Internalized ricin and the plasma membrane glycoprotein MAM-6 colocalize in the trans-Golgi network of T47D human breast carcinoma cells

We have analyzed the intracellular transport of endocytosed ricin in the human breast carcinoma cell line T47D. Cells were incubated with ricin (10 μg/ml) for 1 h at 37 °C. Marked reduction in the protein synthesis did not take place until the end of this period. To detect ricin immunocytochemically, a rabbit anti-ricin serum was used. Gel electrophoresis followed by immunoblotting revealed that the antiserum reacted specifically with ricin and detected both the ricin A-chain and the ricin B-chain. Immunofluorescence experiments showed endocytosed ricin in endosomal and lysosomal vacuoles throughout the cytoplasm, as well as in a typical perinuclear position corresponding to the Golgi region. Using the monoclonal mouse antibody 115D8 directed toward the high-molecular-weight membrane glycoprotein MAM-6 of human breast epithelial cells, we similarly obtained a marked perinuclear fluorescence. In addition, MAM-6 fluorescence was observed in swarms of small vesicles throughout the cytoplasm. To further analyze the apparent colocalization of ricin and MAM-6 in the perinuclear Golgi region, immunogold cytochemistry on ultracryosections was performed. MAM-6 was detected mainly in Golgi stacks and associated trans-Golgi network (TGN) profiles, in 0.1 to 0.2-μm secretory vesicles, and on the cell surface. Ricin was detected on the cell surface, in endosomes and lysosomes, and also in the TGN. Furthermore, by using immunogold double labeling, internalized ricin was found to colocalize with MAM-6 in the TGN.     

Estimation of the amount of internalized ricin that reaches the trans- Golgi network

We have used a protocol for internalization of ricin, a ligand binding to plasma membrane glycoproteins and glycolipids with terminal galactosyl residues, and infection with the vesicular stomatitis virus ts 045 mutant in BHK-21 cells to determine whether internalized plasma membrane molecules tagged by ricin reach distinct compartments of the biosynthetic-exocytic pathway. At 39.5 degrees C newly synthesized G protein of ts 045 was largely prevented from leaving the endoplasmic reticulum. At the same temperature ricin was endocytosed and reached, in addition to endosomes and lysosomes, elements of the Golgi complex. When the temperature was lowered to 19.5 degrees C, no more ricin was delivered to the Golgi complex, but now G protein accumulated in the Golgi stacks and the trans-Golgi network (TGN). Double-labeling immunogold cytochemistry on ultracryosections was used to detect G protein and ricin simultaneously. These data, combined with stereological and biochemical methods, showed that approximately 5% of the total amount of ricin within the cells, corresponding to 6-8 X 10(4) molecules per cell, colocalized with G protein in the Golgi complex after 60 min at 39.5 degrees C. Of this amount approximately 70-80% was present in the TGN. Since most of the ricin molecules remain bound to their binding sites at the low pH prevailing in compartments of the endocytic pathway, the results indicate that a fraction of the internalized plasma membrane molecules with terminal galactose are not recycled directly from endosomes or delivered to lysosomes, but are routed to the Golgi complex. Also, the results presented here, in combination with other recent studies on ricin internalization, suggest that translocation of the toxic ricin A-chain to the cytosol occurs in the TGN.     

Endosome to Golgi Transport of Ricin Is Independent of Clathrin and of the Rab9- and Rab11-GTPases

The plant toxin ricin is transported to the Golgi and the endoplasmic reticulum before translocation to the cytosol where it inhibits protein synthesis. The toxin can therefore be used to investigate pathways leading to the Golgi apparatus. Except for the Rab9-mediated transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network (TGN), transport routes between endosomes and the Golgi apparatus are still poorly characterized. To investigate endosome to Golgi transport, we have used here a modified ricin molecule containing a tyrosine sulfation site and quantified incorporation of radioactive sulfate, a TGN modification. A tetracycline-inducible mutant Rab9S21N HeLa cell line was constructed and characterized to study whether Rab9 was involved in transport of ricin to the TGN and, if not, to further investigate the route used by ricin. Induced expression of Rab9S21N inhibited Golgi transport of mannose 6-phosphate receptors but did not affect the sulfation of ricin, suggesting that ricin is transported to the TGN via a Rab9-independent pathway. Moreover, because Rab11 is present in the endosomal recycling compartment and the TGN, studies of transient transfections with mutant Rab11 were performed. The results indicated that routing of ricin from endosomes to the TGN occurs by a Rab11-independent pathway. Finally, because clathrin has been implicated in early endosome to TGN transport, ricin transport was investigated in cells with inducible expression of antisense to clathrin heavy chain. Importantly, endosome to TGN transport (sulfation of endocytosed ricin) was unchanged when clathrin function was abolished. In conclusion, ricin is transported from endosomes to the Golgi apparatus by a Rab9-, Rab11-, and clathrin-independent pathway.     

Intracellular features of type II procollagen and chondroitin sulfate proteoglycan synthesis in chondrocytes

The intracellular compartments of chondrocytes involved in the synthesis and processing of type II procollagen and chondroitin sulfate proteoglycan (CSPG) monomer were investigated using simultaneous double immunofluorescence and lectin localization reactions. Type II procollagen was distributed in vesicles throughout the cytoplasm, whereas intracellular precursors of CSPG monomer were accumulated in the perinuclear cytoplasm. In this study, cytoplasmic vesicles that stained intensely with antibodies directed against CSPG monomer but did not react with type II collagen antibodies, also were observed. A monoclonal antibody, 5-D-4, that recognizes keratan sulfate determinants was used to identify the Golgi complex (the site of keratan sulfate chain elongation). Staining with 5-D-4 was restricted to the perinuclear cytoplasm. The vesicles outside the perinuclear cytoplasm that stained intensely with antibodies to CSPG monomer did not react with 5-D-4. Fluorescent lectins were used to characterize further subcellular compartments. Concanavalin A, which reacts with mannose-rich oligosaccharides, did not stain the perinuclear region, but it did stain vesicles throughout the rest of the cytoplasm. Because mannose oligosaccharides are added cotranslationally, the stained vesicles throughout the cytoplasm presumably correspond to the rough endoplasmic reticulum. Wheat germ agglutinin, which recognizes N-acetyl-D-glucosamine and sialic acid (carbohydrates added in the Golgi), stained exclusively the perinuclear cytoplasm. By several criteria (staining with the monoclonal antibody 5-D-4 and with wheat germ agglutinin), the perinuclear cytoplasm seems to correspond to the Golgi complex. The cytoplasmic vesicles that react with anti-CSPG monomer and not with anti-type II collagen contain precursors of CSPG monomer not yet modified by Golgi-mediated oligosaccharide additions (because they are not stained with wheat germ agglutinin or with the anti-keratan sulfate antibody); these vesicles may have a unique function in the processing of CSPG.     

Ricin transport in brefeldin A-treated cells: correlation between Golgi structure and toxic effect

Whereas brefeldin A (BFA) protected a number of cell lines against the protein toxin ricin, two of the cell lines tested were not protected but rather sensitized to ricin by BFA. EM studies revealed that upon addition of BFA the Golgi stacks in cells which were protected against the toxin rapidly transformed into a characteristic tubulo-vesicular reticulum connected to the endoplasmic reticulum, and subcellular fractionation experiments showed that galactosyl transferase disappeared from the Golgi fractions where it was normally located. EM and subcellular fractionation also indicated that in contrast to the Golgi stacks, the trans-Golgi network (TGN) remained intact and that internalized ricin was still localized in the TGN both when BFA was added before and after the toxin. Thus, BFA does not prevent fusion of ricin-containing vesicles with the TGN, and unlike resident proteins in Golgi stacks, ricin is not transported back to ER upon treatment of cells with BFA. Two kidney epithelial cell lines, MDCK and PtK2, were not protected against ricin by BFA, and EM studies of MDCK cells revealed that BFA did not alter the morphology of the Golgi complex in these cells. Also, subcellular fractionation revealed that, in contrast to the other cell types tested, the localization of galactosyl transferase in the gradients was not affected by BFA treatment. The data show that there is a correlation between BFA-induced disassembly of the Golgi stacks and protection against ricin, and they demonstrate that the structural organization of the Golgi apparatus is affected by BFA to different extents in various cell lines.     

100-kD proteins of Golgi- and trans-Golgi network-associated coated vesicles have related but distinct membrane binding properties

The 100-110-kD proteins (alpha-, beta-, beta'-, and gamma-adaptins) of clathrin-coated vesicles and the 110-kD protein (beta-COP) of the nonclathrin-coated vesicles that mediate constitutive transport through the Golgi have homologous protein sequences. To determine whether homologous processes are involved in assembly of the two types of coated vesicles, the membrane binding properties of their coat proteins were compared. After treatment of MDBK cells with the fungal metabolite Brefeldin A (BFA), beta-COP was redistributed to the cytoplasm within 15 s, gamma-adaptin and clathrin in the trans-Golgi network (TGN) dispersed within 30 s, but the alpha-adaptin and clathrin present on coated pits and vesicles derived from the plasma membrane remained membrane associated even after a 15-min exposure to BFA. In PtK1 cells and MDCK cells, BFA did not affect beta-COP binding or Golgi morphology but still induced redistribution of gamma-adaptin and clathrin from TGN membranes to the cytoplasm. Thus BFA affects the binding of coat proteins to membranes in the Golgi region (Golgi apparatus and TGN) but not plasma membranes. However, the Golgi binding interactions of beta-COP and gamma-adaptin are distinct and differentially sensitive to BFA. BFA treatment did not release gamma-adaptin or clathrin from purified clathrin-coated vesicles, suggesting that their distribution to the cytoplasm after BFA treatment of cells was due to interference with their rebinding to TGN membranes after a normal cycle of disassembly. This was confirmed using an in vitro assay in which gamma-adaptin binding to TGN membranes was blocked by BFA and enhanced by GTP gamma S, similar to the binding of beta-COP to Golgi membranes. These results suggest the involvement of GTP-dependent proteins in the association of the 100-kD coat proteins with membranes in the Golgi region of the cell.     

Retinoic acid disrupts the Golgi apparatus and increases the cytosolic routing of specific protein toxins

All-trans retinoic acid can specifically increase receptor mediated intoxication of ricin A chain immunotoxins more than 10,000 times, whereas fluid phase endocytosis of ricin A chain alone or ricin A chain immunotoxins was not influenced by retinoic acid. The immunotoxin activation by retinoic acid does not require RNA or protein synthesis and is not a consequence of increased receptor binding of the immunotoxin. Vitamin D3 and thyroid hormone T3, that activate retinoic acid receptor (RAR) cognates, forming heterodimers with retinoid X receptor (RXR), do not affect the potency of immunotoxins. Among other retinoids tested, 13-cis retinoic acid, which binds neither RAR nor RXR, also increases the potency of the ricin A chain immunotoxin. Therefore, retinoic acid receptor activation does not appear to be necessary for immunotoxin activity. Retinoic acid potentiation of immunotoxins is prevented by brefeldin A (BFA) indicating that in the presence of retinoic acid, the immunotoxin is efficiently routed through the Golgi apparatus en route to the cytoplasm. Directly examining cells with a monoclonal antibody (Mab) against mannosidase II, a Golgi apparatus marker enzyme, demonstrates that the Golgi apparatus changes upon treatment with retinoic acid from a perinuclear network to a diffuse aggregate. Within 60 min after removal of retinoic acid the cell reassembles the perinuclear Golgi network indistinguishable with that of normal control cells. C6-NBD-ceramide, a vital stain for the Golgi apparatus, shows that retinoic acid prevents the fluorescent staining of the Golgi apparatus and eliminates fluorescence of C6-NBD-ceramide prestained Golgi apparatus. Electron microscopy of retinoic acid-treated cells demonstrates the specific absence of any normal looking Golgi apparatus and a perinuclear vacuolar structure very similar to that seen in monensin-treated cells. This vacuolization disappears after removal of the retinoic acid and a perinuclear Golgi stacking reappears. These results indicate that retinoic acid alters intracellular routing, probably through the Golgi apparatus, potentiating immunotoxin activity indepedently of new gene expression. Retinoic acid appears to be a new reagent to manipulate the Golgi apparatus and intracellular traffic. As retinoic acid and immunotoxins are both in clinical trials for cancer therapy, their combined activity in vivo would be interesting to examine.     

Polarized expression of an apical membrane glycoprotein is established before functional tight junctions have developed in MCF-7 cells

A milk-fat globule membrane antigen, designated MAM-6 and detected immunocytochemically by the monoclonal antibody 115D8, is expressed apically in confluent MCF-7 monolayer cultures. Immediately after preparation of a single-cell suspension, MAM-6 appears on the entire cell surface. However, polarized apical expression of MAM-6 is restored as early as 2-6 hr after plating of unpolarized cells, before functional tight junctions are established, as judged by freeze-fracture and ruthenium red permeability. Quantitative immunogold cytochemistry reveals that the apical:basal ratio of MAM-6 expression was about 17:1 after 6 hr. Tight junctions developed as late as 12-24 hr after plating. At this time the apical:basal MAM-6 ratio was about 30:1 (as compared to about 50:1 in control monolayers).     

Endocytic routes to the Golgi apparatus

 The endocytic routes of labelled lectins as well as cationic ferritin were studied in cells with a regulated secretion, i.e. pancreatic beta cells, and in constitutively secreting cells, i.e. fibroblasts and HepG2 hepatoma cells, paying particular attention to routes into the Golgi apparatus. Considerable amounts of internalised molecules were taken up into the trans Golgi network (TGN) and into Golgi subcompartments in all three cell types as well as in secretory granules of the pancreatic beta cells. The internalised materials did not pass rapidly the TGN and Golgi stacks, but were still present hours after internalisation, being then particularly concentrated in TGN-elements and in the transmost Golgi cisterna. Endocytosed materials reached forming secretory granules present in the TGN. Further, direct fusion between endocytotic vesicles and mature secretory granules was observed. Golgi subcompartments as well as endocytic TGN containing endocytosed materials were in close apposition to specialised regions of the endoplasmic reticulum. The Golgi apparatus including its parts containing endocytosed materials were transformed into a tubular reticulum upon treatment with the fungal metabolite Brefeldin A. Rarely, internalised material was observed in the lumen of the endoplasmic reticulum, thus providing evidence for an endocytic plasma membrane to endoplasmic reticulum route. Accepted: 9 March 1998     

Involvement of cytoplasmic membranes in the non-lytic infection of K-562 cells by Semliki Forest virus

An analysis of the human leukemia cell line, K-562, infected with Semliki Forest virus, has been made with transmission electron microscopy. In contrast to the usual surface budding of the enveloped virus on the plasma membrane of vertebrate cells leading to cytolysis within 20 h, K-562 cells do not show surface budding, and the cells remain intact for periods of several months. Several unusual features of the infection include: 1) the rough endoplasmic reticulum arranges early into continuous perinuclear chains; 2) during the time of virus replication and release, the nucleocapsids aggregate on the cytoplasmic side of internal vesicles in the region of the cell where the Golgi complex is normally located; and 3) during this same time period, the vesicles are seen to contain enveloped virions and rod-like formations, a result suggesting that budding has occurred into these vesicles. Viruses are presumably released from the cell as these vesicles fuse with the plasma membrane. By 12 days post-infection and thereafter, the intact cells show electron-dense aggregates of chromatin, large vacuoles and lipid inclusions throughout the cytoplasm, and only a few virion-containing vesicles.     

The actin-binding protein comitin (p24) is a component of the Golgi apparatus

Comitin (p24) was first identified in Dictyostelium discoideum as a membrane-associated protein which binds in gel overlay assays to G and F actin. To analyze its actin-binding properties we used purified, bacterially expressed comitin and found that it binds to F actin in spin down experiments and increases the viscosity of F actin solutions even under high-salt conditions. Immunofluorescence studies, cell fractionation experiments and EM studies of vesicles precipitated with comitin-specific monoclonal antibodies showed that comitin was present in D. discoideum on: (a) a perinuclear structure with tubular or fibrillary extensions; and (b) on vesicles distributed throughout the cell. In immunofluorescence experiments using comitin antibodies NIH 3T3 fibroblasts showed a similar staining pattern as D. discoideum cells. Using bona fide Golgi markers the perinuclear structure was identified as the Golgi apparatus. The results were supported by an electron microscopic study using cryosections. Based on these data we propose that also in Dictyostelium the stained perinuclear structure is the Golgi apparatus. In vivo the perinuclear structure was found to be attached to the actin and the microtubule network. Alteration of the actin network or depolymerization of the microtubules led to its dispersal into vesicles distributed throughout the cell. These results suggest that the Golgi apparatus in D. discoideum is connected to the actin network by comitin. This protein seems also to be present in mammalian cells.     

The trans-Golgi network can be dissected structurally and functionally from the cisternae of the Golgi complex by brefeldin A.

TGN38, a transmembrane glycoprotein predominantly localized to the trans-Golgi network, is utilized to study both the structure and function of the trans-Golgi network (TGN). The effects of brefeldin A (BFA) on the TGN were studied in comparison to its documented effects on the Golgi cisternae. During the first 30 min of BFA treatment, the TGN loses its cisternal structure and extends as tubules throughout the cytoplasm. By 60 min, it condenses into a stable structure surrounding the microtubule-organizing center. By electron microscopy, this structure appears as a population of large vesicles, and by immunolabeling, most of these vesicles contain TGN38. TGN38 cycles to the plasma membrane and back, which is shown by addition of TGN38 luminal domain antibodies directly to cell culture media. This results in rapid uptake of antibodies which label the TGN within 30 min, both in its native and BFA-induced conformation. A number of transmembrane proteins have been shown to take this cycling pathway, but TGN38 is unique in that it is the only one predominantly localized to the TGN. To investigate the cycling of TGN38, the endocytic pathway was labeled by internalization of Lucifer Yellow, and in the presence of BFA there was partial colocalization with TGN38. Further studies were carried out in which microtubules were depolymerized, resulting in dispersal of Golgi elements and inhibition of transport from endosomes to lysosomes. TGN38 cycling continues in the absence of microtubules. Taken together, these studies indicate that TGN38 returns from the plasma membrane via the endocytic pathway. We conclude that the TGN is structurally and functionally distinct from the Golgi cisternae, indicating that different molecules control membrane traffic from the Golgi cisternae and from the TGN.     

Brefeldin A and filipin distinguish two globotriaosyl ceramide/verotoxin-1 intracellular trafficking pathways involved in Vero cell cytotoxicity

In verotoxin 1 (VT1)-sensitive cells, globotriaosyl ceramide (Gb3) bound VT1 is endocytosed and transported retrogradely to the Golgi/endoplasmic reticulum (ER). The importance of the Golgi-dependent retrograde transport of VT1 is now shown to vary as a function of both VT1 exposure time and concentration. Following 3 h exposure to < 50 ng/ml VT1, Vero cell cytotoxicity and protein synthesis inhibition is absolutely dependent on intact Golgi structure. However, after 24 h incubation with concentrations of VT1 above 50 ng/ml, a filipin-sensitive (caveolae-dependent) route for cytotoxicity becomes significant. Brefeldin A (BFA), which prevents Golgi-dependent retrograde traffic, protects cells from low VT1 concentrations but not following prolonged toxin exposure at higher VT1 concentrations. Under these conditions, only a combination of BFA and filipin is sufficient to fully protect cells. Intracellular VT1 trafficking monitored using the nontoxic B subunit showed accumulation within BFA-collapsed TGN/endosomes. Considerable VT1 B was retained at the surface of filipin-treated cells, but Golgi targeting was still apparent. Filipin-sensitive VT1 cytotoxicity does not require Golgi access and may involve direct transmembrane signaling. Although cell surface VT1 does not colocalize with caveolin 1, a small fraction of endocytosed VT1 is found within caveolin 1-containing vesicles. These studies indicate both a caveolae-dependent and independent pathway for VT1 access to the TGN/Golgi from the cell surface and two noninterconverting pools of membrane Gb3.     

Subcellular localization analysis of the closely related Fps/Fes and Fer protein-tyrosine kinases suggests a distinct role for Fps/Fes in vesicular trafficking

The subcellular localizations of the Fps/Fes and closely related Fer cytoplasmic tyrosine kinases were studied using green fluorescent protein (GFP) fusions and confocal fluorescence microscopy. In contrast to previous reports, neither kinase localized to the nucleus. Fer was diffusely cytoplasmic throughout the cell cycle. Fps/Fes also displayed a diffuse cytoplasmic localization, but in addition it showed distinct accumulations in cytoplasmic vesicles as well as in a perinuclear region consistent with the Golgi. This localization was very similar to that of TGN38, a known marker of the trans Golgi. The localization of Fps/Fes and TGN38 were both perturbed by brefeldin A, a fungal metabolite that disrupts the Golgi apparatus. Fps/Fes was also found to colocalize to various extents with several Rab proteins, which are members of the monomeric G-protein superfamily involved in vesicular transport between specific subcellular compartments. Using Rabs that are involved in endocytosis (Rab5B and Rab7) or exocytosis (Rab1A and Rab3A), we showed that Fps/Fes is localized in both pathways. These results suggest that Fps/Fes may play a general role in the regulation of vesicular trafficking.     

Redistribution of cellular and herpes simplex virus proteins from the trans-golgi network to cell junctions without enveloped capsids

Herpes simplex virus (HSV) and other alphaherpesviruses assemble enveloped virions in the trans-Golgi network (TGN) or endosomes. Enveloped particles are formed when capsids bud into TGN/endosomes and virus particles are subsequently ferried to the plasma membrane in TGN-derived vesicles. Little is known about the last stages of virus egress from the TGN/endosomes to cell surfaces except that the HSV directs transport of nascent virions to specific cell surface domains, i.e., epithelial cell junctions. Previously, we showed that HSV glycoprotein gE/gI accumulates extensively in the TGN at early times after infection and also when expressed without other viral proteins. At late times of infection, gE/gI and a cellular membrane protein, TGN46, were redistributed from the TGN to epithelial cell junctions. We show here that gE/gI and a second glycoprotein, gB, TGN46, and another cellular protein, carboxypeptidase D, all moved to cell junctions after infection with an HSV mutant unable to produce cytoplasmic capsids. This redistribution did not involve L particles. In contrast to TGN membrane proteins, several cellular proteins that normally adhere to the cytoplasmic face of TGN, Golgi, and endosomal membranes remained primarily dispersed throughout the cytoplasm. Therefore, cellular and viral membrane TGN proteins move to cell junctions at late times of HSV infection when the production of enveloped particles is blocked. This is consistent with the hypothesis that there are late HSV proteins that reorganize or redistribute TGN/endosomal compartments to promote virus egress and cell-to-cell spread.     

A role for small GTPase RhoA in regulating intracellular membrane traffic of lysosomes in invasive rat hepatoma cells

Small GTPase RhoA regulates signal transduction from receptors in the membrane to a variety of cellular events related to cell morphology, motility, cytoskeletal dynamics, cytokinesis, and tumour progression, but it is unclear how RhoA regulates intracellular membrane dynamics of lysosomes. We showed previously by confocal immunofluorescence microscopy that the transfection of dominant active RhoA in MM1 cells causes the dispersal translocation of lysosomes stained for cathepsin D throughout the cytoplasm. Y-27632, a selective inhibitor of p160ROCK, impeded the cellular redistribution of lysosomes and promoted reclustering of lysosomes toward the perinuclear region. Here we have further investigated whether the acidic lysosomal vesicles dispersed throughout the cytoplasm are applied to the early endosomes in the endocytic pathway, and we demonstrate that the dispersed lysosomes were accessible to endocytosed molecule such as dextran, and their acidity was not changed, as determined by increased accumulation of the acidotropic probe LysoTracker Red. Brefeldin A did not induce the tabulation of these dispersed lysosomes, but it caused early endosomes to form an extensive tubular network. The dispersed lysosomes associated with cathepsin D and LIMPII were not colocalized with early endosomes, and these vesicles were not inaccessible to the endocytosed anti-transferrin receptor antibody. Moreover, wortmannin, an inhibitor of phosphatidylinositol 3-kinase, induced a dramatic change in LIMPII-containing structures in which LIMPII-positive swollen large vacuoles were increased and small punctate structures disappeared in the cytoplasm. These swollen vacuoles were not doubly positive for LIMPII and transferrin receptor, and were not inaccessible to the internalized anti-transferrin receptor antibody. Therefore, our novel findings presented in this paper indicate that RhoA activity causes a selective translocation of lysosomes without perturbing the machinery of endocytic pathway.     

Lectin-binding pattern in normal human gastric mucosa. A light and electron microscopic study

Normal human gastric mucosal cells were examined by light and electron microscopy using lectins as a probe. The ABC method was used with biotinylated lectins for light microscopy and HRP-labeled lectins for electron microscopy. The human gastric mucosal cells revealed specific binding patterns for each lectin by light microscopy. Among the lectins tested, in particular, DBA gave a characteristic pattern. It specifically stained the supranuclear region of surface epithelial cells and the perinuclear region of parietal cells. By electron microscopy, the stacked cisternae and the vesicles of the Golgi apparatus of the surface epithelial cells were positive for the DBA staining. These results show that the DBA-positive supranuclear region observed by light microscopy corresponds to the Golgi apparatus. In the parietal cells, DBA, RCA and ConA bound to the intracellular secretory canaliculi which are invaginations of the cell membrane running around the nucleus in the cytoplasm. Therefore, the tubular perinuclear positive region observed by light microscopy corresponds to the membranes of the intracellular secretory canaliculi. In addition, the ConA reagent stained the endoplasmic reticulum, Golgi apparatus, nuclear envelope, and cell membrane of the parietal cell, which explains the diffuse cytoplasmic staining observed at the light microscopic level with this lectin. Lectins have proved to be very useful for the evaluation of in situ cytochemical aspects of the glycoconjugates characteristic to human gastric mucosal cells.     

Immunofluorescence studies on cartilage matrix synthesis : The synthesis of link protein, chondroitin sulfate proteoglycan monomer and type II collagen

A comparison of the synthesis and deposition of fibrous type II collagen and the constituents of chondroitin sulfate proteoglycan (CSPG) aggregates, CSPG monomer and link protein, was made for chicken sternal chondrocytes in culture, using simultaneous double immunofluorescence and lectin localization. Chondrocytes deposited only CSPG constituents--and not type II collagen--into the extracellular matrix (ECM). Intracellular precursors of CSPG monomer were localized primarily in perinuclear regions, but were observed in other cytoplasmic vesicles as well. Link protein antibodies stained the same intracellular structures, but stained the perinuclear cytoplasm less intensely. In contrast, type II procollagen was distributed in vesicles throughout the cytoplasm and was clearly absent from the distinctive, CSPG precursor-containing vesicles. Fluorescence-labelled lectins were used to further identify intracellular membrane compartments. Wheat germ agglutinin (WGA) and Ricinus lectins (which recognize carbohydrates added in the Golgi) stained the perinuclear cytoplasm, while concanavalin A (conA) (which recognizes mannose-rich oligosaccharides added co-translationally) stained vesicles throughout the rest of the cytoplasm and not the perinuclear cytoplasm. The distinctive CSPG-containing vesicles were not stained with WGA or Ricinus agglutinins. Data presented elsewhere demonstrate that the vesicles do not react with monoclonal antibodies which recognize chondroitin sulfate (CS) or keratan sulfate (KS) determinants. Thus, we conclude that the vesicles accumulate CSPG precursors which have not been modified by Golgi-mediated processes. The data indicate that matrix molecules may be segregated selectively prior to transit through the Golgi complex. The co-distribution of link protein and CSPG monomer precursors in vesicles prior to further, Golgi-mediated modification may reflect an as yet undetermined function of these vesicles in the processing or assembly of CSPG.     

The hypolipidemic compound cetaben induces changes in Golgi morphology and vesicle movement

The human hepatoma cell line HepG2 was used to study the effect of cetaben, a non-fibrate hypolipidemic drug, on cell morphology and vesicle distribution. Cetaben treatment correlated with a fragmentation and/or condensation of Golgi cisternae and the appearance of large electron-lucent vesicles. The Golgi apparatus, demonstrated, for example, by fluorescence-lectin histochemistry, was fragmented after cetaben treatment. The lectin-positive remnants were dispersed throughout the cytoplasm, but with a preference for being transported to tips of cells. However, microtubules and the intermediate filaments as well as the actin microfilaments were unchanged after cetaben treatment indicating that changes in Golgi morphology are not caused by alterations in the cytoskeleton. Cetaben decreases the cholesterol content due to inhibition of cholesterol biosynthesis. Changes in the intracellular cholesterol content are known to influence the intracellular vesicle distribution and are most likely responsible for cetaben-induced Golgi alterations, as depletion of cellular cholesterol by starvation or lovastatin and/or cyclodextrin treatment resulted in a similar redistribution of Golgi-derived wheat germ agglutinin vesicles. These lectin-stained vesicles colocalized with lysosomal marker proteins such as Limp-1 and Lamp-2, but not with the early endosomal markers Rab5 and EEA1. Upon removal of cetaben the lectin- and Limp-1/Lamp-2-costained vesicles dissociated and were transported back to the perinuclear region. Thus, cetaben-induced changes such as fragmentation of the Golgi apparatus and the dispersion of lysosomes away from their juxtanuclear location were reversible.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00418-004-0689-6     

Endocytosis in spermatids during spermiogenesis of the mouse

In the course of spermiogenesis in the mouse, spermatid cytoplasm contains numerous membrane pits, vesicles and membranous tubules which are frequently anastomosed. Pale and dense multivesicular bodies (MVB) and secondary lysosome-like structures are also present in the cytoplasm. In order to study the pathway of non-specific adsorptive endocytosis in spermatids, cationic ferritin (CF) was directly microinjected into the lumen of seminiferous tubules, and added to germinal cell culture. Tissue and cultures were fixed at various time intervals after injection. Two-5 hr after microinjection of tracer, CF was found simultaneously in vesicles, tubules, MVB and in lysosome-like bodies present in spermatids at all steps of spermiogenesis. Various membranous components of the Golgi medulla, and the innermost transsaccule of the Golgi cortex were labelled simultaneously. In primary cultures of spermatids, the vesicles contained the marker 5 min after its deposition; 10 min after deposition, CF was evident in tubules; at 30 min, CF was present in pale MVB; at 1 hr, the dense MVB and lysosome-like bodies were labelled. Finally, at 2 hr 30 min, vesicles and tubules of the Golgi medulla contained CF grains. Apparently spermatids are very active cells in the process of adsorptive endocytosis throughout spermiogenesis. Endocytosis in spermatids is probably one of the mechanisms involved in the uptake of material used to build up spermatozoa components. The strong labelling of the Golgi region probably point to its role in recycling endocytosed membranes.