Angel Velasco

A Regulatory Role for cAMP-dependent Protein Kinase in Protein Traffic along the Exocytic Route

The influence of protein kinase A activity on transport of newly synthesized vesicular stomatitis virus G glycoprotein along the exocytic pathway was examined. Transport of vesicular stomatitis virus G glycoprotein to the cell surface was inhibited by N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89), a selective inhibitor of protein kinase A. This block occurred at the exit of the Golgi complex, whereas transport through the Golgi compartments or from the endoplasmic reticulum to the Golgi was decreased in the presence of H-89. As judged by immunofluorescence endoplasmic reticulum to Golgi transport was accelerated in cells incubated with activators of protein kinase A such as isobutylmethylxanthine (IBMX) or forskolin (FK). Treatment with IBMX and FK also increased transport from the trans-Golgi network to the cell surface. During incubation with IBMX and FK, the organization of the Golgi complex was altered showing intercisternae fusion and miscompartmentalization of resident proteins. These structural changes affected both the kinetics of acquisition of endoglycosidase H resistance and transport activities. These data support a differential regulatory role for protein kinase A in different transport steps along the exocytic pathway. In particular, transport from the trans-Golgi network to the cell surface was dependent on protein kinase A activity. In addition, the results suggest the involvement of this enzyme on the maintenance of the Golgi complex organization.

Golgi structural stability and biogenesis depend on associated PKA activity.

The mammalian Golgi complex consists of stacks of cisternae linked laterally into a continuous perinuclear ribbon structure. Protein kinase A is stably associated with the Golgi complex during interphase. To analyze its role in Golgi structural maintenance cells were depleted of protein kinase A regulatory subunits using small interfering RNAs. Under these conditions, the catalytic subunits redistributed to the cytosol and the entire Golgi complex underwent disassembly into multiple juxtanuclear fragments. A similar effect took place following pharmacological inhibition or redistribution of the complete holoenzyme to the cytosol. Golgi fragments maintained their polarization and competence for anterograde protein trafficking. By electron microscopy, they were identified as whorl-like structures composed of concentrically arrayed cisternae. To test a possible role of protein kinase A in Golgi biogenesis we analyzed its involvement during Golgi reassembly from the endoplasmic reticulum. In cells incubated with protein kinase A inhibitors, Golgi reconstruction was arrested at a late step of the reassembly process. This is consistent with the stage of enzyme recruitment from cytosol to emerging Golgi membranes during the reassembly process. We conclude that protein kinase A activity plays a relevant role in the assembly and maintenance of a continuous Golgi ribbon from separated membrane stacks.

PKA-Mediated Golgi Remodeling During cAMP Signal Transmission

Cyclic AMP (cAMP)‐dependent protein kinase A (PKA) is part of the set of signaling proteins that are stably associated to the cytosolic surface of Golgi membranes in mammalian cells. In principle, Golgi‐associated PKA could participate in either signal transduction events and/or the coordination of Golgi transport activities. Here, we show data indicating that although Golgi‐associated PKA is activated fast and efficiently during cell stimulation by an extracellular ligand it does not contribute significantly to cAMP signal transmission to the nucleus. Instead, most of the PKA catalytic subunits Cαderived from the Golgi complex remain localized in the perinuclear cytoplasm where they induce changes in Golgi structural organization. Thus, in stimulated cells the Golgi complex appears collapsed, showing increased colocalization of previously segregated markers and exhibiting merging of different proximal cisternae within a single stack. In contrast, the trans‐Golgi network remains as a separate compartment. Consequently, the rate of protein transport is increased whereas glycan processing is not severely affected. This remodeling process requires the presence of PKA activity associated to the Golgi membranes. Together these data indicate that Golgi‐associated PKA activity is involved in the adaptation of Golgi dynamic organization to extracellular signaling events.

Calphostin C induces selective disassembly of the Golgi complex by a protein kinase C-independent mechanism

Intact cells incubated with calphostin C, an inhibitor of the regulatory domain of protein kinase C, showed fragmentation and dispersal of the Golgi complex by a light-dependent mechanism. At the ultrastructural level Golgi stacks were replaced by clusters of vesicles and short tubules that resembled the Golgi remnants present in control mitotic cells. Vesicle-mediated transport processes along both the exocytic and endocytic routes were also inhibited by calphostin C treatment. Golgi disassembly, however, was not due to protein kinase C inhibition since several inhibitors of the catalytic domain did not cause a similar effect. In contrast, pretreatment with phorbol 12-myristate 13-acetate partly protected the Golgi complex from disassembly by calphostin C. The in vitro effect was shown to be reversible, required both cytosol and ATP and it was inhibited by pretreatment of the Golgi membranes with trypsin but not with high salt. These results suggest the interaction of calphostin C with a structural Golgi protein containing a phorbol ester-binding domain and necessary for the stability of this organelle during interphase.

Ultrastructural demonstration of proteoglycans in adult rat cornea

Proteoglycans in the adult rat cornea were demonstrated at the electron microscope level using two approaches: (a) staining with cuprolinic blue dye in the presence of 0.3 MgCl2, and (b) immunocytochemical localization of glycosaminoglycans with monoclonal antibodies and protein A-gold complexes. In the stroma two kinds of cuprolinic blue-induced filaments were morphologically differentiated and characterized according to their sensitivity to enzymatic degradations as keratan sulphate-rich and chondroitin-dermatan sulphate-rich proteoglycans respectively. Both types were mostly associated with collagen fibres, occupying the whole stroma except in certain areas whose significance is discussed. By immunocytochemistry, anterior and posterior regions of the stroma were found to be richer in chondroitin sulphate than the middle part, whereas keratan sulphate showed an homogeneous distribution throughout the stroma. Glycosaminoglycans were also detected in corneal basement membranes, epithelium and endothelium. The latter localizations are discussed in the light of what is known at present about the production of glycosaminoglycans by corneal cells.

Ultrastructural demonstration of lectin binding sites in the Golgi apparatus of rat epiphyseal chondrocytes.

SummaryBinding sites for wheat germ agglutinin (WGA), Dolichos biflorus agglutinin (DBA), Ricinus communis I agglutinin (RCA I) and Limax flavus agglutinin (LFA) have been ultrastructurally detected in rat epiphyseal chondrocytes by a post-embedding cytochemical technique using colloidal gold as marker. The four lectins labelled exclusively the Golgi apparatus of chondrocytes embedded in Lowicryl K4M resin by two different methods. WGA binding sites were localized in medial and trans cisternae as well as in immature secretory vesicles, whereas those for DBA were seen concentrated in cis and medial cisternae. Labelling with both RCA I and LFA lectins was distributed throughout all the cisternac of the Golgi stack, and the latter also in vesicles and tubules at the trans face. Neuraminidase pretreatment of the sections abolished LFA staining, decreased reaction with WGA and increased that with RCA I, while it did not affect DBA staining. After chondroitinase ABC treatment only the RCA I reaction was modified, revealing new binding sites in the trans Golgi face, secretory granules and extracellular matrix. These results indicate that the distribution of subcompartments in the Golgi apparatus of chondrocytes is different from that in cells secreting glycoproteins as major products.

Light microscopic characterization of glycoconjugates in secretory cells of the carp (Cyprinus carpio) gill epithelium

SummarySecretory products of granular and mucous cells in the gill epithelium of the carp, Cyprinus carpio, were distinguished by their cytochemical reactions with peroxidase-labelled lectins and with the galactose oxidase (GO)-Schiff reagents. Secretory products of granular cells reacted with lectins from Triticum vulgaris (WGA), Arachis hypogaea (PNA), Dolichos biflorus (DBA), Glycine max (SAB), and Lotus tetragonolobus (LTA). They also reacted with GO-Schiff reagents. After sialic acid cleavage with HCl, new binding sites for DBA and SBA appeared, suggesting the terminal sequence sialic acid-N-acetylgalactosamine (SA-GalNAc) for the secretion of this cell type. In mucous cells, binding sites for WGA, DBA, and SBA and, after acid hydrolysis, binding sites for PNA and a positive GO-Schiff reaction were detected. The terminal trisaccharide sialic acid-galactose (β1-3)-N-acetylgalactosamine (SA-Gal-GalNAc) is proposed for the secretion of mucuous cells. These cytochemical differences are discussed in light of the involvement of both cell types in fish mucus elaboration.

Light and electron microscopical localization of concanavalin A lectin binding sites in rat epiphyseal chondrocytes.

SummaryConcanavalin A lectin binding sites have been detected within the cytoplasm of epiphyseal chondrocytes. Correlative light and electron microscopic results were obtained, indicating the presence ofα-d-mannose and/or α-D-glucose residues detected by the lectin in the rough endoplasmic reticulum region. Quantitation of the electron microscopic cytochemical reaction also showed that the specific labelling was almost exclusively localized in the lumen of endoplasmic reticulum cisternae. No significant staining was found in other membrane compartments or extracellular matrix. This labelling pattern could be considered as the cytochemical evidence ofN-glycosylation processes occurring during the biosynthesis of cartilage extracellular matrix components by chondrocytes.

Carbohydrate cytochemistry on hypodermic lymphatic endothelium of the green lizard,Lacerta hispanica

SummaryA cytochemical study on the endothelium of the hypodermic lymphatic capillaries of the green lizard,Lacerta hispanica, has been carried out. The dialysed iron method produced a homogeneous precipitate on the surface of the endothelial cells and on the inside of the endocytic vesicles. The periodic acid-thiocarbohydrazide-silver proteinate, low pH phosphotungstic acid and high iron diamine techniques gave negative results. The carbohydrates in the capillaries thus seem to be glycosaminoglycans with carboxyl groups. The possible role of these glycosaminoglycans in the formation of the endocytic vesicles is discussed.