Differential subcompartmentation of terminal glycosylation in the Golgi apparatus of intestinal absorptive and goblet cells

Two terminal glycosyltransferases, a sialyltransferase and the blood group A alpha 1,3 N-acetylgalactosaminyltransferase, were found to exhibit differential subcompartmentation in the Golgi apparatus of intestinal goblet and absorptive cells. As expected from their role in terminal glycosylation, the two glycosyltransferases and their products, sialic acid residues and blood group A substance, were localized in the trans cisternae of the Golgi apparatus of goblet cells. In contrast, however, they were found throughout the Golgi apparatus stack of adjacent absorptive cells, with the exception of the fenestrated first cis cisterna. The results are in contrast to the general view that enzymes in the glycosylation pathway are arranged in a cis to trans gradient across the Golgi apparatus and that such polarized distributions may instead be cell type-specific.     

The trans-tubular network of the hepatocyte Golgi apparatus is part of the secretory pathway

We have recently demonstrated the presence of sialyltransferase and sialic acid in a trans-tubular network (TTN) continuous with trans Golgi apparatus cisternae of rat liver hepatocytes. Based on these findings, we concluded that this structure, which also exhibited thiamine pyrophosphatase and acid phosphatase activity, is an integral part of the Golgi apparatus and functions in sialylation. In the present study, by comparing the distribution of a major hepatocyte secretory product with that of sialyltransferase, we sought to determine whether the TTN is also part of the secretory pathway. Examination of adjacent serial thin sections labeled for albumin showed its presence throughout the TTN and simultaneously provided new details about the structural complexity of the TTN. Double-immunolabeling with protein A-gold allowed the direct demonstration of albumin throughout the sialyltransferase containing TTN. Additional double staining protocols (combination of preembedding enzyme cytochemistry with postembedding immunolabeling) revealed the presence of albumin in both the thiamine pyrophosphatase and acid phosphatase positive regions of the TTN. These data show that albumin, a nonglycosylated secretory protein, reaches the TTN where terminal glycosylation of glycoproteins occurs. Therefore, it appears that the TTN of rat hepatocytes which functions in terminal glycosylation is also part of the constitutive secretory pathway.     

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

Sialyltransferase (Galβ1,4GlcNAc α2,6 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.     

Lipoxygenase metabolites of arachidonic acid do not induce mucus secretion from rabbit intestinal goblet cells in vitro

Lipoxygenase metabolites of arachidonic acid are effective mucus secretagogues in the respiratory tract but their efficacy in the intestinal tract was unknown. Mucosal explants and sheets of epithelial cells isolated from rabbit small and large intestine were exposed to leukotrienes B4, C4, and D4 and monohydroxyeicosatetraenoic acids 5-HETE, 12-HETE, and 15-HETE. Light and electron microscopic inspection of goblet cells in treated tissues failed to detect evidence of recent compound exocytosis of mucin granules or other morphological evidence of secretory activity. These results indicate that lipoxygenase metabolites are not directly responsible for the increased mucus secretion observed in ulcerative colitis.     

The Golgi apparatus of goblet cells in the mouse descending colon: three-dimensional visualization using a confocal laser scanning microscope

The three-dimensional structure of the Golgi apparatus was studied in goblet cells in lectin-stained sections of the mouse descending colon by using a confocal laser scanning microscope. In the lower part of the crypt, the Golgi apparatus formed a dome- or globe-like structure in the supranuclear region. The wall of the dome had some holes, one of which usually faced toward the nucleus and others toward the apical cytoplasm. Mucous granules seemed to be initially released into the interior of the dome and transported toward the apical cytoplasm through the holes. In the upper part of the crypt, on the other hand, the Golgi apparatus formed a cup- or funnel-like structure with a larger opening toward the cell apex and a smaller opening toward the nucleus. A large mass of mucous granules occupied the inside of the cup to the apical cytoplasm. It is thought that the accumulation of mucous granules enlarges holes at the ceiling of the dome to form a large opening, which makes the configuration of the Golgi apparatus cup-shaped.     

Maturation of the Golgi apparatus in urothelial cells

The differentiation of urothelial cells is characterized by the synthesis of uroplakins and their assembly into the asymmetric unit membrane. The Golgi apparatus (GA) has been proposed to play a central role in asymmetric unit membrane formation. We have studied the distribution and organization of the GA in normal mouse urothelial cells and in the superficial urothelial cells that undergo differentiation following cyclophosphamide-induced regeneration, in correlation with urothelial cell differentiation. In normal urothelium, immature basal cells have a simple GA, which is small and distributed close to the nucleus. In intermediate cells, the GA starts to expand into the cytoplasm, whereas the GA of terminally differentiated umbrella cells is complex, being large and spread over the whole basal half of the cytoplasm. During early stages of regeneration after cyclophosphamide treatment, the GA of superficial cells is simple and no markers of urothelial differentiation (uroplakins or asymmetric unit membranes, discoidal or fusiform vesicles, apical surface covered with microvilli) are expressed. At a later stage, the GA expands and, in the final stage of regeneration, when cells express all markers of terminal urothelial differentiation, the GA become complex once again. Our results show that: (1) GA distribution and organization in urothelial cells is differentiation-dependent; (2) the GA matures from a simple form in partially differentiated cells to a complex form in terminally differentiated superficial cells; (3) major rearrangements of GA distribution and organization correlate with the beginning of asymmetric unit membrane production. Thus, GA maturation seems to be crucial for asymmetric unit membrane formation. The work was supported by the Ministry of Education and Sport, Government of Republic of Slovenia, Slovenia (grant no. 3311-04-831450).     

Buds of the Golgi apparatus in parotid acinar cells

The Golgi apparatus (GA) is a membranous organelle composed of stacked cisterns with associated vesicles. This study was undertaken to determine its origin in rat parotid acinar cells. The morphogenesis of the GA could be recognized in the developmental process as well as in mitotic division of cells. EM studies depicted an aggregation of small vesicles in the early stage of postnatal development or mitosis, that appeared to be the rudimental element of GA. Brefeldin A induced rapid degradation of the cisternal structure to vesicular aggregates. Reconstruction of the GA structure based on these remnant vesicles was observed upon removal of the drug. Similar membranous assembly could be observed after destruction of microtubules. These membranous aggregates presumably corresponded to 'buds of the GA' in parotid acinar cells. However, conventional cytochemical markers for GA were not detected on such immature form of GA. We found that the GA matrix protein GM130 and osmium reductivity (a classical marker for cis-Golgi elements) were consistently localized in the GA elements. Therefore, immunohistochemical distribution of GM130 and osmium impregnation of parotid acinar cells were studied under various dynamic conditions that produced structural modification of the GA.     

Novel localization of Rab3D in rat intestinal goblet cells and Brunner's gland acinar cells suggests a role in early Golgi trafficking

Rab3D is a small GTP-binding protein that associates with secretory granules of endocrine and exocrine cells. The physiological role of Rab3D remains unclear. While it has initially been implicated in the control of regulated exocytosis, recent deletion-mutation studies have suggested that Rab3D is involved in the biogenesis of secretory granules. Here, we report the unexpected finding that Rab3D also associates with early Golgi compartments in intestinal goblet cells and in Brunner's gland acinar cells. Expression of Rab3D in the intestine was demonstrated by SDS-PAGE and Western blot analysis of homogenates prepared from the rat duodenum and colon. Confocal laser scanning microscopy revealed Rab3D immunofluorescence in the Golgi area of goblet cells of the duodenum and colon and in Brunner's gland acinar cells. There was no colocalization between Rab3D and a trans-Golgi network marker, TGN-38. In contrast, Rab3D colocalized partially with a cis-Golgi marker, GM-130, and with a marker of cis-Golgi and coat protein complex I vesicles, beta-COP. Strong colocalization was observed between Rab3D and the lectins Griffonia simplicifolia agglutinin II and soybean agglutinin, which have been described as markers of the medial and cis-Golgi, respectively. Rabphilin, a putative effector of Rab3D, displayed an identical pattern of Golgi localization. Incubation of colon tissue with carbamylcholine or deoxycholate to stimulate exocytosis by goblet cells caused a partial redistribution of Rab3D to the cytoplasm and mucous granule field and a concomitant transformation of the Golgi architecture. Taken together, the present data suggest that Rab3D and rabphilin may regulate the secretory pathway at a much earlier stage than what has hitherto been assumed.     

Cloning of the gene gob-4, which is expressed in intestinal goblet cells in mice

We isolated the novel cDNA gob-4, which was shown to be expressed in intestinal goblet cells. The deduced amino acid sequence is similar to the gene coding for the Xenopus laevis cement gland-specific XAG-2. These sequence and expression data suggest this gene may be involved in the secretory function.     

Tridimensional structure of the Golgi apparatus in type A ganglion cells of the rat

The three-dimensional structure of the whole Golgi apparatus and of its components in type A ganglion cells was examined in thin and thick sections by low- and high-voltage electron microscopy. At low magnification, in 10-micron-thick sections of osmicated cells, the Golgi apparatus formed a broad, continuous perinuclear network. At higher magnification and in thinner sections of cells impregnated with uranyl acetate-lead-copper citrate or postfixed in K-ferrocyanide-reduced osmium, the Golgi apparatus appeared as a heterogeneous structure in which saccular regions characterized by stacks of saccules alternated with intersaccular regions made up of branching membranous tubules which bridged the saccules of adjacent stacks. The saccular regions consisted of the following superimposed elements: a cis-osmiophilic element made up of anastomosing tubules; two or three saccules negative for the phosphatases tested (i.e., nicotinamide adenine dinucleotide phosphatase = NADPase, thiamine pyrophosphatase = TPPase, and cytidine monophosphatase = CMPase); two saccules showing TPPase activity; and one to three trans-sacculotubular elements showing a "peeling-off" configuration, one of which showed CMPase activity. The saccules (phosphatase-negative) on the cis-side of the Golgi stacks showed, in addition to small circular pores, larger perforations in register. The cavities thus formed in the stacks of saccules, called "wells," always associated with small 80-nm vesicles, had a pan shape with the mouth directed toward the cis-face and the bottom closed by a TPPase-positive saccule. In face views of the saccules, the smallest of these perforations showed either a crescent shape, due to the presence of a bud on one side of the perforation, or a circular shape with a single small 80-nm vesicle in the center which was occasionally attached to the saccule by a filiform stalk. Such smaller cavities were considered as the precursors of the larger perforations and eventually of the wells. The small 80-nm vesicles seen in the small cavities or in the wells appeared to form in situ and possibly migrate toward the cisternae of endoplasmic reticulum seen proximal to the cis-face of the stack of saccules. Small 80-nm vesicles were also numerous in the intersaccular regions, along the lateral- and trans-aspects of the Golgi stacks, while larger, 150-to 300-nm vesicles, coated and uncoated, were seen only on the trans-face of the Golgi stacks in proximity to the trans-sacculotubular elements which appear to "peel off" from the Golgi stacks.     

4Pi-microscopy of the Golgi apparatus in live mammalian cells

We report the applicability of 4Pi-microscopy to live mammalian cells. Controlled interference of the counterpropagating wavefronts is possible despite the slight variations in cellular refractive index. Superresolved 3D-fluorescence imaging is exemplified with the first representation of the Golgi apparatus in a live cell at approximately 100 nm resolution.