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PubMed1985

Development of cortical polarity in mouse eggs: involvement of the meiotic apparatus.

Experiments were carried out to determine the origin of cortical polarity in mouse eggs and its possible relation to the meiotic apparatus. Cortices of mature eggs overlying the meiotic apparatus (microvillus-free area) were distinguished by an absence of microvilli and a thickened layer of actin. In contrast, the surfaces of immature oocytes were covered entirely with a dense population of microvilli and were subtended by a uniform layer of actin. When induced to undergo maturation, meiotic spindles formed in the center of immature oocytes and then moved peripherally. Coincident with the cortical localization of the meiotic spindle was the formation of a microvillus-free area, i.e., a loss of microvilli and a thickening of the actin layer associated with this region of the egg cortex. If immature oocytes were incubated in cytochalasin B, meiotic spindles formed; however, they failed to move peripherally and microvillus-free areas did not develop. Oocytes incubated in colchicine did not form meiotic spindles, although the chromosomes condensed and became localized to cortices where microvillus-free areas developed. Cytochalasin B-treated mature eggs maintained intact meiotic spindles and exhibited a disappearance of microvillus-free areas and a reduction in cortical actin. The chromosomes of mature eggs treated with colchicine remained associated with microvillus-free areas despite the disappearance of meiotic spindles. Occasionally, colchicine-treated eggs possessed more than one cortically located mass of chromosomes, each of which was associated with a microvillus-free area. These observations indicate that mechanisms involving the movement of the meiotic spindle to the oocyte cortex and development and maintenance of cortical polarity are cytochalasin B sensitive. Commensurate with the localization of meiotic chromosomes to the egg cortex is the reorganization of cortical actin and the formation of a microvillus-free area.

Animals↗

PubMed1986

An antiserum to the sea urchin 20 S egg dynein reacts with embryonic ciliary dynein but it does not react with the mitotic apparatus.

Unfertilized sea urchin eggs contain one or more dynein-like enzymes which may be able to serve as microtubule translocators during embryonic development. There are at least two interesting possibilities for the function of the egg dynein: the enzyme may be involved in cytoplasmic microtubule movement such as mitotic spindle anaphase motion; or the enzyme may be a stored precursor for the dynein that functions in embryonic cilia, which are expressed and highly motile at the blastula stage of development. In order to determine directly the distribution and possible function of one of the previously described egg dyneins, the latent-activity 20 S egg dynein (Asai and Wilson, 1985), an antiserum was produced which was highly reactive with the important high Mr polypeptides of 20 S dynein. This antiserum reacted in "Western" immunoblots and in dot-blotting experiments with egg dynein and with embryonic ciliary dynein, but it did not react with any component of sperm flagella. Indirect double immunofluorescence microscopy demonstrated that the anti-20 S antiserum could brightly stain embryonic cilia but it did not stain the sperm flagella from the same sea urchin species. Under the same conditions that the antiserum stained cilia, anti-20 S did not stain the mitotic apparatus but it did appear to stain the cortical region of the dividing egg. In a time-course experiment, the antigen reactive with the anti-20 S antiserum gradually accumulated in the developing early sea urchin embryo. The most significant increase in the apparent concentration of the 20 S dynein occurred just prior to embryonic ciliation and during a period when the mitotic activity of the embryo was in decline. These results lead to two conclusions. First, ciliary dynein and sperm flagellar dynein, although derived from very similar organelles and from the same species of sea urchin, are immunologically distinct. Second, the 20 S egg dynein may be a stored precursor of embryonic ciliary dynein and does not appear to be a component of the mitotic apparatus.

Adenosine Triphosphatases↗

PubMed1983

Ultrastructural responses by Golgi apparatus of rat submandibular gland to beta-adrenergic, alpha-adrenergic, and cholinergic stimulation.

Rat submandibular gland tissue pieces were stimulated in vitro for 30 min with a beta-adrenergic agent or a cyclic AMP analog to stimulate protein secretion, or with alpha-adrenergic or cholinergic agents or a Ca2+ ionophore to stimulate fluid secretion. Acinar cells were examined by transmission electron microscopy. In control tissue, acinar cells showed little evidence of secretory activity. The Golgi apparatus was sparse and was associated with a few small, immature secretory granules with fine fibrillar contents. Following secretory granule discharge stimulated by isoproterenol or dibutyryl cyclic AMP, acinar cells were constricted, and had extensive basolateral membrane folding and tightly packed rough endoplasmic reticulum. Golgi complexes were prominent and had multiple small granules with filamentous contents. After stimulation of fluid secretion by alpha-adrenergic agents (epinephrine, phenylephrine), or cholinergic agents (acetylcholine, carbachol, pilocarpine), or a Ca2+ ionophore (A23187), the Golgi apparatus had compact concave cisternae enclosing aggregates of tubulovesicles. Acinar cells were distended, basolateral membranes were expanded, and rough endoplasmic reticulum was dilated and vesiculated.

Acetylcholine↗

PubMed1986

TAME stabilizes the cortex and mitotic apparatus of the sea urchin egg during isolation.

The synthetic substrate p-tosyl-L-arginine methyl ester (TAME) has been included in buffered EGTA media used for the isolation of the mitotic apparatus from clam eggs and also for the isolation of the cortex from sea urchin eggs. In the course of an investigation of the role of actin-fascin and actin-myosin interactions in cytokinesis, the isolation of the sea urchin egg cortex was re-examined and the stability of the cortex to lysis in a buffered EGTA medium near neutrality found to depend directly on the presence of TAME. Lysis of eggs at metaphase in this medium yielded a mixture of cortices and mitotic apparatuses (MA); MA stability under these conditions also required the presence of TAME, although a reduced pH allowed MA isolation in its absence. The action of TAME in stabilizing the actin-based structure of the cortex and the microtubule-based structure of the MA is not duplicated by other proteolysis inhibitors and this compound will also induce actin polymerization and gelation in extracts of the soluble cytoplasmic proteins of the egg under conditions where these are normally inhibited.

Actins↗

PubMed1990

Fragmentation of re-formation of mitotic Golgi apparatus detected by a centrifugal method.

The fragmentation/re-formation process of the Golgi apparatus during mitosis was studied by flotation centrifugation in a stepwise sucrose density gradient. The mitotic Golgi fraction was obtained from Chinese hamster ovary cells synchronized with thymidine and nocodazole. The Golgi apparatus detected by a marker enzyme, galactosyltransferase, was separated into two peaks by the flotation centrifugation. The amount of the Golgi recovered at the lower density peak was less in the mitotic cells than in the interphase cells. The separation profile of the mitotic Golgi returned to that of the interphase Golgi by further incubation of the mitotic cells. The re-formation of the fragmented Golgi was inhibited by nocodazole and vinblastine, but not by actinomycin D and cycloheximide.

Animals↗

PubMed1988

Human proteins IEF 58 and 57a are associated with the Golgi apparatus.

A mouse monoclonal antibody (mAB 22-II-D8B) raised against lysed transformed human amnion cells (AMA) has been characterized. The mAB decorated the Golgi apparatus in growing and quiescent cultured monolayer cells (fibroblasts and epithelial cells) of various species as determined by double immunofluorescence labeling and colocalization with galactosyltransferase antibodies. It reacted with the acidic human proteins IEF 58 (Mr = 29,000) and 57a, respectively (Mr = 30,000) (HeLa protein catalogue number; [(1982) Clin. Chem. 28, 766]), Golgi staining was also observed in BS-C-1 cells microinjected with mAB 22-II-D8B suggesting that the epitopes recognized by the antibody are most likely located on the cytoplasmic face of the membranes. The precise localization of the antigens to the various cisternae of the Golgi apparatus could not be demonstrated by immunogold cytochemistry on ultrathin cryosections due to either weak reactivity of the antibody or low concentration of the antigens. Immunofluorescence staining with mAB 22-II-D8B of lymphoid human Molt-4 cells and some human tissues failed to reveal any significant staining even though these expressed high levels of both IEF 58 and 57a. These results are taken to imply that the epitopes recognized by mAB 22-II-D8B may be masked in some cell types.

Animals↗

PubMed1991

Solubilization of UDP-glucose collagen glucosyltransferase activities from chick embryo liver: Golgi apparatus, smooth and rough endoplasmic reticulum.

1. The choice of a suitable detergent for solubilization of UDP-glucose collagen glucosyltransferase (GGT) activities from chick embryo liver has been investigated. Several detergents were used (zwitterionic detergent as Chaps, and non-ionic detergents as Triton X-100, Nonidet P 40, Brij 35). 2. All the detergents with GGT activities were tested in Golgi apparatus, smooth and rough endoplasmic reticulum (SER, RER). 3. 80-100% GGT Golgi apparatus activity was easily solubilized at low concentrations in surfactant (0.5 mg/ml). 25-78% of SER and RER GGT activities were extracted at this concentration. 4. A higher level of detergent (5 mg/ml) was necessary to release all GGT activities of SER and RER. Protein extraction was identical to GGT activities.

Animals↗

PubMed1992

Studies on the mechanism of different collagen glucosyltransferase reactions (Golgi apparatus, smooth endoplasmic reticulum, rough endoplasmic reticulum) in chick embryo liver.

1. The galactosylhydroxylysylglucosyltransferase (GGT) specific to collagen is located in the RER (rough endoplasmic reticulum), SER (smooth endoplasmic reticulum) and Golgi apparatus for the chick embryo liver. 2. The UDP-glucose collagen glucosyltransferase activities in chick embryo liver were solubilized by Nonidet P-40. 3. The mechanism of collagen glucosyltransferase reaction was studied with enzyme preparation of Golgi apparatus CF2, smooth endoplasmic reticulum CF4 and rough endoplasmic reticulum CF8. 4. For the three fractions, data obtained in experiments were consistent with a sequential ordered mechanism in which the substrates are bound to the enzyme in the following order: Mn2+, collagen and UDP-glucose substrate, with different values for Km and Vmax.

Animals↗

PubMed1985

Vesicles and cisternae in the trans Golgi apparatus of human fibroblasts are acidic compartments.

Recently we demonstrated that low-pH compartments can be visualized with the electron microscope using a basic congener of dinitrophenol, 3-(2,4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP), which concentrates in acidic compartments and can be detected by immunocytochemistry with a monoclonal anti-dinitrophenol antibody. We now report that DAMP also accumulates in cisternae and vesicles associated with the trans face of the Golgi apparatus. DAMP rapidly leaves this compartment when cells are incubated with the ionophore monensin, which indicates that accumulation is due to the acidic pH in this compartment. Using indirect protein A-gold immunocytochemistry, we localized fibronectin, a major secretory protein in fibroblasts, to the trans Golgi vesicles that took up DAMP. Therefore, the trans cisternae of the Golgi apparatus and forming secretory vesicles have an acidic pH.

Animals↗

PubMed1982

Isolation and solubilization of casein kinase from Golgi apparatus of bovine mammary gland and phosphorylation of peptides.

Phosphate incorporation from [gamma-32P]ATP into native and dephosphorylated alpha s1-casein is catalyzed by a casein kinase localized in the Golgi apparatus of lactating bovine mammary gland. Casein kinase from the Golgi is activated with either Mg2+ or Ca2+, and increased specific activity is observed with dephosphorylated casein as the substrate. The casein kinase can be solubilized from Golgi apparatus by the non-ionic detergent, Triton X-100. Gel permeation chromatography on Sepharose CL-4B yields a Stokes radius of 10 nm for the detergent-solubilized casein kinase. Dephosphorylated beta-peptide, the amino-terminal peptide from beta-casein, is a good substrate for the solubilized casein kinase. With dephosphorylated beta-peptide, the maximal velocity is 9.1 and 12.0 nmol/min per mg protein with Mg2+ and Ca2+ activation, respectively. The Michaelis constant for beta-peptide is greater with Ca2+ than with Mg2+ (4.8 mg/ml compared to 0.97 mg/ml). However, the Michaelis constant for ATP is not greatly influenced by these metal ions. The Triton X-100-solubilized Golgi enzyme can also catalyze the phosphorylation of peptides, such as fibrinopeptide A and alpha-melanocyte stimulating hormone.

Animals↗

PubMed1990

Retinoid modulation of cell-free membrane transfer between endoplasmic reticulum and Golgi apparatus.

Cell-free transfer of radiolabeled membrane proteins from part-rough, part-smooth transitional elements of the endoplasmic reticulum to Golgi apparatus immobilized to nitrocellulose in the presence of nucleoside triphosphate, an ATP-regenerating system and a cytosol fraction was promoted by retinol. At an optimum concentration of 1 microgram/ml, the rate and amount of transfer was approximately doubled over 1 to 2 h of incubation in the cell-free system. The transition vesicles induced to form in the cell-free system were concentrated by preparative free-flow electrophoresis in order to study separately the steps of vesicle formation from transitional endoplasmic reticulum and the steps of vesicle fusion with Golgi apparatus. The retinol effect was on vesicle formation as evidenced by an approx. 2-fold increase in transition vesicle numbers, as determined by electron microscope morphometry, and amount from protein determinations on the isolated fractions enriched in transition vesicles. The retinol response in the complete transfer could be eliminated by addition of concentrated cytosol, including cytosol depleted of retinol. An interaction of retinol with some component of the vesicle formation process, possibly involving guanine nucleotides, is indicated.

Adenosine Triphosphate↗

PubMed1991

Glucose-6-phosphatase activity of endoplasmic reticulum and Golgi apparatus in spermatocytes and spermatids of the rat: an electron microscopic cytochemical study.

The glucose-6-phosphatase (G6Pase) activity of cytoplasmic components of spermatocytes and spermatids of the rat was examined by electron microscope cytochemistry using cerium chloride as a capture agent. G6Pase activity, a recognized ER-resident enzyme, was present in all ER cisternae of spermatocytes. In spermatids, while some ER cisternae were G6Pase-reactive, others were negative or only slightly reactive, indicating an unequal distribution of the enzymatic activity throughout the network of ER cisternae in these cells. In spermatocytes, the cis- and trans-elements of the stacks of Golgi saccules were slightly but significantly reactive for G6Pase. In the Golgi apparatus of spermatids, the cis-element, 4 or 5 underlying saccules, as well as one or two thick trans Golgi elements were G6Pase reactive. The G6Pase activity of the various Golgi elements, like that of the ER cisternae was not affected by the pH of the medium and was completely inhibited by Na-vanadate, a known G6Pase inhibitor. Sertoli and Leydig cells, submitted to the same cytochemical conditions, showed complete G6Pase reactivity of their ER; however in Sertoli cells, all Golgi components were consistently negative while in Leydig cells the cis- and trans-elements of the Golgi stacks were slightly reactive, as in spermatocytes. Thus, the G6Pase reactivity of Golgi elements, appeared variable from one cell type to another. The compact juxtanuclear Golgi apparatuses of spermatocytes and spermatids were both associated with numerous G6Pase reactive ER cisternae; some were present at their surface, others crossed their cortices between Golgi stacks and formed elaborate networks in their cores.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1976

Morphological and biochemical changes in the hepatic endoplasmic reticulum and golgi apparatus of male Xenopus laevis after induction of egg-yolk protein synthesis by oestradiol-17 beta.

This report describes morphological and biochemical changes accompanying oestrogen induced synthesis of the egg-yolk protein precursor, vitellogenin, in male Xenopus liver. Extensive proliferation of the rough and smooth endoplasmic reticulum and the Golgi apparatus occurs between 3 and 9 days after administration of oestradiol-17 beta. Subcellular fractionation showed that microsomal fractions have an increased number of ribosomes available for protein synthesis, hormone treatment enhances the in vitro protein synthetic capacity per unit of RNA; both in microsome and ribosome preparations. Polypeptides synthesized in vitro by ribosome preparations show an enrichment in serine content after hormone treatment and an increased proportion of ribosomes can be immunoprecipitated by antibodies directed against vitellogenin. Our data are consistent with the proposal that vitellogenin is synthesized on the ribosomes of the rough endoplasmic reticulum and processed and packaged for secretion in the smooth endoplasmic reticulum and Golgi apparatus. Response to hormonal induction of vitellogenin involves an early phase in which membrane proliferation occurs in order to increase the cellular capacity to synthesize, process and secrete large quantities of egg-yolk protein precursor.

Animals↗

PubMed1988

Wheat germ agglutinin fracture-label of Golgi apparatus membranes in proliferating cells.

The thin section fracture-label technique has been recently used to analyze the distribution and compartmentalization of fully glycosylated components on intracellular membranes. Labelling with the lectin wheat germ agglutinin over the freeze-fractured membranes of Golgi apparatus in various secretory and non-secretory cells as well as in human peripheral lymphocytes was always very weak or absent even over the trans-most cisternae. In order to investigate if the labelling density may reflect the cellular activity in membrane biogenesis, we used in this study wheat germ agglutinin fracture-label of rapidly proliferating cells and mitogen-activated lymphocytes. Labelling over the fractured cisternae of the medial and trans portions of the Golgi apparatus was intense. Treatment with cycloheximide of proliferating cells induced a drastic reduction of the labelling over the Golgi cisternae.

Cell Division↗

PubMed2006

The mitotic phosphatase cdc25C at the Golgi apparatus.

cdc25C is a phosphatase which regulates the activity of the mitosis promoting factor cyclin B/cdk1 by dephosphorylation, thus triggering G(2)/M transition. The activity of cdc25C is regulated by phosphorylation which by itself is implicated in regulating the subcellular localization. It is well accepted that cdc25C has to enter the nucleus to activate the cyclin B/cdk1 complex at G(2)/M transition. Here, we will show that cdc25C is located in the cytoplasm at defined dense structures which by immunofluorescence analysis as well as by biochemical subfractionation turned out to be the Golgi apparatus. It will be further shown that cdc25C at the Golgi fraction is an active phosphatase suggesting an additional and new role of cdc25C at the Golgi apparatus.

Cell Cycle Proteins↗

PubMed2006

Traffic between the plant endoplasmic reticulum and Golgi apparatus: to the Golgi and beyond.

Significant advances have been made in recent years that have increased our understanding of the trafficking to and from membranes that are functionally linked to the Golgi apparatus in plants. New routes from the Golgi to organelles outside the secretory pathway are now being identified, revealing the importance of the Golgi apparatus as a major sorting station in the plant cell. This review discusses our current perception of Golgi structure and organization as well as the molecular mechanisms that direct traffic in and out of the Golgi.

Endoplasmic Reticulum↗

PubMed2003

"Slip, sliding away": phospholipase D and the Golgi apparatus.

Phospholipase D enzymes (PLDs) constitute a family of phosphodiesterases that catalyze the hydrolysis of phosphatidylcholine (PtdCho) to generate choline and phosphatidic acid (PtdOH), a potent lipid signaling molecule implicated in numerous physiological processes. Mammalian PLDs have been localized to multiple organelles, including the nucleus, Golgi apparatus, lysosomes, secretory granules and plasma membrane. However, the detailed mechanisms that govern targeting of PLDs to different organelles, how their local activity is controlled or indeed the nature of PA effectors are not well understood. Here, we discuss recent observations on PLD localization to the Golgi apparatus and how members of this enzyme family might play a role in regulating the structure of this organelle.

Animals↗

PubMed2003

Overexpression of gK in gK-transformed cells collapses the Golgi apparatus into the endoplasmic reticulum inhibiting virion egress, glycoprotein transport, and virus-induced cell fusion.

Intracellular transport and egress of alphaherpesviruses require the coordinate function of multiple proteins and glycoproteins. Recently, we showed that gK is expressed on infected cell surfaces and that gK cell-surface expression required the presence of the UL20 protein [J. Virol. 77 (2003), 499]. Overexpression of gK by gK-transformed cells blocked transport of enveloped virions from perinuclear spaces and inhibited virus-induced cell fusion caused by gK syncytial mutants [J. Virol. 69 (1995), 5401]. Therefore, we investigated whether altered synthesis and transport of gK was responsible for the observed gK-mediated interference phenomena. HSV-1 infection of the gK-transformed cell line Vero (gK9) caused a profound entrapment of gK in the endoplasmic reticulum and total inhibition of gK cell surface expression. In addition, gK drastically inhibited intracellular transport and maturation of gD and caused substantial defects in Golgi-dependent glycosylation of gB. Visualization of intracellular organelles via confocal microscopy revealed a profound collapse of the Golgi apparatus into the endoplasmic reticulum. These results were analogous to those observed in the presence of brefeldin A, a known Golgi disruptor. Therefore, virion entrapment within perinuclear spaces and inhibition of glycoprotein transport are due to gK-mediated collapse of the Golgi apparatus.

Animals↗