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PubMed1992

Swelling response of Golgi apparatus cisternae in cells treated with monensin is reduced by cell injury.

The effect of mechanical stress on Golgi apparatus was examined in thin slices of rat liver. The findings should be of relevance both to electron microscopists who routinely mince tissue, and to biochemists who homogenize tissues to isolate membranous components. The swelling response of Golgi apparatus to monensin was used as an assay because the swelling response is distinct and is thought to result from a well-characterized metabolic process, namely the acidification of vesicles. The results showed that the swelling response was compromised by monensin as far away as 6-7 cells from a cut surface even though other aspects of cell ultrastructure were not altered from normal. The monensin-induced swelling response was also evaluated in isolated Golgi apparatus and found to be similar to that with tissue. Thus, mechanical stress such as commonly used to mince tissue or isolate tissue components, appears to markedly alter Golgi apparatus function compared to the situation in vivo. In this example, the altered response of Golgi apparatus to monensin indicated that some aspects associated with the ATP-dependent proton-pumping machinery of the trans-most cisternae and trans Golgi network were compromised.

Animals↗
PubMed2003

Biochemical sub-fractionation of the mammalian Golgi apparatus.

We have exploited the breakdown of the Golgi apparatus that occurs during mitosis to isolate subfractions using immuno-affinity methods. Rat liver Golgi stacks were treated with mitotic cytosol from HeLa cells, and the fragments were then incubated with antibodies immobilized on magnetic beads. Antibodies against the cis-Golgi marker, GM130, bound membranes that were depleted in the trans-Golgi network marker, TGN38, whereas antibodies against the cytoplasmic tail of TGN38 did the reverse. A range of other Golgi enzymes, SNAREs and tethers were also tested and were found to bind to anti-GM130 antibodies to an extent that reflected their proximity to cis-cisternae as determined by other techniques. This method should provide a useful complement to the immuno-EM methods presently used to map the Golgi apparatus.

Animals↗
PubMed1986

RCA I-binding patterns of the Golgi apparatus.

The distribution in the Golgi apparatus of binding sites for the galactose-specific Ricinus communis I lectin (RCA I) was studied in differently specialized cells, including goblet cells and absorptive enterocytes of the rat small intestine as well as acinar cells of the rat embryonic pancreas and submandibular gland. For the purpose of localizing the binding reactions, a pre-embedment method using horseradish peroxidase for electron microscopic visualization, and a post-embedding technique making use of the colloidal gold system were employed. The reactions obtained, localizing cell constituents which contain saccharides with terminal or internal beta-D-galactosyl residues, labeled diverse Golgi subcompartments. The goblet cells showed intense RCA I staining of the cisternae of the trans side of the Golgi stacks. The reaction was weak in the medial cisternae and the cis side of the stacks mostly was devoid of label. In the absorptive cells, in addition to the RCA I reaction of trans Golgi elements, binding sites for this lectin were concentrated in the stacks' medial section. In the embryonic acinar cells, accessible galactosyl residues were either confined to the trans and/or medial cisternae, or distributed across elements of all the stacked saccules. In the latter stacks, the reactions mostly were weak in the cis cisternae and increased in intensity towards the trans side. As regards the respective labeling patterns, similar percentages were calculated for the early and late stages of development: they were approximately 62% for the pattern which showed RCA I label limited to trans/medial cisternae and approximately 38% for the "cis-to-trans"-distributed RCA I reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1998

Environmental factors modulate the size and the secretory activity of the notochord. A study of the Golgi apparatus in avian embryos.

In this study we examined the Golgi apparatus of avian notochord transplants excised from 2-day-old (E2) chick embryos and grafted isochronically into a chick host either in a medial-ventral position, next to the host notochord, or in a superficial position under the ectoderm laterally or dorsally to the neural tube. The operated embryos were examined from E2 to E8. The diameters, the cytoplasmic vacuolization and the immunostained Golgi apparatus were identical between the endogenous and ventrally grafted notochords, as well as between host and superficially transplanted notochords when observed at E2. In contrast, from E4 to E8, the size of the notochords grafted dorsally or laterally to the neural tube significantly smaller than the host, while the cytoplasmic vacuolization and the degree of fragmentation of the Golgi apparatus were significantly less than in the host notochords. These results show that environmental and position-specific factors influence the developmental program and the secretory activity of the notochordal cells.

Animals↗
PubMed1979

The lipids of the Golgi apparatus subfractions from rat liver.

Golgi apparatus were isolated from untreated rat liver and separated into three fractions. One consisted mainly of vesicles, a second of tubular particles (dictyosomes) and the third was a mixed fraction. Large differences between these fractions could be seen in the electron microscope and by enzyme analysis. The total lipid content of the vesicles was 3.5-times greater than that of the dictyosomes and the neutral lipid value was 7-times greater. The ratio of phospholipids to protein was approximately the same in the three fractions. However, the phospholipid patterns differed between the vesicle and dictyosome fractions.

Animals↗
PubMed1969

Cellulosic wall component produced by the golgi apparatus of Pleurochrysis scherffelii.

The Golgi apparatus of a marine chrysophycean alga Pleurochrysis scherffelii Pringsheim produces wall fragments (circular-to-ellipsoidal "scales") which are released to the periphery by an exocytotic process involving the fusion of cisternae and the plasma membrane. The cellulosic component of the scales is a complex network of fibrils (10 to 25 angstroms in diameter) that resist treatment with strong alkali. Untreated washed scales yield galactose, ribose, arabinose, and traces of glucose; alkali-purified scales yield much more glucose. The fibrillar scale constituent shows a positive iodine dichroism of the intact wall, a positive zinc chloride-iodine reaction, breakage sites characteristic of highly crystalline cellulose, and solubility in Schweizer's reagent.

Cellulose↗
PubMed1991

Localization of components involved in protein transport and processing through the yeast Golgi apparatus.

Saccharomyces cerevisiae sec7 mutants exhibit pleiotropic deficiencies in the transit of proteins through the Golgi apparatus, and elaborate an array of Golgi apparatus-like cisternae at a restrictive growth temperature (37 degrees C). The SEC7 gene encodes an essential high-molecular weight protein (227 kD) that is phosphorylated in vivo. In cell lysates, Sec7 protein (Sec7p) is recovered in both sedimentable and soluble fractions. A punctate immunofluorescent pattern of Sec7p-associated structures seen in SEC cells coalesces in sec14 mutant yeast that accumulate exaggerated Golgi cisternae at 37 degrees C. Sec7p may function as a peripheral membrane protein that cycles between a soluble, cytosolic pool and a sedimentable, membrane-associated complex for its essential role in vesicular traffic through the Golgi apparatus. The transmembrane Kex2 protease, which processes precursors of secreted peptides within the yeast secretory pathway, is also localized by indirect immunofluorescence to multiple structures in the yeast cell (Redding, K., and R. Fuller, manuscript submitted for publication). In double-immunofluorescence labeling experiments, significant colocalization of Sec7 and Kex2 proteins was found. Colocalization of the two antigens, one implicated in protein transport through the Golgi apparatus and the other in processing within a late Golgi compartment, supports the conclusion that we have visualized the yeast Golgi apparatus.

Biological Transport↗
PubMed1997

Influence of LEPK on biochemical activity and morphology in situ of liver Golgi apparatus from control and streptozotocin-diabetic rats.

This paper presents yields of Golgi-rich membrane isolation, the activity of galactosyltransferase (GalT), the marker enzyme of Golgi apparatus as well as the morphology of the organelle from the livers in situ, in two groups of rats. One group consisted of control rats injected twice intraperitoneally with LEPK. Second group consisted of rats injected with LEPK and additionally after 24hrs given streptozotocin (SZ) to induce experimental diabetes. The results were compared with our previous investigations in control and diabetic rats. In the latter the activity of GalT was diminished, therefore diminishing glycosylation ability, and destructing Golgi apparatus morphology. This experiment shows that two-fold injection of LEPK prior to SZ does not prevent from changes in such biochemical parameters as free blood glucose level, yield of liver Golgi membranes isolation or total activity of GalT. For the first time in c. 30% of investigated rats the inactive enzyme of Golgi apparatus was found in the rats treated with LEPK+SZ. Morphological investigations of liver Golgi apparatus in rats treated with LEPK show slightly increased secretory activity with similar to untreated control rats morphological structure of this organelle. In the rats treated with LEPK and SZ the same morphological changes as in diabetic liver were found, however, such dramatic alterations as in SZ-diabetic rats were never found, irrespective of active or inactive GalT.

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↗
PubMed1976

The synthesis of apoproteins of very low density lipoproteins isolated from the Golgi apparatus of rat liver.

The incorporation of [3H]leucine in vivo into very low density lipoproteins (VLDL) from the rat hepatic Golgi apparatus and serum was studied. A Golgi-rich fraction isolated on a discontinuous sucrose gradient between 0.5 and 1.1 M was found to contain VLDL having common antigenic determinants with serum VLDL. The incorporation of the [3H]leucine into the Golgi VLDL and serum VLDL suggested a precursor-product relationship. Analysis of the apoproteins of the Golgi VLDL by polacrylamide gel electrophoresis revealed protein bands with similar mobility to those of serum VLDL, except that the former contained virtually no rapidly migrating peptides with the mobility of serum apo-C-II and apo-C-III. The pattern of incorporation of the [3H]leucine into the apoproteins was similar in VLDL from Golgi apparatus and serum, except for the absence of radioactivity in the area of the gel of Golgi apo-VLDL corresponding to apo-C-II and apo-C-III. The radioactive amino acid was incorporated predominantly into the Golgi apo-VLDL bands with similar mobility to apo-B and an apoprotein or group of apoproteins containing the arginine-rich peptide of serum VLDL. In vitro incubation of the Golgi VLDL with [3H]leucine-labeled HDL resulted in the acquisition of a number of proteins, including the rapidly migrating proteins. Administration of colchicine prior to the injection of [3H]leucine resulted in the appearance of gel bands and radioactivity in the apo-C-II and apo-C-III areas of Golgi apo-VLDL, suggesting that these can be acquired if secretion of VLDL is slowed or inhibited. The hepatic Golgi apparatus was then divided into fractions of predominantly forming face (GF3) or secretory granules (GF1). After polyacrylamide gel electrophoresis of the apo-VLDL from GF, no visible bands or incorporation of [3H]leucine was found in the region of apo-C-II or apo-C-III. However VLDL from GF1, showed visible and radioactive bands in the apo-C-II and apo-C-III area although they represented a much smaller proportion of the total apoprotein than was found in the corresponding serum apo-VLDL. In the isolated perfused liver the percentage incorporation of [3H]leucine into the rapidly migrating apoproteins of Golgi VLDL was considerably less than that found in the corresponding apoproteins of perfusate VLDL, where circulating C lipoproteins are virtually absent. The data indicate that nascent VLDL begins to acquire the C-II and C-III apoproteins during its passage through the Golgi apparatus but that the main acquisition occurs during or after secretion into the space of Disse.

Animals↗
PubMed1999

The discovery of the Golgi apparatus.

The existence of the cell organelle which is now known as Golgi apparatus or Golgi complex, or simply as 'the Golgi", was first reported by Camillo Golgi in 1898, when he described in nerve cells an 'internal reticular apparatus' impregnated by a variant of his chromoargentic staining. It soon became clear that the newly-identified cytoplasmic structure occurred in a variety of cell types. However, the reality of the organelle was questioned for decades, until it was finally ascertained with electron microscopy. The Golgi apparatus was destined to become a protagonist of the research in cytology and cell biology pursued in the second half of the twentieth century.

Cell Biology↗
PubMed1994

Cell-free transfer of phospholipids between the endoplasmic reticulum and the Golgi apparatus of leek seedlings.

The transfer of lipids between the endoplasmic reticulum and the Golgi apparatus was investigated in vitro using a cell-free system from leek seedlings. Lipids of the donor membranes (endoplasmic reticulum) were radiolabeled either by incubating leek seedlings with [1-14C]acetate or [3H]acetate. Acceptor membranes (Golgi apparatus) were unlabeled and immobilized on nitrocellulose strips. The assay measured the lipid transfer resulting from both an ATP-independent process and an ATP- and cytosol-dependent process. A significant ATP- and cytosol-dependent lipid transfer was observed only in the case of the endoplasmic reticulum as donor and the Golgi apparatus as acceptor. Lipids transferred in an ATP-dependent manner were chiefly phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine. The stimulation of lipid transfer by ATP as compared to the ATP-independent process was +79% (PC), +123% (PS) and +69% (PE). On the other hand, PI was not transferred in an ATP-dependent manner (the stimulation by ATP was only 20%). This supports the theory that a sorting of phospholipids takes place in the donor membrane. Moreover, a formation of lysoPC was observed only in the presence of ATP (+330%). The ATP-dependent lipid transfer was inhibited by N-ethylmaleimide, indicating the involvement of cytosolic (but no phospholipid transfer proteins) or membrane proteins in the transfer process. The ATP-dependent transfer of lipids was also diminished at 12 degrees C showing the sensitivity to low temperatures of the transfer of lipids between the endoplasmic reticulum and the Golgi apparatus.

Adenosine Triphosphate↗
PubMed2002

GS15 forms a SNARE complex with syntaxin 5, GS28, and Ykt6 and is implicated in traffic in the early cisternae of the Golgi apparatus.

The subcellular localization, interacting partners, and function of GS15, a Golgi SNARE, remain to be established. In our present study, it is revealed that unlike proteins (Bet1 and the KDEL receptor) cycling between the Golgi and the intermediate compartment (IC, inclusive of the ER exit sites), GS15 is not redistributed into the IC upon incubation at 15 degrees C or when cells are treated with brefeldin A. Immuno-electron microscopy (immuno-EM) reveals that GS15 is mainly found in the medial-cisternae of the Golgi apparatus and adjacent tubulo-vesicular elements. Coimmunoprecipitation experiments suggest that GS15 exists in a distinct SNARE complex that contains SNAREs (syntaxin5, GS28, and Ykt6) that are implicated in both ER-to-Golgi and intra-Golgi transport but not with SNAREs involved exclusively in ER-to-Golgi traffic. Furthermore, components of COPI coat can be selectively coimmunoprecipitated with GS15 from Golgi extracts. Overexpression of mutant forms of GS15 affects the normal distribution of cis- and medial-Golgi proteins (GS28, syntaxin 5, and Golgi mannosidase II), whereas proteins of the trans-Golgi and TGN (Vti1-rp2/Vti1a and syntaxin 6) and Golgi matrix/scaffold (GM130 and p115) are less affected. When the level of GS15 is reduced by duplex 21-nt small interfering RNA (siRNA)-mediated knockdown approach, diverse markers of the Golgi apparatus are redistributed into small dotty and diffuse labeling, suggesting an essential role of GS15 in the Golgi apparatus.

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↗
PubMed1988

Post-Golgi apparatus localization and regional expression of rat intestinal sialyltransferase detected by immunoelectron microscopy with polypeptide epitope-purified antibody.

During studies on the Golgi apparatus immunolocalization of beta-galactoside alpha 2,6-sialyltransferase in intestinal cells, immunostaining of a number of post-Golgi apparatus structures including mucus droplets and plasma membrane were observed. In order to determine if this labeling was in fact due to sialyltransferase and not carbohydrate-specific antibodies in the polyclonal antiserum preparation, fusion protein to sialyltransferase was used to epitope purify polypeptide-specific antibodies. The affinity purification was performed on a column containing a beta-galactosidase-sialyltransferase fusion protein expressed in Escherichia coli. Using such antibodies we present evidence that in intestinal cells sialyltransferase is not only present in the Golgi apparatus cisternal stack but also its transtubular network and various post-Golgi apparatus structures. In absorptive enterocytes, post-Golgi apparatus vesicles, the brush border and basolateral plasma membrane, multivesicular bodies, and lysosome-like structures were labeled. In goblet cells the limiting membrane and lumen of forming and mature mucus droplets as well as the plasma membrane exhibited label for sialyltransferase. The results provide evidence for "ecto-sialyltransferase" in the plasma membranes of these cells, and suggest that most of the sialyltransferase is released from the Golgi membranes and becomes secreted with the goblet cell mucus. In addition, the polypeptide epitope-purified antibody was also used to examine regional expression of sialyltransferase in the rat intestinal epithelium. Immunolabel was restricted to the large intestine and not found in duodenum, jejunum, and ileum. Direct measurement of the enzyme activity was found to correlate with the immunoelectron microscopic data. This observation suggests that there is regional specific expression of the beta-galactoside alpha 2,6-sialyltransferase.

Animals↗
PubMed1987

Prenatal exposure to alcohol alters the Golgi apparatus of newborn rat hepatocytes: a cytochemical study.

The effect of prenatal exposure to ethanol on the Golgi apparatus of newborn rat hepatocytes has been studied cytochemically using several trans-Golgi markers (thiamine pyrophosphatase, uridine diphosphatase, inosine diphosphatase, acid phosphatase, and 5'-nucleotidase) as well as a cis-side marker (osmium impregnation). The amount of cerium phosphate formed in the cytochemical reactions was roughly quantitated by stereologic methods. The Golgi apparatus of about 40% of the hepatocytes appeared disorganized after alcohol treatment, and in the other 60%, the electron density of reaction product deposits for all phosphatases investigated was decreased. 5'-Nucleotidase was completely absent in cisternae of Golgi apparatus of treated cells. In control cells impregnated with osmium tetroxide, reduced osmium compounds were observed in most Golgi cisternae and in nearby vesicles. In contrast, only small vesicles appeared positive in treated hepatocytes. These results suggest that prenatal alcohol exposure alters some Golgi functions. Thus, the decrease in nucleoside diphosphatase and 5'-nucleotidase cytochemical activities after ethanol exposure strongly suggests that this treatment could affect glycosylation in the Golgi apparatus of newborn rat hepatocytes.

5'-Nucleotidase↗
PubMed1995

Cationic amphiphilic drugs inhibit the internalization of cholera toxin to the Golgi apparatus and the subsequent elevation of cyclic AMP.

Cholera toxin (CT) consists of a pentameric B subunit which binds with high affinity to ganglioside GM1, and an A subunit which stimulates adenylate cyclase, resulting in the elevation of cAMP. We now examine the effect of cationic amphiphilic drugs (CADs) on the internalization of rhodamine (Rh)-CT in cultured hippocampal neurons. CADs have recently been shown to inhibit receptor recycling by disrupting the assembly-disassembly of clathrin at the plasma membrane and on endosomes (Wang, L.-H., Rothberg, K. G., and Anderson, R. G. W. (1993) J. Cell Biol. 123, 1107-1117). Rh-CT was internalized by an energy- and temperature-dependent (presumably vesicular) mechanism to the Golgi apparatus. Internalization to the Golgi apparatus was completely but reversibly blocked by CADs, and the ability of CT to stimulate the elevation of cAMP was significantly reduced. In control cells, cAMP levels were elevated 2.3-fold after 20 min of incubation with CT, but in CAD-treated cells cAMP levels were only elevated 1.3-fold. The effect of CADs on CT internalization was not due to a direct effect of CADs on the Golgi apparatus. Our data demonstrate that CADs inhibit vesicular transport of CT to the Golgi apparatus and imply that the sorting of CT to the Golgi apparatus occurs in the same endosomal compartment involved in sorting recycling receptors to the plasma membrane, since both pathways are inhibited by CADs.

Animals↗
PubMed1969

Characterization of lipoprotein particles isolated from the Golgi apparatus of rat liver.

It has been proposed that particles within tubules and vesicles of the Golgi apparatus of liver cells are precursors of very low density lipoproteins in blood plasma. To characterize these particles we isolated a cell fraction rich in Golgi apparatus and associated particles from rat liver in quantities sufficient for analysis. Particles freed from the membranes of the Golgi apparatus and floated at d = 1.006 were studied by chemical analysis, immunodiffusion, and paper electrophoresis. The lipid composition of the Golgi particles was similar to that of very low density lipoproteins from the same rats. The protein content was about 10% of dry weight for both the Golgi particles and plasma very low density lipoproteins. The Golgi particles formed lines of identity with plasma very low density lipoproteins during immunodiffusion against antiserum to plasma very low density lipoproteins. On paper electrophoresis, however, many Golgi particles remained near the origin, with only a few migrating to the pre-beta position. It was concluded that the lipoproteins in the Golgi apparatus are the precursors of plasma very low density lipoproteins.

Animals↗