Overlapping distribution of two glycosyltransferases in the Golgi apparatus of HeLa cells

Thin, frozen sections of a HeLa cell line were double labeled with specific antibodies to localize the trans-Golgi enzyme, beta 1,4 galactosyltransferase (GalT) and the medial enzyme, N- acetylglucosaminyltransferase I (NAGT I). The latter was detected by generating a HeLa cell line stably expressing a myc-tagged version of the endogenous protein. GalT was found in the trans-cisterna and trans- Golgi network but, contrary to expectation, NAGT I was found both in the medial- and trans-cisternae, overlapping the distribution of GalT. About one third of the NAGT I and half of the GalT were found in the shared, trans-cisterna. These data show that the differences between cisternae are determined not by different sets of enzymes but by different mixtures.     

Fragmentation and partitioning of the Golgi apparatus during mitosis in HeLa cells.

Osmium impregnation was used to determine the number of Golgi apparatus in both interphase and mitotic HeLa cells. The number was found to increase substantially during mitosis to the point where random partitioning alone would explain the nearly equal numbers found in each daughter cell.     

Evidence that globular Golgi clusters in mitotic HeLa cells are clustered tubular endosomes.

By conventional electron microscopy we observed in mitotic HeLa cells the structures termed Golgi clusters by Lucocq et al. (J. Cell Biol. 104, 865-874 (1987)) and interpreted by them as clusters of vesicular remnants of the Golgi apparatus. Golgi clusters consist of tubular and vesicular profiles about 50 nm in diameter, sometimes associated with larger 250 nm vesicles. When cultures of HeLa cells were incubated for 60 min or 120 min with medium containing high specific activity horseradish peroxidase (HRP) at 10 mg/ml we found that the membrane-bound compartments in the Golgi clusters in mitotic cells contained heavy deposits of HRP reaction product. Neither interphase nor mitotic HeLa cells contain an endogenous peroxidase activity. We concluded that Golgi clusters are an endocytic compartment and confirmed this by showing that Golgi clusters could be labeled with two other endocytic tracers--bovine serum albumin conjugated to colloidal gold and transferrin conjugated to HRP. When cultures were incubated with HRP for only 15 min most of the Golgi clusters in the mitotic cells were either unlabeled or consisted of a mixture of HRP-labeled and unlabeled profiles. Since during mitosis endocytosis is inhibited this was the expected result. When interphase HeLa cells were incubated with Brefeldin A (BFA), the Golgi apparatus disassembled and immunofluorescence microscopy showed that 1,4 beta galactosyltransferase had relocated to the endoplasmic reticulum. When cells in the presence of BFA and lacking the Golgi apparatus were allowed to endocytose HRP and then entered mitosis, typical HRP-labeled Golgi clusters were seen in the mitotic cells. It is therefore highly unlikely that these structures contain membrane derived from the Golgi cisternae that are sensitive to BFA, including in HeLa cells those containing galactosyltransferase. Finally, we found that interphase HeLa cells incubated with okadaic acid contain structures that are morphologically indistinguishable from Golgi clusters but can be labeled by endocytic tracer. Taken together, this evidence indicates that most, if not all, of the membrane-bound compartments in Golgi clusters are tubular early endosomes.     

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.     

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).     

Sphingolipid transport in mitotic HeLa cells.

Mitotic and interphase HeLa cells were labeled with [3H]serine. Ceramide and its derivatives, lactosylceramide and sphingomyelin, were biosynthetically labeled under both conditions. Only in the absence of nocodazole, as the cells entered telophase, was an additional glycosphingolipid synthesized which was identified as GA2 (GalNAc(beta 1,4)Gal(beta 1,4)Glc(beta 1,1)Cer). Ceramide, the basic sphingolipid precursor, is synthesized in the endoplasmic reticulum, whereas its immediate derivatives are synthesized in early Golgi compartments. Transport of newly synthesized proteins from the endoplasmic reticulum to the Golgi is inhibited in mitotic cells while ceramide acquires early Golgi modifications under the same conditions, suggesting that ceramide can be delivered to the Golgi by a different route. Since GA2 is synthesized in late Golgi, its absence in mitotic cells strongly argues for an in vivo inhibition of intra-Golgi transport, an observation with important implications for the mechanism of Golgi division.     

A transmembrane form of the prion protein is localized in the Golgi apparatus of neurons

(Ctm)PrP is a transmembrane version of the prion protein that has been proposed to be a neurotoxic intermediate underlying prion-induced pathogenesis. In previous studies, we found that PrP molecules carrying mutations in the N-terminal signal peptide (L9R) and the transmembrane domain (3AV) were synthesized exclusively in the (Ctm)PrP form in transfected cell lines. To characterize the properties of (Ctm)PrP in a neuronal setting, we have utilized cerebellar granule neurons cultured from Tg(L9R-3AV) mice that developed a fatal neurodegenerative illness. We found that about half of the L9R-3AV PrP synthesized in these neurons represents (Ctm)PrP, with the rest being (Sec)PrP, the glycolipid anchored form that does not span the membrane. Both forms contained an uncleaved signal peptide, and they are differentially glycosylated. (Sec)PrP was localized on the surface of neuronal processes. Most surprisingly, (Ctm)PrP was concentrated in the Golgi apparatus, rather in the endoplasmic reticulum as it is in transfected cell lines. Our study is the first to analyze the properties of (Ctm)PrP in a neuronal context, and our results suggest new hypotheses about how this form may exert its neurotoxic effects.     

Evidence that the entire Golgi apparatus cycles in interphase HeLa cells: sensitivity of Golgi matrix proteins to an ER exit block.

We tested whether the entire Golgi apparatus is a dynamic structure in interphase mammalian cells by assessing the response of 12 different Golgi region proteins to an endoplasmic reticulum (ER) exit block. The proteins chosen spanned the Golgi apparatus and included both Golgi glycosyltransferases and putative matrix proteins. Protein exit from ER was blocked either by microinjection of a GTP-restricted Sar1p mutant protein in the presence of a protein synthesis inhibitor, or by plasmid-encoded expression of the same dominant negative Sar1p. All Golgi region proteins examined lost juxtanuclear Golgi apparatus-like distribution as scored by conventional and confocal fluorescence microscopy in response to an ER exit block, albeit with a differential dependence on Sar1p concentration. Redistribution of GalNAcT2 was more sensitive to low Sar1p(dn) concentrations than giantin or GM130. Redistribution was most rapid for p27, COPI, and p115. Giantin, GM130, and GalNAcT2 relocated with approximately equal kinetics. Distinct ER accumulation could be demonstrated for all integral membrane proteins. ER-accumulated Golgi region proteins were functional. Photobleaching experiments indicated that Golgi-to-ER protein cycling occurred in the absence of any ER exit block. We conclude that the entire Golgi apparatus is a dynamic structure and suggest that most, if not all, Golgi region-integral membrane proteins cycle through ER in interphase cells.     

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.     

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.     

Specific organization of Golgi apparatus in plant cells

Microtubules, actin filaments, and Golgi apparatus are connected both directly and indirectly, but it is manifested differently depending on the cell organization and specialization, and these connections are considered in many original studies and reviews. In this review we would like to discuss what underlies differences in the structural organization of the Golgi apparatus in animal and plant cells: specific features of the microtubule cytoskeleton organization, the use of different cytoskeleton components for Golgi apparatus movement and maintenance of its integrity, or specific features of synthetic and secretory processes. We suppose that a dispersed state of the Golgi apparatus in higher plant cells cannot be explained only by specific features of the microtubule system organization and by the absence of centrosome as an active center of their organization because the Golgi apparatus is organized similarly in the cells of other organisms that possess the centrosome and centrosomal microtubules. One of the key factors determining the Golgi apparatus state in plant cells is the functional uniformity or functional specialization of stacks. The functional specialization does not suggest the joining of the stacks to form a ribbon; therefore, the disperse state of the Golgi apparatus needs to be supported, but it also can exist “by default”. We believe that the dispersed state of the Golgi apparatus in plants is supported, on one hand, by dynamic connections of the Golgi apparatus stacks with the actin filament system and, on the other hand, with the endoplasmic reticulum exit sites distributed throughout the endoplasmic reticulum.     

Calcium release from the Golgi apparatus and the endoplasmic reticulum in HeLa cells stably expressing targeted aequorin to these compartments

Extracellular agonists mobilize Ca2+ from SERCA-comprising intracellular Ca2+ stores located in both the Golgi apparatus and the endoplasmic reticulum. Ca2+ release from both these compartments was studied in HeLa cells stably expressing the luminescent Ca2+ indicator aequorin specifically targeted to these compartments. Changes in lumenal [Ca2+] as detected by the aequorin measurements were correlated with parallel changes in total Ca2+ content of the stores. The latencies and initial rates of Ca2+ release from the Golgi apparatus and the endoplasmic reticulum were quite similar. However, maximal Ca2+ release measured with Golgi-targeted aequorin terminated faster than that from the endoplasmic reticulum. The rate and extent of Ca2+ depletion from both compartments correlated well with the peak amplitude of the cytosolic [Ca2+] rise. Time-course experiments further revealed that the peak of the cytosolic Ca2+ response occurred before the lumenal [Ca2+] reached its lowest level. We conclude that both the Golgi apparatus and the endoplasmic reticulum contribute to the rise in cytosolic [Ca2+] upon agonist stimulation, but the kinetics of the Ca2+ release are different.     

Regulation of protein synthesis in mitotic HeLa cells.

Mitotic HeLa cells (M cells) synthesize protein at about 25% of the rate of S phase cells. This decrease in protein synthesis is due to a reduction in the rate of initiation. However, extracts prepared from M cells are almost as active in protein synthesis as S cell extracts. Both cell extracts are quite active in in vitro initiation of protein synthesis. Moreover, two steps in initiation, binding of Met-tRNAf to 40S ribosomal subunits and binding of mRNA to ribosomes, show similar activity in both extracts. The difference in protein synthesizing activity observed in vivo is largely eliminated in the preparation of cell-free systems. The ribosomes of M cells contain small mol wt RNA, which inhibits protein synthesis in vitro. This RNA, which has possibly a nuclear origin, may be a cause of the reduction in the rate of protein synthesis in M cells.     

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.     

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.     

The vesicular compartment of the mitotic apparatus in mammalian cells

Intracellular membranes might play an eminent role in regulating several events during mitosis: In this paper the appearance and changing configurations of the vesicular compartment of the mitotic apparatus of HeLa cells was studied from anaphase to telophase. In early prophase electron opaque and transparent membranous vesicles are found in the pericentriolar region outside the nucleus. During prometaphase when the nuclear envelope opens and starts to disappear, an increasing number of these vesicles appears in the mitotic apparatus near the chromosomes. During metaphase vesicles are spread all over the mitotic apparatus, the number of electron opaque vesicles decreases while the total amount of vesicles does not change significantly. Anaphase shows the same pattern of distribution in the half-spindle and in the midbody. In telophase the amount of electron opaque vesicles increases again. They are now found around vacuoles and near the newly appearing Golgi-cisternae. We assume that the electron opaque vesicles are derived from the Golgi- apparatus which disintegrates during prophase and reappears in late telophase. The change in the appearance of the different types of vesicles during metaphase coincides with drastic changes in the ionic milieu in the mitotic apparatus (Wolniak et al., 1983).