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PubMed1982

Polarization of the Golgi apparatus and the microtubule-organizing center in cultured fibroblasts at the edge of an experimental wound.

We have used the technique of experimental wounding of confluent monolayers of normal fibroblasts to induce essentially unidirectional and synchronous cell movement at the edge of the wound. The intracellular location of the Golgi apparatus and the microtubule-organizing center was determined by double indirect immunofluorescence microscopy, using antibodies specific for the membranes of the Golgi apparatus and antibodies specific for tubulin, respectively. In cells at the wound edge, the immunolabeled Golgi apparatus and microtubule-organizing center were in close proximity to one another and located predominantly forward of the cell nucleus facing the wound. In the same cultures in cells removed from the wound, the two organelles were also coordinately located; however, they were randomly oriented with respect to the wound edge. This reorientation of the two organelles in cells at the wound edge was evident within minutes after wounding and persisted as cell extension subsequently occurred into the wound. These results suggest that both the Golgi apparatus and the microtubule-organizing center may participate in directing cell movement. The possible mechanisms involved are discussed in the light of previous hypotheses and experimental evidence concerning cell motility.

Animals↗
PubMed1984

The Golgi apparatus and GERL during postnatal differentiation of rat parotid acinar cells: an electron microscopic cytochemical study.

Morphological and cytochemical changes in the Golgi apparatus and GERL of differentiating parotid acinar cells were examined in Sprague-Dawley rats from 5 days to young adult. At day 5, the Golgi apparatus consisted of 3-6 narrow saccules, with short segments of GERL lying adjacent to the trans Golgi saccule. As the glands matured, the Golgi apparatus increased in size and the saccules became broadened and fenestrated reaching a maximum from days 15-20. The saccules subsequently narrowed slightly and by day 25 resembled those seen in young adults. Numerous cisternae of GERL could be seen at the trans face during this period. While the glands were maturing, marked changes occurred in the distribution of thiamine pyrophosphatase (TPPase) activity in the Golgi saccules. In the immature cells, TPPase activity was restricted to 1 or 2 trans Golgi saccules. However, by day 10 TPPase could also be localized in immature secretory granules and in GERL-like cisternae. Unreactive segments of GERL were also present. This pattern of localization persisted until day 20, after which the TPPase activity in the GERL-like cisternae diminished gradually until by day 40 TPPase again was localized in 1-2 trans Golgi saccules and an occasional immature secretory granule. Acid phosphatase (AcPase) activity was localized primarily in lysosomes in the very young animals and increased in GERL with age up to day 15. From days 15 to 20 there was a decrease in the amount of activity seen in GERL, but from day 20 on, the AcPase activity increased until it reached that seen in young adult animals. These results indicate that the presence of TPPase activity in GERL-like cisternae and immature secretory granules may be dependent upon the developmental as well as the physiologic state of the acinar cells and lend further support to the suggestion that GERL is derived from the trans Golgi saccules.

Acid Phosphatase↗
PubMed1980

Biosynthesis of chondroitin sulphate by a Golgi-apparatus-enriched preparation from cultures of mouse mastocytoma cells.

Mouse mastocytoma cells grown in suspension culture produce chondroitin 4-sulphate. A Golgi-apparatus-enriched fraction from these cells was prepared and examined for chondroitin-synthesizing activity. When Golgi-apparatus-enriched fractions were incubated with UDP-[14C]glucuronic acid and UDP-N-acetylgalactosamine, they demonstrated a greater than 13-fold increase in chondroitin-synthesizing activity over cell homogenates. Similar incubations with the addition of a pentasaccharide from chondroitin sulphate resulted in a greater than 40-fold increase in [14C]glucuronic acid-incorporating activity over cell homogenates. Other membrane fractions had much less activity, suggesting that the Golgi apparatus is the most active location for chondroitin biosynthesis. Products of the incubations indicated the formation of [14C]chondroitin glycosaminoglycan on endogenous primers and formation of [14C]-hexasaccharide and somewhat larger [14C]oligosaccharides on exogenous pentasaccharide acceptors. There was, however, a significant amount of large [14C]-chondroitin glycosaminoglycan formed on pentasaccharide, indicating that some pentasaccharide did serve as a true primer for polysaccharide synthesis.

Animals↗
PubMed1994

The Golgi apparatus and acid phosphatase-negative cisternal portions of the trans-Golgi network: ultrastructural and cytochemical studies of secretory epithelial cells in the rat lateral prostate.

Ultrastructural and enzyme-cytochemical studies were performed on the Golgi apparatus in secretory cells of the lateral prostate of normal adult rats using serial ultra-thin sections. In the trans-Golgi area, a unique membrane complex composed of tubular portions and cisternal portions showing a rigid appearance is found. This corresponds to the GERL (Novikoff 1964) or the trans-Golgi network (Griffiths and Simons 1986). From their structural similarities, the cisternal portion found in this study is considered to be the same structure as the plate-like cisterna reported by Inoue and Kurosumi (1989). As we reported previously (Kimura and Ichihara 1985), there are at least two types of acid phosphatase (AcPase) in secretory cells of the rat lateral prostate: one is located only in the structural components involved in their secretory functions and reacts readily with naphthol AS-BI phosphate; the other is a lysosomal type, which reacts well with beta-glycerophosphate. Lysosomal AcPase activity demonstrated by Gomori's method (1952) was found in a few middle- to trans-Golgi cisternae and in lysosomes. The AcPase detectable with Gomori's method and thiamine pyrophosphatase seemed to exist in part in the same cisterna on the trans side. With Robinson and Karnovsky's method (1983) for lysosomal AcPase, however, the reaction products were found only in lysosomes that were occasionally tubular in shape. On the other hand, any activity of AcPases tested could not be detected in the cisternal portion with the rigid appearance. Thus, in secretory cells of the adult rat lateral prostate in normal condition, it is considered that the cisternal portion of GERL, or the trans-Golgi network, has no relation to the processing and/or transport of AcPases.

Acid Phosphatase↗
PubMed1998

Influence of bis(maltolato)oxovanadium(IV) on activity of galactosyltransferase (GalT) and morphology of rat liver Golgi apparatus in control and streptozotocin diabetes.

The relation between bis(maltolato)oxovanadium(IV) (BMOV) influencing the biochemical activity of rat liver Golgi apparatus and the morphology of this organelle was studied in normal and streptozotocin-diabetic rat livers. Ultrastructural examinations revealed marked differences in the morphology of Golgi apparatus in three groups of animals. In the control rats treated only with 0.5% NaCl we did not find any biochemical and morphological changes. Marked changes were found in the rat liver after 1.8 mmol BMOV in 0.5% NaCl (as drinking solution) applied for 7 days, so-called "control" group for vanadium. In this group Golgi apparatus seemed shorter than in the diabetic animals. Finally, the same treatment of rats with previously induced SZ-diabetes, showed relatively small morphological alterations. The ultrastructural observation was compatible with the activity of galactosyltransferase (GalT), the Golgi marker enzyme. In diabetic rats treated with BMOV the activity of this enzyme was almost the same as in controls. Summing up dramatic alterations, previously found in diabetic-untreated rats [22], normalized after orally applied BMOV solution, even after a short time.

Animals↗
PubMedDate not supplied

Tubules of the trans Golgi apparatus visualized by immunoelectron microscopy.

Tubules constitute an integral part of the Golgi apparatus and have been shown to form a complex and dynamic network at its trans side. We have studied in detail structural features of the trans Golgi network and its relationship with the cisternal stack in thin sections of Lowicryl K4M embedded human absorptive enterocytes by immunolectron microscopy. Immunoreactive sites for alpha1,3 N-acetylgalactosaminyltransferase and blood group A substance were detectable throughout the cisternal stack and the entire trans Golgi network. Furthermore, the entire trans Golgi network was reactive for CMPase activity. Evidence for two kinds of tubules at the trans side of the Golgi apparatus was found: tubules that laterally connect adjacent and distant cisternal stacks, and others extending from central and lateral portions of trans cisternae to form the complex and extensive trans Golgi network. Trans cisternae showed often the peeling-off phenomenon and were continuous with the trans Golgi network. Both, trans cisternae and tubules of the trans Golgi network exhibited regionally buds and vesicles with a lace-like, non clathrin coat, previously reported by others in NRK cells, which contained glycoproteins with terminal N-acetylgalactosamine residues. These buds and vesicle are therefore involved in constitutive exocytosis.

Acid Phosphatase↗
PubMed2003

Golgins in the structure and dynamics of the Golgi apparatus.

Golgins are a family of coiled-coil proteins associated with the Golgi apparatus necessary for tethering events in membrane fusion and as structural supports for Golgi cisternae. Recent work has shown that golgins such as GM130, golgin-45 and p115 bind to Rab GTPases via their coiled-coil domains, and that GM130, rather than being part of a static structural matrix, is in dynamic exchange between the membrane surface and the cytoplasm. Golgins such as bicaudal-D1 and -D2 bind to Rab6, but, rather than tethering membranes together, link vesicles to the cytoskeleton, thus adding a new function for this class of proteins. Other golgins containing the Golgi targeting GRIP domain, rather than binding Rabs, interact with and are recruited to membranes by another class of GTPase, the Arls. Current evidence therefore suggests that golgins function in a variety of membrane-membrane and membrane-cytoskeleton tethering events at the Golgi apparatus, and that all these are regulated by small GTPases of the Rab and Arl families.

Animals↗
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↗
PubMed1986

NADP phosphatase as a marker in free-flow electrophoretic separations for cisternae of the Golgi apparatus midregion.

Based on cytochemical analysis, the enzyme NADP phosphatase is most concentrated in the so-called intercalary cisternae from the mid-region of the Golgi apparatus stack. Using free-flow electrophoresis to separate different Golgi regions of rat liver Golgi apparatus, the NADP phosphatase activity, based on estimation of the rate of release of inorganic phosphate from NADP under standard conditions, was similarly localized to membrane fractions from the center of electrophoretic separations. Peak specific activities for both a putative cis marker (NADH-cytochrome c reductase) and an established trans marker (galactosyltransferase) coincided with minima in NADP phosphatase activity, in agreement with the cytochemical observations. The pattern of distribution of enzyme activity for NADP phosphatase differed from that of both acid phosphatase and glucose-6-phosphatase. The pH optimum was 5.0, the Km for NADP was 0.6 mM and a corresponding production of NAD and inorganic phosphorus was shown. Taken together with other markers for free-flow electrophoresis separation, the NADP phosphatase will provide considerable utility as a specific marker to help identify intercalary cisternae of the mammalian Golgi apparatus and to monitor electrophoretic separations.

Animals↗
PubMed1991

Calcium sequestration in the Golgi apparatus of cultured mammalian cells revealed by laser scanning confocal microscopy and ion microscopy.

Co-localization of the elements calcium, potassium, sodium and magnesium with sequestering organelles has been achieved by application of two microscopy techniques on the same cell. Organelles were first localized by laser scanning confocal microscopy (LSCFM) using fluorescent organelle stains. The same cells were then analyzed for elemental distribution with ion microscopy. This approach has identified a perinuclear region of prominent total calcium concentration with the Golgi apparatus. Live cells were fluorescently stained with C6-NBD-ceramide for labeling the Golgi apparatus prior to cryogenic preparation and freeze-drying, and imaged with LSCFM for Golgi localization; identical cells were then analyzed with ion microscopy to image subcellular distributions of total calcium, potassium, sodium and magnesium. In three cell lines, LLC-PK1 porcine kidney epithelial cells, Swiss 3T3 mouse fibroblast cells and L5 rat myoblast cells, the Golgi regions contained significantly higher total calcium concentrations than any other region of the cell (as measured at the spatial resolution of ion microscopy of about 0.5 micron). Intracellular potassium, sodium and magnesium were homogeneously distributed throughout the cell and did not show this pattern. Measurements of depletion of calcium by exposure to calcium-free medium showed that the Golgi apparatus was substantially more resistant to calcium depletion than all other regions of these cells, but sequestered Ca2+ could be released from the Golgi by exposing the cells to calcium ionophore A23187. The Golgi apparatus appears to sequester about 5% of the total cell calcium in LLC-PK1 cells, about 2.5% in 3T3 cells and L5 cells.

4-Chloro-7-nitrobenzofurazan↗
PubMed1994

The effector domain of Rab6, plus a highly hydrophobic C terminus, is required for Golgi apparatus localization.

C-terminal lipid modifications are essential for the interaction of Ras-related proteins with membranes. While all Ras proteins are farnesylated and some palmitoylated, the majority of other Ras-related proteins are geranylgeranylated. One such protein, Rab6, is associated with the Golgi apparatus and has a C-terminal CXC motif that is geranylgeranylated on both cysteines. We show here that farnesylation alone cannot substitute for geranylgeranylation in targeting Rab6 to the Golgi apparatus and that whereas Ras proteins that are farnesylated and palmitoylated are targeted to the plasma membrane, mutant Rab proteins that are both farnesylated and palmitoylated associate with the Golgi apparatus. Using chimeric Ras-Rab proteins, we find that there are sequences in the N-terminal 71 amino acids of Rab6 which are required for Golgi complex localization and show that these sequences comprise or include the effector domain. The C-terminal hypervariable domain is not essential for the Golgi complex targeting of Rab6 but is required to prevent prenylated and palmitoylated Rab6 from localizing to the plasma membrane. Functional analysis of these mutant Rab6 proteins in Saccharomyces cerevisiae shows that wild-type Rab6 and C-terminal mutant Rab6 proteins which localize to the Golgi apparatus in mammalian cells can complement the temperature-sensitive phenotype of ypt6 null mutants. Interestingly, therefore, the C-terminal hypervariable domain of Rab6 is not required for this protein to function in S. cerevisiae.

Amino Acid Sequence↗
PubMed1994

A possible role for stable microtubules in intracellular transport from the endoplasmic reticulum to the Golgi apparatus.

The intracellular transport of secretory proteins involves at an early stage the formation of vesicles from transitional elements of the endoplasmic reticulum (ER) containing these proteins and the transfer of these vesicles to the cis-face of the Golgi apparatus. We propose that the latter transfer process does not occur by random diffusion, but is instead mediated by tracking along stable microtubules. To test this proposal, we have carried out double immunoelectron microscopic labeling experiments on frozen sections of HepG2 hepatoma cells secreting the protein human serum albumin (HSA). By a cycloheximide treatment protocol, the stage during which the transfer of newly synthesized HSA from the ER to the Golgi apparatus occurs in vivo was determined. Sections of the cells were then double immunolabeled using primary antibodies to HSA and to glu-tubulin, the latter specifically detecting stable microtubules. We observed a significantly high frequency of HSA-containing structures between the ER and the Golgi apparatus with which stable microtubules were closely associated. These results support the proposal that stable microtubules may play a critical role in directing the transfer process from the ER to the Golgi apparatus.

Biological Transport, Active↗
PubMed2003

Capacity of the golgi apparatus for biogenesis from the endoplasmic reticulum.

It is unclear whether the mammalian Golgi apparatus can form de novo from the ER or whether it requires a preassembled Golgi matrix. As a test, we assayed Golgi reassembly after forced redistribution of Golgi matrix proteins into the ER. Two conditions were used. In one, ER redistribution was achieved using a combination of brefeldin A (BFA) to cause Golgi collapse and H89 to block ER export. Unlike brefeldin A alone, which leaves matrix proteins in relatively large remnant structures outside the ER, the addition of H89 to BFA-treated cells caused ER accumulation of all Golgi markers tested. In the other, clofibrate treatment induced ER redistribution of matrix and nonmatrix proteins. Significantly, Golgi reassembly after either treatment was robust, implying that the Golgi has the capacity to form de novo from the ER. Furthermore, matrix proteins reemerged from the ER with faster ER exit rates. This, together with the sensitivity of BFA remnants to ER export blockade, suggests that presence of matrix proteins in BFA remnants is due to cycling via the ER and preferential ER export rather than their stable assembly in a matrix outside the ER. In summary, the Golgi apparatus appears capable of efficient self-assembly.

Animals↗
PubMed1988

Excess vitamin A decreases the specific activity of galactosyltransferase in Golgi apparatus of rat liver.

This study investigated the effects of vitamin A excess on hepatic galactosyltransferase (EC 2.4.1.13) activity in livers of rats achieved either by feeding of high levels of retinyl palmitate for 16 wk or gavaging with retinol in olive oil for 3 d. Both hypervitaminotic conditions were characterized by hepatic lipid accumulation. Golgi apparatus fractions were isolated and purity of the fractions was monitored by marker-enzyme analyses and electron microscopy. The quality of the fractions isolated from livers of rats receiving vitamin A excess was not different from that of fractions from control rats. An increase in fat-storing cells in liver, observed in vitamin A excess, coincided with the presence of a floating lipid layer present during isolation of the Golgi apparatus. Galactosyltransferase specific activity (with ovomucoid as acceptor) of Golgi apparatus of rats fed excess vitamin A was 27% of control with chronic feeding and 59% of control with administration by gavage. Activity of another luminally oriented protein, uridine 5'-diphosphate phosphatase, was increased under both in vivo regimens. Vitamin A content of Golgi apparatus, as determined by high performance liquid chromatography, correlated negatively with galactosyltransferase activity after both chronic and acute administration of excess vitamin A.

Acid Anhydride Hydrolases↗
PubMed1990

Identification of an intermediate compartment involved in protein transport from endoplasmic reticulum to Golgi apparatus.

We have studied the role of a previously described tubulovesicular compartment near the cis-Golgi apparatus in endoplasmic reticulum (ER)-to-Golgi protein transport by light and immunoelectron microscopy in Vero cells. The compartment is defined by a 53-kDa transmembrane protein designated p53. When transport of the vesicular stomatitis virus strain ts045 G protein was arrested at 39.5 degrees C, the G protein accumulated in the ER but had access to the p53 compartment. At 15 degrees C, the G protein was exported from the ER into the p53 compartment which formed a compact structure composed of vesicular and tubular profiles in close proximity to the Golgi. Upon raising the temperature to 32 degrees C, the G protein migrated through the Golgi apparatus while the p53 compartment resumed its normal structure again. These results establish the p53 compartment as the 15 degrees C intermediate of the ER-to-Golgi protein transport pathway.

Animals↗
PubMed1990

Microtubule depolymerization inhibits transport of cathepsin D from the Golgi apparatus to lysosomes.

Lysosomes as well as a prelysosomal compartment rich in the mannose 6-phosphate receptor are clustered close to the Golgi apparatus in the perinuclear region of the microtubule organizing center in interphase human skin fibroblasts. The spatial organization of these organelles depends on an intact microtubule network. Depolymerization of the microtubules by treatment of cells with nocodazole leads to random scattering of Golgi elements, the prelysosomal compartment, and lysosomes throughout the cytoplasm. To test whether microtubules and the spatial organization of these organelles are important for efficient transport of lysosomal enzymes, the effect of microtubule depolymerization on the maturation of newly synthesized cathepsin D was studied. An up to fivefold inhibition of proteolytic maturation of cathepsin D was observed in drug-treated cells. This effect was due to a decreased rate of transport of cathepsin D from the Golgi apparatus to lysosomes. Depolymerization of microtubules did not inhibit transport of cathepsin D from the endoplasmic reticulum to the trans-Golgi network. Furthermore, synthesis of the phosphomannosyl marker present on cathepsin D was not affected by nocodazole. These results suggest that efficient transport of cathepsin D from the Golgi apparatus to a prelysosomal compartment and lysosomes is facilitated by microtubules and the spatial organization of these organelles.

Biological Transport↗
PubMed2002

Interorganellar regulation of lysosome positioning by the Golgi apparatus through Rab34 interaction with Rab-interacting lysosomal protein.

We present evidence to suggest the existence of a regulatory pathway for the Golgi apparatus to modulate the spatial positioning of otherwise distantly located lysosomes. Rab34, a new member of the Rab GTPase family, is associated primarily with the Golgi apparatus. Expression of wild-type or GTP-restricted but not GDP-restricted versions of Rab34 causes spatial redistribution of lysosomes from the periphery to the peri-Golgi region. The regulation of lysosomal positioning by Rab34 depends on its association with the membrane mediated by prenylation and its direct interaction with Rab-interacting lysosomal protein (RILP). This biological activity, mediated by Rab34-RILP interaction, is dependent on Lys82 in the switch I region. Our results have uncovered a novel mechanism for the Golgi apparatus to regulate the spatial distribution of another organelle.

Adaptor Proteins, Signal Transducing↗
PubMed1983

Alkaline phosphatase biosynthesis in the endoplasmic reticulum and its transport through the Golgi apparatus to the plasma membrane: cytochemical evidence.

Enzyme induction of HeLa cell placental alkaline phosphatase with various agents such as prednisolone, sodium butyrate, hyperosmolality (NaCl), or combination of these inducers resulted in the appearance of enzyme activity in the rough endoplasmic reticulum, nuclear envelope, Golgi apparatus, and plasma membrane. In the Golgi apparatus, intense reaction product deposits tended to be concentrated on its trans side, with small vesicles and granules also being positively stained. Inhibition of protein synthesis with cycloheximide was followed by the disappearance of enzyme activity from these cytoplasmic organelles but not from the plasma membrane. Treatment with monensin, a secretory protein transport inhibitor, uniformly increased activity in the rough endoplasmic reticulum while causing marked dilatation of the intensely positive Golgi cisternae. These results suggest that intracellular alkaline phosphatase is newly synthesized in the endoplasmic reticulum and then passes en route through the Golgi apparatus to the plasma membrane. Accordingly, the present system could represent the biosynthesis, transport, and incorporation of the model cell surface enzyme protein to add to the vesicular stomatitus virus glyco-1 (VSV-G) protein and acetylcholine receptor model systems for studying the dynamics of cell surface protein genesis, transport, and membrane integration.

Alkaline Phosphatase↗