Internalization of lectins in neuronal GERL

Conjugates of ricin agglutinin and phytohemagglutinin with horseradish peroxidase (HRP) were used for a cytochemical study of internalization of their plasma membrane "receptors" in cultured isolated mouse dorsal root ganglion neurons. Labeling of cells with lectin-HRP was done at 4 degrees C, and internalization was performed at 37 degrees C in a culture medium free of lectin-HRP. 15-20 min after incubation at 37 degrees C, lectin-HRP receptor complexes were seen in vesicles or tubules located near the plasma membrane. After 1-3 h at 37 degrees C, lectin-HRP-receptor complexes accumulated in vesicles and tubules corresponding to acid phosphatase-rich vesicles and tubules (GERL) at the trans aspect of the Golgi apparatus. A few coated vesicles and probably some dense bodies contained HRP after 3-6 h of incubation at 37 degrees C. Soluble HRP was not endocytosed under the conditions of this experiment or when it was present in the incubation medium at 37 degrees C. Internalization of lectin-HRP-receptor conjugates was decreased or inhibited by mitochondrial respiration inhibitors but not by cytochalasin B or colchicine. These studies indicate that lectin- labeled plasma membrane moieties of neurons are endocytosed primarily in elements of GERL.     

Vesicular tubular clusters between the ER and Golgi mediate concentration of soluble secretory proteins by exclusion from COPI-coated vesicles.

We have determined the concentrations of the secretory proteins amylase and chymotrypsinogen and the membrane proteins KDELr and rBet1 in COPII- and COPI-coated pre-Golgi compartments of pancreatic cells by quantitative immunoelectron microscopy. COPII was confined to ER membrane buds and adjacent vesicles. COPI occurred on vesicular tubular clusters (VTCs), Golgi cisternae, the trans-Golgi network, and immature secretory granules. Both secretory proteins exhibited a first, significant concentration step in noncoated segments of VTC tubules and were excluded from COPI-coated tips. By contrast, KDELr and rBet1 showed a first, significant concentration in COPII-coated ER buds and vesicles and were prominently present in COPI-coated tips of VTC tubules. These data suggest an important role of VTCs in soluble cargo concentration by exclusion from COPI-coated domains.     

Study on the origin of apical tubules in ileal absorptive cells of suckling rats using concanavalin-A as a membrane-bound tracer

Summary The ileal absorptive cells of suckling rats exhibit high levels of endocytic activity being engaged in nonselective uptake of macromolecules from the intestinal lumen. The apical cytoplasm usually contains an extensive network of small, membrane-limited tubules (apical tubules: AT), in addition to newly formed endocytic vesicles and large endocytic vacuoles. To determine whether the AT are directly involved in the endocytic process by carrying the tracer into the cell, we have analysed movements of the apical cell membrane of the ileal absorptive cells by using a membrane-bound tracer (horseradish peroxidase-labelled cancanavalin-A: Con-A HRP). The ileal absorptive cells were exposed in vitro to Con-A HRP for 10 min at 4° C, incubated for different times in Con-A free medium at 37° C, and prepared for electron microscopy. After 1 min incubation at 37° C, invaginations of the apical cell membrane, including coated pits, and endocytic vesicles were labelled with HRP-reaction product, whereas the AT and large endocytic vacuoles were negative. After 2.5 min, almost all the large endocytic vacuoles were labelled with reaction product, which was seen in their vacuolar lumen and along the luminal surface of their limiting membrane. A few AT with reaction product were seen in the apical cytoplasm; they were in frequent connection with the reaction-positive large endocytic vacuoles. With increasing incubation time, the number of the labelled AT increased. Thus, after 15 min at 37° C, the apical cytoplasm was fully occupied by the reaction-positive AT. The ends of these AT were often continuous with small spherical coated vesicles. No reaction product was detected in the Golgi complex at any time after incubation. These observations indicate that the AT located in the apical cytoplasm probably originate by budding off from the large endocytic vacuoles, rather than being involved in the process of endocytosis.     

Internalization of horseradish peroxidase isozymes by pancreatic acinar cells in vitro

We examined the uptake and fate of four horseradish peroxidase (HRP) isozymes (Type VI, VII, VIII, and IX) in isolated pancreatic acinar cells. The pattern of uptake was similar for all the isozymes examined, with the exception of Type IX. Very little Type IX HRP was internalized by the cells, and what endocytosis did occur was primarily from the apical cell surface in coated vesicles. In contrast, HRP Type VI, VII, and VIII appeared to be endocytosed largely at the basolateral cell surface. Initially, the tracer was found in smooth vesicles and tubules near the plasma membrane. The tubules resembled the basal lysosomes known to be present in these cells. At the early time points, HRP reaction product was also present in multivesicular bodies (MVBs). By 60 min, the HRP was localized in MVBs, vesicles, and tubules adjacent to the Golgi apparatus. By 12 hr after exposure to the isozymes, the tracer was present in small apical vesicles. At no time could reaction product be localized in the rough endoplasmic reticulum, Golgi saccules, or secretory granules. The results of this study suggest that the charge of a soluble-phase marker has little effect on its uptake or intracellular distribution.     

Endocytosis of cholera toxin in GERL-like structures of murine neuroblastoma cells pretreated with GM1 ganglioside. Cholera toxin internalization into Neuroblastoma GERL

Cholera toxin (CT), covalently attached to horseradish peroxidase (HRP), is a specific cytochemical marker for GM1 ganglioside (GM1) and retains the ability of the native toxin to raise levels of cyclic AMP in avian erythrocytes. Using a cytochemical stain for HRP, we found that 9% of control cultured murine neuroblastoma cells bound cholera toxin-horseradish peroxidase conjugates (CT-HRP) on their surfaces after incubations for 1 h at 4 degrees C. Exogenous GM1, the natural receptor of CT, becomes associated in the culture medium with the plasma membranes of these cells so that 96% of cells are stained. Cells preincubated with GM1 at 4 degrees C were exposed to CT-HRP for 1 h at 4 degrees C. After washing, cells were incubated at 37 degrees C for 30 min-24 h. Endocytosis of CT-HRP occurred within 30 min and CT-HRP remained, throughout the 24-h period, in tubules, vesicles, and cisternae often found near the Golgi apparatus; this aggregate of peroxidase-positive elements probably corresponds to Golgi apparatus-endoplasmic reticulum-lysosomes (GERL) of neurons. In metaphase cells, CT-HRP was observed in aggregates of vesicles and tubules clustered near the centriole. Conjugates of HRP with subunit B, the GM1 binding component of CT, were internalized by cells pretreated with GM1 as was CT-HRP. The 9% of neuroblastoma cells binding CT-HRP in the absence of exogenous GM1 internalized the ligand in a manner indistinguishable from that of the treated cells. These findings indicate that, in neuroblastoma cells, a system of vesicles, tubules, and cisternae, analogous to GERL of neurons, is the primary recipient of adsorptive endocytosis of CT bound to endogenous or exogenously introduced GM1.     

Endosomes and Golgi vesicles in adsorptive and fluid phase endocytosis

We studied with morphometric methods the endocytosis by pheochromocytoma cells of a conjugate of wheat germ agglutinin with ferritin (WGA-Ft) and of horseradish peroxidase (HRP). Quantitative studies indicated that WGA-Ft was cleared slowly from cell surfaces and that it was not recycled to the surface. Cells labeled with WGA-Ft for 15 min at room temperature were washed and incubated in medium containing HRP for 15 or 30 min at 37 degrees C. The greatest proportion of labeled vesicles and tubules contained only WGA-Ft (83.4% at 15 min and 85.3% at 30 min). A very small fraction of labeled vesicles and tubules contained only HRP (0.2% at 15 min and 0.9% at 30 min). Vesicles and tubules at the Golgi apparatus were labeled almost exclusively with WGA-Ft (97% at 15 min and 30 min); the rest had both labels. Most labeled lysosomes contained both labels (80.1% at 15 min and 80.8% at 30 min). Of the remainder more contained WGA-Ft alone (20% at 15 min and 10.9% at 30 min), then HRP alone (none at 15 min and 8.2% at 30 min). In contrast to the various and varying patterns of labeling with WGA-Ft and HRP of the other organelles studied, the vast majority of endosomes contained both markers (94.1% at 15 min and 100% at 30 min); the rest contained WGA-Ft only. These results demonstrate that endosomes are recipients of both fluid phase and adsorptive endocytosis markers; these findings are consistent with the hypothesis that endosomes mediate the sorting out and subsequent intracellular traffic of membrane bound and fluid phase markers. Cisterns of the Golgi apparatus did not contain WGA-Ft; in sharp contrast, when WGA-HRP was used, the cisterns of the Golgi apparatus consistently contained HRP.     

Origin of melanosome structures and cytochemical localizations of tyrosinase activity in differentiating epidermal melanocytes of newborn mouse skin

The mode of differentiation of epidermal melanocytes was studied by ultrastructural cytochemistry in the skin of newborn mice of strain C57BL/10J. From observations of epidermal melanoblasts and melanocytes, stage I melanosomes, including both unit membranes and inner matrices, appear to be formed from Golgi vacuoles or rough endoplasmic reticulum (RER). Stage I melanosomes were positive to ammoniacal silver-nitrate reaction in the melanoblasts of 1-day-old mice. All stages of melanosomes were similarly positive in the differentiating melanocytes of 2-day-old mice. However, Golgi apparatus, RER, and vesicles were negative. Therefore, it is conceivable that structural proteins, originated from Golgi vacuoles or RER, are developed into specialized proteins and are detected by this reaction in stage I melanosomes. Stage I melanosomes were dopa-negative in the melanoblasts. Stage I and II melanosomes were similarly negative in the differentiating melanocytes. Thus, the melanoblasts are thought to begin production of stage I melanosomes prior to the onset of tyrosinase activity. In the differentiating melanocytes, dopa-melanin depositions were observed in stage III and IV melanosomes, trans Golgi saccules, and small vesicles derived from these saccules, but not in RER. These vesicles were in contact with, or fused to, melanosomes. These findings suggest that tyrosinase may be transferred by Golgi vesicles into stage I and II melanosomes originating from Golgi vacuoles or RER.     

Endocytic pathways at the lateral and basal cell surfaces of exocrine acinar cells

In parotid acinar cells, horseradish peroxidase (HRP) administered via the main excretory duct is endocytosed from the apical cell surface in smooth C- or ring-shaped vesicles (Oliver, C. and A. R. Hand. 1979. J. Cell Biol. 76:207). These vesicles ultimately fuse with lysosomes adjacent to the Golgi apparatus. The present investigation extends these findings and examines the uptake and fate of intravenously injected HRP from the lateral and basal cell surfaces of resting and stimulated parotid and pancreatic acinar cells from rats and mice. Isoproterenol and pilocarpine were used to stimulate the parotid gland and the pancreas, respectively. HRP was internalized in smooth and coated vesicles primarily in areas of membrane infoldings. Both the number of coated vesicles and the amount of tracer internalized increased markedly following secretagogue administration. In both resting and stimulated cells, the HRP was rapidly sequestered in a unique system of basally located lysosomes that possess trimetaphosphatase activity, but not acid phosphatase activity. At 1-3 h after HRP administration, reaction product was also found in multivesicular bodies, vesicles, and lysosomes adjacent to the Golgi apparatus. With time, more HRP was localized in Golgi-associated lysosomes. By 6-7 h, tubules in the apical cytoplasm of stimulated cells contained HRP reaction product. When native ferritin was administered retrogradely and HRP injected intravenously, both tracers could be localized in the same lysosome after 4-5 h, indicating that material taken in from all cell surfaces mixes in Golgi-associated lysosomes. The results of this study suggest that two separate and distinct endocytic pathways exist in exocrine acinar cells: one involves membrane retrieval from the apical cell surface; and the other is a stimulation-dependent process at the lateral and basal cell surfaces.     

gp74 a membrane glycoprotein of the cis-Golgi network that cycles through the endoplasmic reticulum and intermediate compartment

A monoclonal antibody CC92 (IgM), raised against a fraction of rat liver enriched in Golgi membranes, recognizes a novel Endo H-resistant 74-kD membrane glycoprotein (gp74). The bulk of gp74 is confined to the cis-Golgi network (CGN). Outside the Golgi gp74 is found in tubulovesicular structures and ER foci. In cells incubated at 37 degrees C the majority of gp74 is segregated from the intermediate compartment (IC) marker p58. However, in cells treated with organelle perturbants such as low temperature, BFA, and [AIF4]- the patterns of the two proteins become indistinguishable. Both proteins are retained in the Golgi complex at 20 degrees C and in the IC at 15 degrees C. Incubation of cells with BFA results in relocation of gp74 to p58 positive IC elements. [AIF4]- induces the redistribution of gp74 from the Golgi to p58-positive vesicles and does not retard the translocation of gp74 to IC elements in cells treated with BFA. Disruption of microtubules by nocodazol results in the rapid disappearance of the Golgi elements stained by gp74 and redistribution of the protein into vesicle-like structures. The responses of gp74 to cell perturbants are in sharp contrast with those of cis/middle and trans-Golgi resident proteins whose location is not affected by low temperatures or [AIF4]-, are translocated to the ER upon addition of BFA, and stay in slow disintegrating Golgi elements in cells treated with nocodazol. The results suggest that gp74 is an itinerant protein that resides most of the time in the CGN and cycles through the ER/IC following the pathway used by p58.     

Temperature and energy dependence of secretory protein transport in the exocrine pancreas.

Pancreatic lobules pulse-labeled with [3H]leucine have been incubated at temperatures between 0 and 37 degrees C in the presence or absence of ongoing oxidative phosphorylation. Subcellular fractionation methods and electron microscopic autoradiography have been used to monitor the progress of intracellular transport of newly synthesized secretory proteins. Over the period studied, exit from the rough endoplasmic reticulum (RER) occurs only at greater than 10 degrees C while traversal of the Golgi complex and entry into condensing vacuoles requires greater than 22 degrees C. Both steps of transport require ongoing ATP production. Incubation at 10 or 20 degrees C does not diminish ATP levels, relative to 37 degrees C controls. Remarkable and unprecedented alterations of the ultrastructure of transitional elements of the RER accompany the arrest of exit from the RER: at 10 degrees C transitional elements are much more numerous and longer than in controls; in the absence of ATP production they are essentially absent. These observations are interpreted in terms of a cyclic model of RER-to-Golgi vesicular traffic. Inhibition of ATP production also causes an increase in the rigid cisternae and coated elements in the distal Golgi area.     

Distribution of calcitonin gene-related peptide-containing fibers in the urinary bladder of the rat and their origin

Summary Using horseradish peroxidase (HRP) as a tracer, we have investigated if the so-called apical tubules (AT) in the kidney proximal tubule cells are directly involved in the endocytic process by carrying the tracer into the cells, or if they are derived from the intracellular membrane compartments. Rat kidney was fixed by vascular perfusion at different time intervals after intravenous injection of HRP and prepared for electron microscopy. An analysis revealed that 0.5 min after injection, invaginations of the plasma membrane and small apical endocytic vesicles, including coated vesicles, were labelled with reaction product, whereas almost all large apical endocytic vacuoles and the AT were negative. The endocytic vacuoles and about 18% of the AT were labelled 1 min after injection. The reaction product in the large endocytic vacuoles was usually seen along the luminal surface of the vacuoles. The AT with reaction product appeared as a branched network, and were frequently connected with the labelled endocytic vacuoles. Three min after injection, reaction product was detected in about 38% of the AT, and thereafter, the percentage increased to about 74% after 7 min. No reaction product was detected in the Golgi complex at any time after HRP-injection. These findings indicate that the AT are probably formed by budding off from the large endocytic vacuoles, rather than being directly involved in the endocytic process.     

Effects of Brefeldin A on the Golgi complex, endoplasmic reticulum and viral envelope glycoproteins in murine erythroleukemia cells

This report concerns the effects of Brefeldin A (BFA): i) on the Golgi complex and the ER of retrovirus-transformed murine erythroleukemia (MEL) cells and, ii) on the viral proteins these cells express. Golgi complexes were extensively disorganized by BFA. Within 5 min, most stacked cisternae were converted to vesicles scattered throughout the centrosphere region. By 30 min, the Golgi complexes were completely disassembled. Only clusters of small vesicles ("Golgi remnants") persisted in the vicinity of the centrioles and microtubule-organizing centers. Some of these small vesicles had a simple coat structure on their membranes. Over the next 1 to 2 h of BFA treatment, the number of vesicles in the Golgi area decreased concomitantly with the expansion of a predominantly smooth membrane portion of the ER, consisting of a network of dilated tubules in continuity with regular RER cisternae, annulate lamellae and the nuclear envelope. By electron microscopy, viral glycoproteins appeared to accumulate on the membranes of this network, and immature virions were found to bud preferentially into its cisternal space. Viral accumulations increased with time under BFA. The rest of the RER appeared normal, apparently unaffected by the drug. Preferential virion budding suggests that this expanding network is a chemically differentiated part of the ER. By immunofluorescence, antibodies to viral envelope proteins gave a punctate staining at the surface of control cells, presumably in the areas of virion budding, whereas relatively large intracellular masses of antigens were found in BFA-treated cells. We assume that these masses represent the differentiated parts of the ER. Taken together, these findings suggest that BFA blocks intracellular transport of newly synthesized cellular and viral proteins immediately distal to the distinct compartment of the ER in which virion budding preferentially occurs. BFA effects are rapidly and fully reversible. Within 1 min of the removal of the drug, stacks of Golgi cisternae began to reappear in the vicinity of the centrioles, and by 30 min, Golgi complexes regained their normal structural appearance.     

Quantitative ultrastructural, autoradiographic evidence for the magnitude and early involvement of the Golgi apparatus complex in the endocytosis of wheat germ agglutinin by cultured neuroblastoma

Several ligands undergo endocytosis into the Golgi apparatus. We have examined with a quantitative ultrastructural, autoradiographic method the sequential endocytosis of tritiated wheat germ agglutinin (3H-WGA) by cultured murine neuroblastoma cells. Cells were incubated with 3H-WGA for 1 hour at 4 degrees C, washed, and incubated in complete medium without ligand at 37 degrees C for 5, 15, 30, and 120 minutes. At 5 minutes, the optimized sources/micron 2 of neuroblastoma cell area, which represented the grain density of each compartment, were as follows: smooth vesicles and tubules, 1.03 +/- 0.88; Golgi-associated vesicles, i.e., clusters of vesicles within a 1 micron radius of the Golgi cisterns, 1.03 +/- 0.31; Golgi cisterns, less than 0.01; and lysosomes, 0.26 +/- 0.16. At 15 minutes grain densities were: smooth vesicles and tubules, 0.9 +/- 0.34; Golgi-associated vesicles, 1.41 +/- 0.28; Golgi cisterns, 0.73 +/- 0.41; and lysosomes, 0.1 +/- 0.09. At 30 minutes grain densities were: smooth vesicles and tubules, 0.46 +/- 0.46; Golgi-associated vesicles, 1.78 +/- 0.34; Golgi cisterns, 0.89 +/- 0.78; and lysosomes, 0.39 +/- 0.14. At 2 hours, smooth vesicles, tubules, and Golgi cisterns were not labeled, Golgi-associated vesicles were still labeled (0.71 +/- 0.1), and lysosomes were heavily labeled (2.17 +/- 0.22). These results are consistent with the hypotheses that either the Golgi complex (cisterns and associated vesicles) is an early and intermediate step of the endocytosis of 3H-WGA into lysosomes or that it constitutes part of a separate and quantitatively significant pathway of endocytosis of this ligand.     

Virus-neuron interactions in the mouse brain infected with Japanese encephalitis virus

The virus-host interactions between Japanese encephalitis (JE) virus and mouse brain neurons were analyzed by electron microscopy. JE virus replicated exclusively in the rough endoplasmic reticulum (RER) of neurons. In the early phase of infection, the perikaryon of infected neurons had relatively normal-looking lamellar RER whose cisternae showed focal dilations containing progeny virions and characteristic endoplasmic reticulum (ER) vesicles. The reticular RER, consisted of rows of ribosomes surrounding irregular-shaped, membrane-unbounded cisternae and resembled that observed in JE-virus-infected PC12 cells, were also seen adjacent to the lamellar RER. The appearance of the reticular RER indicated that RER morphogenesis occurred in infected neurons in association with the viral replication. The fine network of Golgi apparatus was extensively obliterated by fragmentation and dissolution of the Golgi membranes and their replacement by the electron-lucent material. As the infection progressed, the lamellar RER was increasingly replaced by the hypertrophic RER which had diffusely dilated cisternae containing multiple progeny virions and ER vesicles. The Golgi apparatus, at this stage, was seen as coarse, localized Golgi complexes near the hypertrophic RER. In the later phase of infection, RER of infected neurons showed a degenerative change, with the cystically dilated cisternae being filled with ER vesicles and virions. Small, localized Golgi complexes frequently showed vesiculation, vacuolation, and dispersion. The present study, therefore, indicated that during the viral replication the normal lamellar RER which synthesized neuronal secretory and membrane proteins was replaced by the hypertrophic RER which synthesized the viral proteins. The hypertrophic RER eventually degenerated into cystic RER whose cisternae were filled with viral products. The constant degenerative change which occurred in the Golgi apparatus during the viral replication suggested that some of the viral proteins transported from RER to the Golgi apparatus were harmful to the Golgi apparatus and that increasing damage to the Golgi apparatus during the viral replication played the principal role in the pathogenesis of JE-virus-infected neurons in the central nervous system.     

The coiled-coil membrane protein golgin-84 is a novel rab effector required for Golgi ribbon formation

Fragmentation of the mammalian Golgi apparatus during mitosis requires the phosphorylation of a specific subset of Golgi-associated proteins. We have used a biochemical approach to characterize these proteins and report here the identification of golgin-84 as a novel mitotic target. Using cryoelectron microscopy we could localize golgin-84 to the cis-Golgi network and found that it is enriched on tubules emanating from the lateral edges of, and often connecting, Golgi stacks. Golgin-84 binds to active rab1 but not cis-Golgi matrix proteins. Overexpression or depletion of golgin-84 results in fragmentation of the Golgi ribbon. Strikingly, the Golgi ribbon is converted into mini-stacks constituting only approximately 25% of the volume of a normal Golgi apparatus upon golgin-84 depletion. These mini-stacks are able to carry out protein transport, though with reduced efficiency compared with a normal Golgi apparatus. Our results suggest that golgin-84 plays a key role in the assembly and maintenance of the Golgi ribbon in mammalian cells.     

Dynamics of Golgi matrix proteins after the blockage of ER to Golgi transport

When the ER to Golgi transport is blocked by a GTP-restricted mutant of Sar1p (H79G) in NRK-52E cells, most Golgi resident proteins are transported back into the ER. In contrast, the cis-Golgi matrix proteins GM130 and GRASP65 are retained in punctate cytoplasmic structures, namely Golgi remnants. Significant amounts of the medial-Golgi matrix proteins golgin-45, GRASP55 and giantin are retained in the Golgi remnants, but a fraction of these proteins relocates to the ER. Golgin-97, a candidate trans-Golgi network matrix protein, is retained in Golgi remnant-like structures, but mostly separated from GM130 and GRASP65. Interestingly, most Sec13p, a COPII component, congregates into larger cytoplasmic clusters soon after the microinjection of Sar1p(H79G), and these move to accumulate around the Golgi apparatus. Sec13p clusters remain associated with Golgi remnants after prolonged incubation. Electron microscopic analysis revealed that Golgi remnants are clusters of larger vesicles with smaller vesicles, many of which are coated. GM130 is mainly associated with larger vesicles and Sec13p with smaller coated vesicles. The Sec13p clusters disperse when p115 binding to the Golgi apparatus is inhibited. These results suggest that cis-Golgi matrix proteins resist retrograde transport flow and stay as true residents in Golgi remnants after the inhibition of ER to Golgi transport.     

Morphological characterization of the pathway of endocytosis and intracellular processing of transferrin and alpha-fetoprotein in human T lymphocytes stimulated with phytohemagglutinin (PHA)

Covalent conjugates of transferrin (Tf) and alpha-fetoprotein (AFP) with horseradish peroxidase (HRP) have been used to follow, at the ultrastructural level, the uptake and the intracellular pathway of these proteins in peripheral blood human lymphocytes stimulated by phytohemagglutinin (PHA) to blast formation. Both proteins enter specifically the cells via vesicles (60-70 nm in diameter) and endosomes. They are then observed in multivesicular bodies and tubular vesicular elements in the Golgi region. AFP is thus found in the same subcellular compartments as Tf and is probably also recycled, as most of the 125I-labeled protein leaves the cells undegraded. Unstimulated lymphocytes do not internalize significantly AFP-HRP. The uptake of a noncovalent conjugate of AFP-HRP and [3H]-arachidonic acid [3H-(20:4)] is usually poor, at 37 degrees C, in unstimulated lymphocytes as well as, at 4 degrees C, in lymphocytes stimulated for 72 h. Stimulated lymphocytes incubated at 37 degrees C with the radioactive conjugate show a heavy labeling of cell organelles and more particularly of lipid droplets. AFP could regulate the intracellular delivery of fatty acid molecules.     

Serial section analysis of clathrin-coated pits in rat liver sinusoidal endothelial cells

Electron microscopy and serial sections were used to examine the shape of clathrin-coated pits in sinusoidal endothelial cells of rat livers. Livers were perfused at 4 degrees C with either concanavalin A-horseradish peroxidase (conA-HRP), or HRP alone, followed by warm-up to 37 degrees C and fixation with glutaraldehyde. Alternatively, the livers were perfused with HRP at 37 degrees C, followed by fixation. All tissue was preserved using a membrane contrast enhancement technique (R-OTO) consisting of sequential osmium-ferrocyanide, thiocarbohydrazide, and osmium-ferrocyanide treatment. Peroxidase reaction product was used to identify structures participating in endocytosis. One hundred and ninety-three clathrin-coated structures were examined. Sixty-six were from livers perfused with conA-HRP at 4 degrees C, 63 were from livers perfused with only HRP at 4 degrees C, and 64 were from livers perfused with HRP at 37 degrees C. These coated structures were morphologically classified into three categories: (a) flat pits; (b) cup-shaped pits; (c) pits with a narrow neck. No isolated coated vesicles were found. In cells perfused at 4 degrees C followed by warming to 37 degrees C, the percentage of coated pits found connected to the cell surface by narrow necks was 31%, using conA-HRP, and 27% using HRP alone. In cells perfused continuously at 37 degrees C, the percentage of coated pits with narrow neck connections was 21% using HRP alone. These results suggest that the formation of coated pits connected to the surface by narrow necks is not an artifact of cell type, of experimental protocol or of incubation with a lectin.     

Beta-COP localizes mainly to the cis-Golgi side in exocrine pancreas

We examined the distribution of the non-clathrin-coated vesicle- associated coat protein beta-COP in rat exocrine pancreatic cells by immunogold cytochemistry. Labeling for beta-COP was found in the Golgi region (48%) where it was associated with vesicles and buds of approximately 50 nm, showing a characteristic approximately 10-nm-thick coat. The other half of the label was present in the cytoplasm, not associated with visible coats or membranes, with a minor fraction present on small clusters of tubules and vesicles. Clathrin-coated vesicles were typically located at the trans-side of the Golgi complex, and showed a thicker coat of approximately 18 nm. Of the total beta-COP labeling over the Golgi region, 68% occurred on the cis-side, 6% on the cisternae, 17% on the rims of the cisternae, and only 9% on the trans- side. For clathrin these figures were 16, 2, 4, and 78%, respectively. At the cis-Golgi side beta-COP was present in transitional areas (TA), on so-called peripheral elements (PE), consisting of tubules and vesicles located between the cup-shaped transitional elements (TE) of the RER and the cis-most Golgi cisternae. Label for Sec23p was also present in TA but was located closer to the TE, while beta-COP labeled PE were located near the cis-Golgi cisternae. Upon energy depletion, Golgi associated beta-COP was almost exclusively (86%) in spherical aggregates of 200-500 nm in diameter, whereas the cis-side (6%), the cisternae (1%), the rims (4%) and trans-side (3%) of the Golgi complex, were barely labeled; 50% of the total label remained in the cytoplasm. The aggregates were predominantly located at the cis-side of the Golgi stack, next to, but distinct from the Sec23p positive TA, that were devoid of beta-COP and had only a few recognizable vesicles left. Incubation with aluminum fluoride resulted in fragmentation of the Golgi complex into large clusters of beta-COP positive vesicles, while 50% of the label remained in the cytoplasm, as in control cells. After 10 min of Brefeldin A treatment 91% of beta-COP was cytoplasmic and only 7% associated with membranes of the Golgi complex. The total label for beta-COP over exocrine cells remained unchanged during the incubation with either of the drugs, indicating that the drugs induce reallocation of beta-COP. Our data suggest that beta-COP plays a role in membrane transport at the cis-side of the Golgi complex.     

Transport into and out of the Golgi complex studied by transfecting cells with cDNAs encoding horseradish peroxidase

We have developed a novel technique with which to investigate the morphological basis of exocytotic traffic. We have used expression of HRP from cDNA in a variety of cells in combination with peroxidase cytochemistry to outline traffic into and out of the Golgi apparatus at the electron microscopic level with very high sensitivity. A secretory form of the peroxidase (ssHRP) is active from the beginning of the secretory pathway and the activity is efficiently cleared from cells. Investigation of the morphological elements involved in the itinerary of soluble ER proteins using ssHRP tagged with the ER retention motif (ssHRPKDEL) shows that it progresses through the Golgi stack no further than the cis-most element. Traffic between the RER and the Golgi stack as outlined by ssHRPKDEL occurs via vesicular carriers as well as by tubular elements. ssHRP has also been used to investigate the trans side of the Golgi complex, where incubation at reduced temperatures outlines the trans-Golgi network with HRP reaction product. Tracing the endosomal compartment with transferrin receptor in double-labeling experiments with ssHRP fails to show any overlap between these two compartments.