chemical composition of the Golgi LD particles and serum LD lipoproteins, it appears unlikely that the LD particles are directly secreted into the serum pool.

Animals↗
PubMed1995

The Brefeldin A-induced retrograde transport from the Golgi apparatus to the endoplasmic reticulum depends on calcium sequestered to intracellular stores.

Ribophorin I is a type I transmembrane glycoprotein specific to the rough endoplasmic reticulum. We have previously shown that, when expressed in transfected HeLa cells, a carboxyl-terminally truncated form of ribophorin I that contains most of the luminal domain (RI332) is, like the native protein, retained in the endoplasmic reticulum (ER). Brefeldin A (BFA) treatment of these HeLa cells leads to O-glycosylation of RI332 by glycosyltransferases that are redistributed from the Golgi apparatus to the ER (Ivessa, N. E., De Lemos-Chiarandini, C., Tsao, Y.-S., Takatsuki, A., Adesnik, M., Sabatini, D. D., and Kreibich, G. (1992) J. Cell Biol. 117, 949-958). Using the state of glycosylation of RI332 as a measure for the BFA-induced backflow of enzymes of the Golgi apparatus to the ER, we now demonstrate that the retrograde transport is inhibited when cells are treated with various agents that affect intracellular Ca2+ concentrations, such as the dipeptide benzyloxycarbonyl (Cbz)-Gly-Phe-amide, the Ca2+ ionophore A23187, and thapsigargin, an inhibitor of the Ca(2+)-transporting ATPase of the ER. These treatments prevent the BFA-induced O-glycosylation of RI332. Immunofluorescence localization of the Golgi markers, MG-160 and galactosyltransferase, shows that when BFA is applied in the presence of Ca2+ modulating agents, the markers remain confined to the Golgi apparatus and are not redistributed to the ER, as is the case when BFA alone is used. Cbz-Gly-Phe-amide does not, however, interfere with the BFA-induced release of beta-COP from the Golgi apparatus. We conclude that the maintenance of a Ca2+ gradient between the cytoplasm and the lumen of the ER and the Golgi apparatus is required for the BFA-induced retrograde transport from the Golgi apparatus to the ER to occur.

Biological Transport↗
PubMed1999

Transport of bacterial lipopolysaccharide to the golgi apparatus.

Addition of lipopolysaccharide (LPS) to cells in the form of LPS-soluble (s)CD14 complexes induces strong cellular responses. During this process, LPS is delivered from sCD14 to the plasma membrane, and the cell-associated LPS is then rapidly transported to an intracellular site. This transport appears to be important for certain cellular responses to LPS, as drugs that block transport also inhibit signaling and cells from LPS-hyporesponsive C3H/HeJ mice fail to exhibit this transport. To identify the intracellular destination of fluorescently labeled LPS after its delivery from sCD14 into cells, we have made simultaneous observations of different organelles using fluorescent vital dyes or probes. Endosomes, lysosomes, the endoplasmic reticulum, and the Golgi apparatus were labeled using Texas red (TR)-dextran, LysoTrackertrade mark Red DND-99, DiOC6(3), and boron dipyrromethane (BODIPY)-ceramide, respectively. After 30 min, LPS did not colocalize with endosomes, lysosomes, or endoplasmic reticulum in polymorphonuclear leukocytes, although some LPS-positive vesicles overlapped with the endosomal marker, fluorescent dextran. On the other hand, LPS did appear to colocalize with two markers of the Golgi apparatus, BODIPY-ceramide and TRITC (tetramethylrhodamine isothiocyanate)-labeled cholera toxin B subunit. We further confirmed the localization of LPS in the Golgi apparatus using an epithelial cell line, HeLa, which responds to LPS-sCD14 complexes in a CD14-dependent fashion: BODIPY-LPS was internalized and colocalized with fluorescently labeled Golgi apparatus probes in live HeLa cells. Morphological disruption of the Golgi apparatus in brefeldin A-treated HeLa cells caused intracellular redistribution of fluorescent LPS. These results are consistent with the Golgi apparatus being the primary delivery site of monomeric LPS.

Biological Transport↗
PubMed1983

Ultrastructure and cytochemistry of the Golgi apparatus and related organelles of the secretory ameloblasts of the rat incisor.

Glutaraldehyde-fixed rat incisors were either post-fixed in ferrocyanide-reduced osmium impregnated with the Ur-Pb-Cu technique or incubated in the medium for acid-phosphatase (AcPase) reaction. The Golgi apparatus of the secretory ameloblast was composed of 4-7 cisternae, small vesicles and condensing vacuoles. It formed an elongated, continuous membrane system over from one- to two-thirds of the supranuclear cytoplasm. Condensing vacuoles seemed to be produced from the dilated margins of both Golgi cisternae and GERL. AcPase activity was demonstrated in the inner 2 or 3 Golgi cisternae, in GERL and in some condensing vacuoles and secretory granules within the Tomes process. It thus seemed that primary lysosomes originate from both the Golgi apparatus and GERL. Whereas many lysosomal bodies appeared in the supranuclear cytoplasm, autophagic vacuoles were rare. A well-developed Golgi apparatus and GERL were, therefore, considered to be involved in the digestion of exogeneous materials as well as in the formation of the precursor of enamel matrix. The simple Golgi apparatus consisting of only compactly stacked cisternae and small vesicles, were sometimes observed in the supranuclear cytoplasm and having no clear relationship with rough-surfaced endoplasmic reticula or GERL, may serve as a source of the plasma membranes necessary for continuous renewal of the cisternae of the well-developed Golgi apparatus.

Acid Phosphatase↗
PubMed1981

Newly biosynthesized cytochrome P-450 associated with the golgi apparatus from livers of rats induced with phenobarbital.

1. Cytochrome P-450 has been detected in preparations of golgi apparatus from the livers of phenobarbital-induced rats. 2. Newly biosynthesized cytochrome P-450 is also present in preparations of golgi apparatus. By using three different techniques to fractionate the golgi into vesicle contents and membrane components it was found that newly biosynthesized cytochrome P-450 is associated solely with the membrane fraction. 3. By investigating the susceptibility of cytochrome P-450, present in the golgi apparatus, to the action of trypsin it was found that the majority was oriented on the cytosolic face of the membrane.

Animals↗
PubMed2001

S-adenosylmethionine reverses ilimaquinone's vesiculation of the Golgi apparatus: a fluorescence study on the cellular interactions of ilimaquinone.

The marine sponge metabolite ilimaquinone has a wide range of biological activities, including vesiculation of the Golgi apparatus and interference with intracellular protein trafficking. Some of these activities may arise from ilimaquinone's influence on the activated methyl cycle. To visualize the morphological effects of ilimaquinone on the Golgi apparatus, NRK (normal rat kidney) cells were labeled with fluorescent wheat germ agglutinin and treated with ilimaquinone in the presence and absence of the methylating agent S-adenosylmethionine (SAMe). While ilimaquinone alone fragments the Golgi apparatus, the organelle remains intact when SAMe is included in the incubation mixture. This observation supports ilimaquinone's interaction with methylation enzymes as the cause of Golgi vesiculation. The examination of a fluorescently labeled ilimaquinone analogue in NRK cells suggests that the cellular interactions of ilimaquinone are not localized to the Golgi apparatus.

Adenosylhomocysteinase↗