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PubMed1985

Demonstration of an extensive trans-tubular network continuous with the Golgi apparatus stack that may function in glycosylation.

Sialyltransferase (Gal beta 1,4GlcNAc alpha 2,6 sialyltransferase) was localized by immunoelectron microscopy in rat liver hepatocytes using affinity-purified antibodies. Immunoreactivity for sialyltransferase was found in the Golgi apparatus, where it was restricted to an interconnected system consisting of the trans-cisternae and the trans-tubular network. This region of the Golgi apparatus exhibited both TPPase and CMPase activity and was the intracellular site where sialic acid residues bound to glycoprotein were detected using the Limax flavus lectin. Sialyltransferase and sialic acid residues were not detected in medial and cis-cisternae of the Golgi apparatus. These findings suggest that in rat hepatocytes sialylation of N-linked glycoproteins occurs in the complex formed by the trans-cisternae and the trans-tubular network of Golgi apparatus.

Animals↗

PubMed1989

Microinjection of antibodies to a 62 kd mitotic apparatus protein arrests mitosis in dividing sea urchin embryos.

Previously, we described a 62 kd protein that is a component of the isolated sea urchin mitotic apparatus. This protein is a substrate for an endogenous, calcium/calmodulin-dependent protein kinase also associated with the mitotic apparatus. Phosphorylation of the 62 kd protein directly correlates with the depolymerization of microtubules in isolated mitotic apparatuses. Here we report a test of the function of the 62 kd protein in vivo. Double labeling studies using a monoclonal antibody to tubulin and an affinity purified antibody specific for the 62 kd protein reveal that the 62 kd protein co-localizes with mitotic apparatus microtubules. When affinity purified antibodies to the 62 kd protein were microinjected into dividing sea urchin embryos, mitosis was blocked in a stage-specific manner. The results are discussed with respect to the role of the 62 kd protein in the metaphase-anaphase transition.

Animals↗

PubMed1992

Beta-amyloid precursor protein localization in the Golgi apparatus in neurons and oligodendrocytes. An immunocytochemical structural and ultrastructural study in normal and axotomized neurons.

We have used a polyclonal antibody raised against a synthetic peptide from the carboxyl terminal of the beta-amyloid precursor protein (APP) to examine the cellular and subcellular localization of this protein in the rat brain. Light and electron microscopic immunocytochemical techniques were used. Immunoreactivity was found throughout the brain in all the neurons examined as well as in oligodendrocytes. At the light microscopic level, a perinuclear filamentous distribution was seen in neurons, suggesting a concentration of the protein to the Golgi apparatus. Axotomy of motor neurons of the facial nucleus produced a decrease in choline acetyltransferase (ChAT) activity and an increase in the perineuronal microglial nucleoside diphosphatase (NDPase)-positive cells in addition to a hypertrophy of the GFAP immunoreactive astrocytes. On the other hand, increased APP-like immunoreactivity all over the neuronal cell bodies accompanied by a dispersion ('rete dispersion') of the Golgi apparatus labeling was demonstrated. In contrast, reactive microglia and hypertrophic astrocytes in axotomized facial nucleus were not immunolabeled. Oligodendrocytes showed a punctate APP immunoreactivity corresponding to the Golgi apparatus in both operated and control facial nucleus. This was further demonstrated by electron microscopic immunolabeling. These results show that the main localization of the C-terminal containing forms of the APP in the rat brain is the Golgi apparatus in both neurons and oligodendroglia and further supporting the secretory nature of these proteins. The increased synthesis of this protein after axotomy is suggestive of a role of the APPs in growth and/or regeneration.

Alzheimer Disease↗

PubMed1995

Effects of 1,2-dichloroethane intoxication on dolichol levels and glycosyltransferase activities in rat liver microsomes and Golgi apparatus.

Rat intoxication with a single dose of 1,2-dichloroethane (DCE) (50 microliters/100 g b.w) is able to induce a significant modification of protein glycosylation in the liver endoplasmic reticulum and Golgi apparatus. HPLC analysis shows that within 5-60 min after DCE-intoxication, the levels of total dolichol, free dolichol and dolichyl phosphate strongly decreased in the microsomes and Golgi apparatus. Particularly in total microsomes, dolichyl phosphate, which is rate-limiting for the biosynthesis of the N-linked oligosaccharide chains, drops to values significantly lower than in the control group 15 min after DCE poisoning. In the Golgi apparatus, the total dolichol, essential to enhance the fluidity and permeability of these membranes, early and significantly decreases already 5 min after DCE poisoning. Moreover, in the Golgi apparatus galactosyl- and sialyltransferase activities, the main enzymatic activities of terminal protein glycosylation, are significantly reduced, as measured 15 min after DCE intoxication. These data suggest that the impairment of glycoprotein synthesis, maturation and secretion may be involved in the pathogenesis of liver injury induced by acute DCE-intoxication.

Alanine Transaminase↗

PubMed1983

Labelling of an intermediate saccule of the Golgi apparatus and of parts of the endoplasmic reticulum by a lectin (soybean agglutinin) in the chick ciliary ganglion.

A lectin (soybean agglutinin or SBA) conjugated with horseradish peroxidase was used to identify the intracellular membranes containing galactose or alpha-N-acetylgalactosamine in young chick ciliary ganglions. The label is found on one or, less frequently, on two saccules of the Golgi apparatus in the ciliary and choroid neurones. The labelled intermediate saccules are always located either near the cisface or the middle region of the Golgi apparatus. This observation confirms that distinct compartments are present in the Golgi apparatus. Labelling is also frequently observed at the end of the rough endoplasmic reticulum cisternae in the neurones. It is also sometimes observed in a few vesicles. The lectin-peroxidase labelling, however, was never observed in Schwann cell Golgi apparatus or in their rough endoplasmic reticulum.

Acetylgalactosamine↗

PubMed1981

Energy-dependent calcium sequestration activity in a Golgi apparatus fraction derived from lactating rat mammary glands.

A fraction isolated from lactating rat mammary glands was shown by marker enzyme assays to be rich in Golgi apparatus vesicles. This Golgi apparatus-rich fraction was shown to accumulate calcium in the presence of ATP but not in its absence. Other nucleoside triphosphates were only partially effective in promoting calcium transport. Mg2+ was required for the uptake which was also temperature and pH dependent. The uptake was sustained by the use of oxalate and phosphate as intravesicular trapping agents. In the presence of 10 mM oxalate the apparent Km for calcium uptake was 0.24 muM ionized calcium. The V was 4.45 nmol calcium/min per mg protein. Preloaded calcium could be rapidly released by the addition of the ionophore A23187 indicating an intravesicular location for the sequestered ion. Addition of ethylene glycol bis(beta-aminoethylether)-N,N'-tetraacetic acid resulted in a slower release of preaccumulated calcium, indicating the existence of one or more efflux routes by which calcium leaves the vesicles in the presence of MgATP. Ruthenium red partially inhibited the uptake but lanthanum and particularly the sulphydryl inhibitor p-hydroxy-mercuribenzoate were much more effective. The properties of the calcium-sequestering system in the Golgi apparatus-rich fraction were similar to those reported for other non-muscular tissues and lend support to the hypothesis that calcium is secreted into milk via the Golgi apparatus of the mammary gland secretory gland.

Animals↗

PubMed1984

Monodehydroascorbate as an electron acceptor for NADH reduction by coated vesicle and Golgi apparatus fractions of rat liver.

Coated vesicles were isolated from rat liver in about 80% fraction purity as determined from electron microscopy and analyses of marker enzymes and compared with Golgi apparatus and other membrane fractions isolated in parallel. The fractions were enriched in NADH-monodehydroascorbate reductase, ascorbate oxidase and ascorbic acid. The NADH-monodehydroascorbate reductase and ascorbate oxidase of the Golgi apparatus and coated vesicles differed from that of the endoplasmic reticulum in being inhibited by the sodium selective ionophore, monensin, at physiological concentrations while these activities were stimulated by ethylenediaminetetraacetic acid in coated vesicles but not in Golgi apparatus. Activities of both coated vesicles and Golgi apparatus fractions depleted in the coat protein, clathrin, were activated by the addition of clathrin-rich supernatant fractions. The results are discussed in the context of monodehydroascorbate as an acceptor for electron transport-mediated transfer of electrons from NADH by coated vesicles as part of a possible mechanism to drive membrane translocations or to acidify the interiors of vesicles.

Animals↗

PubMed1984

Three-dimensional ultrastructure of the Golgi apparatus in bovine mammary epithelial cells during lactation.

The three-dimensional ultrastructure of the Golgi apparatus in milk secreting epithelial cells of bovine mammary gland was explored. From computer-aided reconstructions of serial thin sections, it was determined that the Golgi apparatus was composed of a single set of stacked cisternae. The three-dimensional shape of the dictyosome varied from cell to cell, but the overall shape was that of a hollow cone, cylinder, or bowl. The cis and trans surfaces of the dictyosome were arranged in three-dimensional space such that the cis face was located on the outer surface of the hollow structure and the trans face on the inner surface. The cytoplasmic channel (secretory channel) that traversed the longitudinal axis of the hollow dictyosome contained secretory vesicles. Densely stacked cisternae of rough endoplasmic reticulum surrounded the dictyosome, and microvesicles appeared to fuse with, or bud from, cisternae of both organelles. These findings suggest that Golgi apparatus of the lactating epithelial cell is highly organized and that the Golgi apparatus and secretory channel are essentially an independent compartment within the cell.

Animals↗

PubMed1990

Ultrastructural distribution of NADPase within the Golgi apparatus and lysosomes of mammalian cells.

Cytochemical studies with over 40 different mammalian cell types have indicated that NADPase activity is associated with the Golgi apparatus and/or lysosomes of all cells. In the majority of cases, NADPase is restricted to saccular elements comprising the medial region of the Golgi stack and an occasional lysosome. There is often weak NADPase activity in other Golgi compartments such as the trans Golgi saccules and/or elements of the trans Golgi network. In some cells, however, strong NADPase activity is found within these latter compartments, either exclusively in trans Golgi saccules or elements of the trans Golgi network, or in combination with medial Golgi saccules and each other including (1) medial Golgi saccules + trans Golgi saccules, (2) medial Golgi saccules + trans Golgi saccules + trans Golgi network, or (3) trans Golgi saccules + trans Golgi network. In some rare cases, no NADPase activity is detectable in either Golgi saccules or elements of the trans Golgi network, but it is observed in an occasional lysosome or throughout the lysosomal system of these cells. It is unclear at present if these variations in the distribution of NADPase across the Golgi apparatus, and between the Golgi apparatus and lysosomal system, are due to differences in targeting mechanisms or to the existence of "bottlenecks" in the natural flow of NADPase along the biosynthetic pathway toward lysosomes. While no clear pattern in the association of strong NADPase activity with lysosomes was apparent relative to the ultrastructural distribution of NADPase activity in Golgi saccules or elements of the trans Golgi network, the results of this investigation suggested that cells having NADPase localized predominantly toward the trans aspect of the Golgi apparatus (in trans Golgi saccules or elements of the trans Golgi network or both) have few NADPase-positive lysosomes. The only exception is hepatocytes which were classified as predominantly trans but had noticeable NADPase activity within medial Golgi saccules and elements of the trans Golgi network as well, and highly reactive lysosomes. Other cells showing highly reactive lysosomes including (1) Kupffer cells of liver and those forming the proximal convoluted tubules of the kidney, both of which also had strong NADPase activity within medial and trans Golgi saccules and elements of the trans Golgi network, (2) Leydig cells of the testis and interstitial cells of the ovary, which also showed strong NADPase activity within medial Golgi saccules, and (3) macrophages from lung, spleen and testis, and Sertoli cells from the testis all of which showed no Golgi associated NADPase activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1998

1898: the Golgi apparatus emerges from nerve cells.

A century ago, Camillo Golgi discovered in neurons an intracellular network of anastomosing threads, impregnated by the chromoargentic reaction he had devised to stain the nervous tissue. This structure, designated by Golgi as 'internal reticular apparatus', was soon detected in a wide variety of eukaryotic cells. However, skepticism arose on the existence of the Golgi apparatus in the first decades of this century, when it was fiercely debated whether this structure represented a genuine new cell constituent or an artifact due to the deposit of metallic impregnation on diverse cytoplasmic structures. The reality of the Golgi apparatus became established unequivocally only with the application of electron microscopy; with the visualization of its fine structure, the apparatus finally achieved the status of cytoplasmic organelle, and thus, linked with Golgi's name, entered the modern era of investigation on its components, chemistry and function.

Animals↗

PubMed1999

Alpha-mannosidases involved in N-glycan processing show cell specificity and distinct subcompartmentalization within the Golgi apparatus of cells in the testis and epididymis.

The Golgi apparatus is enriched in specific enzymes involved in the maturation of carbohydrates of glycoproteins. Among them, alpha-mannosidases IA, IB and II are type II transmembrane Golgi-resident enzymes that remove mannose residues at different stages of N-glycan maturation. alpha-Mannosidases IA and IB trim Man9GlcNAc2 to Man5GlcNAc2, while alpha-mannosidase II acts after GlcNAc transferase I to remove two mannose residues from GlcNAcMan5GlcNAc2 to form GlcNAcMan3GlcNAc2 prior to extension into complex N-glycans by Golgi glycosyltransferases. The objective of this study is to examine the expression as well as the subcellular localization of these Golgi enzymes in the various cells of the male rat reproductive system. Our results show distinct cell-and region-specific expression of the three mannosidases examined. In the testis, only alpha-mannosidase IA and II were detectable in the Golgi apparatus of Sertoli and Leydig cells, and while alpha-mannosidase IB was present in the Golgi apparatus of all germ cells, only the Golgi apparatus of steps 1-7 spermatids was reactive for alpha-mannosidase IA. In the epididymis, principal cells were unreactive for alpha-mannosidase II, but they expressed alpha-mannosidase IB in the initial segment and caput regions, and alpha-mannosidase IA in the corpus and cauda regions. Clear cells expressed alpha-mannosidase II in all epididymal regions, and alpha-mannosidase IB only in the caput and corpus regions. Ultrastructurally, alpha-mannosidase IB was localized mainly over cis saccules, alpha-mannosidase IA was distributed mainly over trans saccules, and alpha-mannosidase II was localized mainly over medial saccules of the Golgi stack. Thus, the cell-specific expression and distinct Golgi subcompartmental localization suggest that these three alpha-mannosidases play different roles during N-glycan maturation.

Animals↗

PubMed1998

The history of the Golgi apparatus in neurones from its discovery in 1898 to electron microscopy.

We celebrate in 1998 the centenary of the discovery of the Golgi apparatus. Neurones have played a special role in the history of this cell organelle, primarily because the endocellular apparatus was discovered in nerve cells by Camillo Golgi when he was studying the cerebellum of an owl. In the following years, the apparatus of the nerve cell represented the prototype of this cell component which, however, was soon also detected in many other cell types. During the 1920s and 1930s the original idea of a Golgi endocellular "network" was refused and research was focused on the constituents and function of the organelle, using various tissues and techniques. However, the real existence of the apparatus was seriously questioned until the organelle was finally identified by electron microscopy in the mid-1950s. The studies performed on nerve cells during these decades is briefly reviewed here.

Animals↗

PubMed2003

The Golgi apparatus at the cell centre.

In non-polarised mammalian cells, the Golgi apparatus is localised around the centrosome and actively maintained there. Microtubules and molecular motor activity are required for determining both the localisation and organisation of the Golgi apparatus. Other factors, however, also appear necessary for regulating both the static steady-state distribution of this organelle and its relationship with microtubule minus-end-anchoring activities of the centrosome. Several non-motor microtubule-binding proteins have now been found to be associated with the Golgi apparatus. Recent advances suggest that, in addition to important roles in cell motility, polarisation and differentiation, the interplay between Golgi apparatus and centrosome could participate in other physiological processes such as intracellular signalling, mitosis and apoptosis.

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↗

PubMed2005

Maintenance of the diacylglycerol level in the Golgi apparatus by the Nir2 protein is critical for Golgi secretory function.

The level of diacylglycerol (DAG) in the Golgi apparatus is crucial for protein transport to the plasma membrane. Studies in budding yeast indicate that Sec14p, a phosphatidylinositol (PI)-transfer protein, is involved in regulating DAG homeostasis in the Golgi complex. Here, we show that Nir2, a peripheral Golgi protein containing a PI-transfer domain, is essential for maintaining the structural and functional integrity of the Golgi apparatus in mammalian cells. Depletion of Nir2 by RNAi leads to substantial inhibition of protein transport from the trans-Golgi network to the plasma membrane, and causes a reduction in the DAG level in the Golgi apparatus. Remarkably, inactivation of cytidine [corrected] 5'-diphosphate (CDP)-choline pathway for phosphatidylcholine biosynthesis restores both effects. These results indicate that Nir2 is involved in maintaining a critical DAG pool in the Golgi apparatus by regulating its consumption via the CDP-choline pathway, demonstrating the interface between secretion from the Golgi and lipid homeostasis.

Calcium-Binding Proteins↗

PubMed2004

Retrograde transport of cholera toxin from the plasma membrane to the endoplasmic reticulum requires the trans-Golgi network but not the Golgi apparatus in Exo2-treated cells.

Cholera toxin (CT) follows a glycolipid-dependent entry pathway from the plasma membrane through the trans-Golgi network (TGN) to the endoplasmic reticulum (ER) where it is retro-translocated into the cytosol to induce toxicity. Whether access to the Golgi apparatus is necessary for transport to the ER is not known. Exo2 is a small chemical that rapidly blocks anterograde traffic from the ER to the Golgi and selectively disrupts the Golgi apparatus but not the TGN. Here we use Exo2 to determine the role of the Golgi apparatus in CT trafficking. We find that under the condition of complete Golgi ablation by Exo2, CT reaches the TGN and moves efficiently into the ER without loss in toxicity. We propose that even in the absence of Exo2 the glycolipid pathway that carries the toxin from plasma membrane into the ER bypasses the Golgi apparatus entirely.

Animals↗

PubMed1999

Low cytoplasmic pH causes fragmentation and dispersal of the Golgi apparatus in human hepatoma cells.

The centrosomal localization of the Golgi apparatus in interphase cells is thought to be maintained by retrograde microtubule-based motility. It is well established that, when intracellular pH is lowered, lysosomes and endosomes, also showing pericentrosomal localization, translocate towards the plus ends of microtubules within 15 min. In this study, we found that prolonged incubation in low pH medium (pH 6.6) with 20 mM Na acetate induced the fragmentation and dispersal of the Golgi apparatus in the human hepatoma cell line PLC/PRF/5. The fraction of Golgi-dispersed cells increased in a time-dependent manner, and reached over 60% after the 16-h incubation. The cytoplasmic pH was dropped to approximately 7.10. Replacement with normal pH medium restored the structure and localization of the apparatus within 30 min. In the low pH condition, the microtubular network and endoplasmic reticulum appeared normal, and cytoplasmic dynein was still bound to the fragmented Golgi membranes. These findings suggest that low cytoplasmic pH suppresses the retrograde movement of the Golgi apparatus as well as that of lysosomes and endosomes.

Carcinoma, Hepatocellular↗

PubMed2005

Dynamic transport of SNARE proteins in the Golgi apparatus.

Localization of a membrane protein in a subcellular compartment can be achieved by its retention in the compartment or by its continuous transport toward this compartment. Previous results have suggested that specific enzymes are localized in the Golgi apparatus at least in part by selective retention and exclusion from transport vesicles. However, the function of some Golgi SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins is not compatible with their exclusion from transport vesicles. To help understand the mechanism accounting for the localization of SNARE proteins in the Golgi apparatus, we analyzed their lateral distribution in the Golgi cisternae and their incorporation into transport vesicles. According to our results, all SNARE proteins are efficiently incorporated into transport vesicles, indicating that the localization of SNARE proteins in the Golgi apparatus is not based on a static retention mechanism. Detailed analysis suggested that incorporation into transport vesicles was more efficient for SNARE proteins restricted to the cis face of the Golgi as compared with SNAREs present at the trans face. Furthermore, overexpression of a cis-Golgi SNARE protein altered concomitantly its incorporation in transport vesicles and its intra-Golgi localization. These observations suggest that, contrary to resident Golgi enzymes, SNARE proteins are localized in the Golgi apparatus as the result of a dynamic transport equilibrium.

Animals↗