Intracellular localization and endocytosis of brush border enzymes in the enterocyte-like cell line Caco-2.

Immunoelectron microscopy was used to localize the brush border hydrolases sucrase-isomaltase (SI) and dipeptidylpeptidase IV (DPPIV) in the human colon carcinoma cell line Caco-2. Both enzymes were detected at the microvillar membrane, in small vesicles and multivesicular bodies (MVBs), and in lysosomal bodies. In addition, DPPIV was found in the Golgi apparatus, a variety of apical vesicles and tubules, and at the basolateral membrane. To investigate whether the hydrolases present in the lysosomal bodies were endocytosed from the apical membrane, endocytic compartments were marked with the endocytic tracer cationized ferritin (CF). After internalization from the apical membrane through coated pits, CF was first recovered in apical vesicles and tubules, and larger electronlucent vesicles (early endosomes), and later accumulated in MVBs (late endosomes) and lysosomal bodies. DPPIV was localized in a subpopulation of both early and late endocytic vesicles, which contained CF after 3 and 15 min of uptake, respectively. Also, internalization of the specific antibody against DPPIV and gold labeling on cryosections showed endocytosed DPPIV in both early and late endosomes. However, unlike CF, no accumulation of DPPIV was seen in MVBs or lysosomal bodies after longer chase times. The results indicate that in Caco-2 cells the majority of brush border hydrolases present in lysosomal bodies are not endocytosed from the brush border membrane. Furthermore, the labeling patterns obtained, suggest that late endosomes may be involved in the recycling of endocytosed DPPIV to the microvilli.     

Pinocytosis in mouse L-fibroblasts: ultrastructural evidence for a direct membrane shuttle between the plasma membrane and the lysosomal compartment

Mouse L-fibroblasts internalized large amounts of cationized ferritin (CF) by pinocytosis. Initially (60-90 s after addition of CF to cell monolayers at 37 degrees C), CF was found in vesicles measuring 100-400 nm (sectioned diameter) and as small clusters adhering to the inner aspect of the limiting membrane of a few large (greater than 600 nm) vacuoles. After 5-30 min, CF labeling of large vacuoles was pronounced and continuous. Moreover, 70-80% of all labeled structures were tiny (less than 100 nm) vesicles. However, the absolute frequency of tiny vesicles increased more than twofold from 5 min to 30 min. When the cells were incubated with CF for 30 min, then washed and further incubated for 3 h without CF, almost all CF was present in dense bodies (100-500 nm). When L-cells were first incubated with horseradish peroxidase (HRP), then washed and incubated with CF, double-labeled vacuoles were observed. Tiny vesicles also contained HRP-CF, and small HRP-CF patches were localized on the cell surface. Distinct labeling of stacked Golgi cisterns was not observed in any experiment. These observations suggest that the numerous tiny vesicles are not endocytic but rather pinch off from the large vacuoles and move towards the cell surface to fuse with the plasma membrane. Thus, ultrastructural evidence is provided in favor of a direct membrane shuttle between the plasma membrane and the lysosomal compartment.     

Endocytotic uptake of cationized ferritin tracer into glomus cells dissociated from the adult rat carotid body

Summary Glomus cells from carotid bodies of adult rats dissociated by means of collagenase or collagenase + trypsin were used to study by electron microscopy the endocytotic uptake of cationized ferritin (CF) tracer into subcellular compartments. The glomus cells were incubated with the tracer (1) in a basic salt medium (BM), or (2) in the BM into which calcium ionophore A23187 had been added, or (3) in a potassium-rich medium.Incubation of the cells in BM containing CF for 30 min resulted in attachment of the tracer to the cell membrane and uptake of a few solitary tracer particles into small vesicles and multivesicular bodies. No uptake into the cisternae of the Golgi apparatus was observed. Further incubation in BM containing CF for another 30 min resulted in increased uptake of the tracer into small vesicles and multivesicular bodies. A similar pattern of uptake was observed when the dissociated glomus cells were first preincubated in BM with CF for 30 min and then incubated for 1 min or 30 min in the BM solution containing both the ionophore and CF. Upon such incubation, CF particles were seen to penetrate into coated pits and sites of exocytosis at the cell surface. When the 30-min preincubation in BM was followed by incubation in a CF-containing potassium-rich medium for 15–30 min, uptake into vesicles, small lysosomes and occasionally also into profiles of the smooth endoplasmic reticulum was seen. Endocytotic mechanisms of the glomus cells are outlined.     

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.     

Comparison of the intracellular pathways of transferrin recycling and vesicular stomatitis virus membrane glycoprotein exocytosis by ultrastructural double-label cytochemistry

Transferrin is taken up by receptor-mediated endocytosis into intracellular vesicles and tubules, and then recycles rapidly to the plasma membrane (diacytosis). We applied double-label cytochemistry to study whether the recycling structures containing transferrin fuse with the intracellular membranous structures that deliver newly synthesized membrane glycoproteins from the ER to the plasma membrane (exocytosis) or whether they remain independent. KB and Vero cells were infected with the temperature-sensitive transport mutant 0-45 of vesicular stomatitis virus (VSV). Temperature-regulated exocytosis of membrane glycoprotein "G" occurred simultaneously with diacytosis of transferrin. The exocytic "G" protein, as detected by immunoperoxidase electron microscopy, passed through the cisternal Golgi stacks and vacuolar, tubular, vesicular, and pit-like structures of the Golgi system. A transferrin-ferritin conjugate used in ultrastructural double-label experiments was detected in diacytic vesicles and tubules that accumulated in the proximal (trans-reticular) Golgi area of the cell. The ferritin-labeled vesicles/tubules were often close to and intermixed with the VSV-"G" containing membranous structures, but in most cases at early times (15-20 min) the transferrin and VSV-"G" containing vesicular structures remained distinct. At later times (30-45 min), the two labels were occasionally found in the same structures. These results indicate that rapid recycling of endocytosed materials and exocytosis of membrane glycoproteins to the cell surface usually occur in distinct vesicles, possibly along the same general morphologic exit pathway.     

Internalization of cationized ferritin by isolated pancreatic acinar cells

The internalization of cationized ferritin (CF) was studied in isolated pancreatic acinar cells in vitro. Horseradish peroxidase (HRP) was used in conjunction with CF to compare internalization of soluble-phase and membrane-bound tracers. The mode of internalization of CF was dependent upon tracer concentration and origin of the plasma membrane (apical vs. lateral-basal). At the lower tracer concentrations (0.19 and 0.38 mg/ml), internalization from the apical cell surface occurred via small vesicles. The tracer then appeared in multivesicular bodies, in tubules, and in irregular membrane-bound structures. After 15 min, CF particles were seen in many small vesicles near the Golgi apparatus, but not in the Golgi saccules. In contrast, at the lateral-basal cell surface the CF particles tended to form clusters. These clusters were more pronounced at higher CF concentrations (0.76 and 1.5 mg/ml) and were associated with elongated cellular processes, which seemed to engulf CF accumulations in a phagocytic manner. Once internalized, CF was found primarily in large irregular structures which appeared to migrate slowly toward the nucleus, reaching a juxtanuclear position after approximately 30 min. CF was observed in lysosomes after 30-45 min and by 90 min most of the CF was confined to large vacuoles and to trimetaphosphatase-positive lysosomes. Similar routes were observed when cells were double-labeled with CF and HRP, where endocytic structures showed co-localization of both tracers. The results of this study indicate the importance of the Golgi region in the intracellular sorting of internalized apical membrane. Furthermore, this work confirms the presence of distinct endocytic pathways at the apical and lateral-basal cell surfaces.     

Desmosome assembly in MDCK cells: transport of precursors to the cell surface occurs by two phases of vesicular traffic and involves major changes in centrosome and Golgi location during a Ca(2+) shift

Desmosome formation in MDCK cells was investigated using a Ca(2+) shift. Following preliminary treatment with cycloheximide at 37 degrees C, continued surface transport and subsequent endocytosis were minimized by incubating cells at 19 degrees C to trap nascent glycoproteins within the Golgi body. Release into high Ca(2+) medium (HCM) at 37 degrees C resulted in junction formation as well as relocation of the Golgi body and centrosomes to a subapical location. Desmosome formation occurred in two stages over 2 h, the first occurring within 30 min of the shift to HCM, in 60-nm vesicles containing chiefly Dsc2 and lower concentrations of Dsg and E-cadherin distributed to the entire cell surface. Much of this material was subsequently endocytosed. The second stage involved transport of Dsg, E-cadherin, plakoglobin, and beta-catenin, in more complex vesicles some 200 nm in size, directed to possible nucleation sites on the developing basolateral surface. Plaque proteins such as desmoplakin I/II were added subsequently. Stage-two vesicles, but possibly not those of stage one, were accessible to endocytic markers via retrograde transport from multivesicular bodies prelabeled at 19 degrees C.     

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     

Estimation of Golgi membrane flow rates in ovary glands of aptenia cordifolia using cytochalasin B

Ovary gland cells of Aptenia cordifolia were exposed to 100 micrograms/ml cytochalasin B (cyt B) for 30 or 60 min during the phase of granulocrine polysaccharide secretion. The drug caused a congestion of Golgi vesicles around the dictyosomes, probably resulting from an inhibition of the vesicle migration towards the plasma membrane. The ultrastructural feature of the Golgi apparatus in control and cyt B treated cells was analyzed using stereological methods in order to estimate the mean area of vesicular membrane produced by a single dictyosome during a 30 min period of effective cyt B action. Assuming that the rate of vesicle congestion can be equated with the rate of vesicle production, the 236 dictyosomes found to be present in the non-growing ovary gland cells form 7517 vesicles in total, or approximately 32 vesicles each within a period of 30 min. This corresponds to a membrane turnover rate of 70.4 micrometers/min (this equals approximately 10% of the total plasma membrane area per min), since the mean vesicle surface area was calculated to be 0.281 microns2. The turnover time of a single Golgi cisterna was determinated to be 7.34 min, and the average vesicle life time to be 8.86 min. Discussion focuses upon the way by which the relatively high amount of vesicular membrane material incorporated into the plasmalemma is recycled into the endomembrane system. Since a bulk membrane retrieval in the form of vesicles, as well as a bulk vesicle migration from the ER to the dictyosomes could not be observed, we suggest that a transfer of membrane subunits is involved in the maintenance of membrane equilibrium in the Golgi apparatus.     

Concanavalin A-Induced Morphological Changes and Its Binding Sites in Starfish Follicle Cells as Revealed by Electron Microscopy

Concanavalin A (Con A) stimulates the production in starfish follicle cells of 1-methyladenine, a hormone which induces oocyte maturation. We have therefore investigated Con A-induced morphological changes and Con A-binding sites in the follicle cell using native Con A and horseradish peroxidase- or ferritin-labeled Con A (HRP-Con A, Fer-Con A). After isolated follicle cells were incubated with Con A (1 mg/ml), vacuoles, the Golgi complex and multivesicular body-like organelles (MVBs) became prominent in most of the cells. After follicle cells were prefixed and then incubated with Fer-Con A for 60 min, tagged ferritin was diffusely and randomly distributed as single or small clustered particles on the cell surface. The incubation of isolated follicle cells with Fer-Con A for 10 min before fixation resulted in numerous ferritin particles localized along the internalized membrane, and also in vacuoles, MVBs and small lysosome-like structures. After 60 min incubation with Fer-Con A, ferritin was further located in large lysosome-like structures and in vesicles near and in the Golgi area as well as in the organelles described above. HRP-Con A binding sites were also observed in vacuoles and MVBs of the intact cells.
These results suggest that Con A binds at first to the cell surface and causes rapid internalization and that membrane-bound Con A is easily endocytosed into vacuoles, MVBs and lysosome-like structures, and is later incorporated in some vesicles in the Golgi area.     

COP-coated vesicles are involved in the mitotic fragmentation of Golgi stacks in a cell-free system

Rat liver Golgi stacks fragmented when incubated with mitotic but not interphase cytosol in a process dependent on time, temperature, energy (added in the form of ATP) and cdc2 kinase. The cross-sectional length of Golgi stacks fell in the presence of mitotic cytosol by approximately 50% over 30 min without a corresponding decrease in the number of cisternae in the stack. The loss of membrane from stacked and single cisternae occurred with a half-time of approximately 20 min, and was matched by the appearance of both small (50-100 nm in diameter) and large (100-200 nm in diameter) vesicular profiles. Small vesicular profiles constituted more than 50% of the total membrane after 60 min of incubation and they were shown to be vesicles or very short tubules by serial sectioning. In the presence of GTP gamma S all of the small vesicles were COP-coated and both the extent and the rate at which they formed were sufficient to account for the production of small vesicles during mitotic incubation. The involvement of the COP-mediated budding mechanism was confirmed by immunodepletion of one of the subunits of COP coats (the coatomer) from mitotic cytosol. Vesicles were no longer formed but highly fenestrated networks appeared, an effect reversed by the readdition of purified coatomer. Together these experiments provide strong support for our hypothesis that the observed vesiculation of the Golgi apparatus during mitosis in animal cells is caused by continued budding of COP-coated transport vesicles but an inhibition of their fusion with their target membranes.     

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 in nonciliated epithelial cells of the ductuli efferentes in the rat

The nonciliated cells lining the ductuli efferentes presented three distinct cytoplasmic regions. The apical region contained, in addition to cisternae of endoplasmic reticulum and mitochondria, two distinct membranous elements. The tubulovesicular system consisted of dilated tubules connected to the apical plasma membrane and subjacent distended vesicular profiles. The apical tubules, not connected to the cell surface, consisted of numerous densely stained tubules of small size which contain a compact, finely granulated material. The supranuclear region, in addition to a Golgi apparatus and ER cisternae, contained dilated vacuoles, pale and dense multivesicular bodies, as well as numerous dense granules identified cytochemically as lysosomes. The basal region contained the nucleus and many lipid droplets. The endocytic activity of these cells was investigated using cationic ferritin (CF) and concanavalin-A-ferritin (Con-A-ferritin) as markers of adsorptive endocytosis; and native ferritin (NF), concanavalin-A-ferritin in the presence of alpha-methyl mannoside, and horseradish peroxidase or albumin bound to colloidal gold for demonstrating fluid-phase endocytosis. These tracers were injected separately into the rete testis, and animals were sacrificed at various time intervals after injection. At 1 min, CF or Con-A-ferritin were seen bound to the apical plasma membrane, to the membrane of microvilli, and to the membrane delimiting elements of the tubulovesicular system. Between 2 and 5 min, these tracers accumulated in the densely stained apical tubules and at 15 min in the dilated vacuoles. Between 30 min and 1 hr, the tracers appeared in multivesicular bodies of progressively increasing density, whereas at 2 hr and later time intervals, many dense lysosomal elements became labeled. The tracers for fluid-phase endocytosis showed a distribution similar to that for CF or Con-A-ferritin except that they did not bind to the apical plasma membrane, microvilli, or membrane delimiting the tubulovesicular system. At no time interval were any of the tracers observed in the abluminal spaces. Thus, the nonciliated epithelial cells of the ductuli efferentes are actively involved in fluid-phase and adsorptive endocytosis, both of which result in the sequestration of endocytosed material within the lysosomal apparatus of the cell.     

Post Golgi apparatus structures and membrane removal in plants

Summary In nongrowing secretory cells of plants, large quantities of membrane are transferred from the Golgi apparatus to the plasma membrane without a corresponding increase in cell surface area or accumulation of internal membranes. Movement and/or redistribution of membrane occurs also in trans Golgi apparatus cisternae which disappear after being sloughed from the dictyosome, and in secretory vesicles which lose much of their membrane in transit to the cell surface. These processes have been visualized in freeze-substituted corn rootcap cells and a structural basis for membrane loss during trafficking is seen. It involves three forms of coated membranes associated with the trans parts of the Golgi apparatus, with cisternae and secretory vesicles, and with plasma membranes. The coated regions of the plasma membrane were predominantly located at sites of recent fusion of secretory vesicles suggesting a vesicular mechanism of membrane removal. The two other forms of coated vesicles were associated with the trans cisternae, with secretory vesicles, and with a post Golgi apparatus tubular/vesicular network not unlike the TGN of animal cells. However, the trans Golgi network in plants, unlike that in animals, appears to derive directly from the trans cisternae and then vesiculate. The magnitude of the coated membrane-mediated contribution of the endocytic pathway to the formation of the TGN in rootcap cells is unknown. Continued formation of new Golgi apparatus cisternae would be required to maintain the relatively constant form of the Golgi apparatus and TGN, as is observed during periods of active secretion.     

Giardia muris: ultrastructural analysis of in vitro excystation

Giardia muris cysts were examined by transmission electron microscopy before treatment, after induction, and at timed intervals during the incubation phase of in vitro excystation. Untreated G. muris cysts had a thick cyst wall composed of a fibrous outer wall and a thin, electron-dense inner membrane which extended from the trophozoite plasma membrane. The cytoplasm was devoid of endoplasmic reticulum, Golgi bodies,and mitochondria. Numerous large vacuoles were present within the ectoplasm just beneath the plasma membrane in untreated cysts. Following induction these cysts lacked ectoplasmic vacuoles. Concurrently, numerous membrane bound vesicles were seen in the peritrophic space closely adhering to the surface of the trophozoite. These vesicles appear to be of cytoplasmic origin. The cytoplasm of fully excysted trophozoites lacked ectoplasmic vacuoles but displayed well-developed ribbons of microtubular bodies, probably precursors of ventral disk, lateral flange, and median bodies and also contained extensive granular endoplasmic reticulum. No more than two nuclei were observed within each organism. The earliest excysted organisms were observed 0-5 min after incubation had begun and most organisms had excysted within 10 min. Cytokinesis occurred only after excystation was complete.     

Redistribution and recycling of internalized membrane in seminal vesicle secretory cells

This study was performed to clarify the fate of membrane constituents internalized from the apical domain in secretory cells, in particular their possible recycling and the compartments involved in it. Glycoproteins of the apical membrane of seminal vesicle secretory cells from guinea-pig were covalently labeled in vitro (0 degrees C, 20 min) with 3H-galactose and the epithelium incubated for 15 min (37 degrees C, first incubation) to allow endocytosis. The label which was not internalized was then exposed to enzymatic hydrolysis (0 degrees C, 30 min) and the epithelium re-incubated to allow membrane movement for 15 and 30 min (37 degrees C, 2nd incubation). After each step of the protocol, tissue pieces were fixed and processed for electron microscope autoradiography and the results studied by morphometric analysis. Following labeling, 99% of the silver grains were associated with the apical domain of the cell membrane (AD). After the 1st incubation at 37 degrees C, 30% of the grains were inside the cells in association with the cytoplasmic vesicles (Cyt ves), secretory vacuoles (SV), Golgi vesicles (GV), Golgi cisternae (GC), multivesicular bodies (MVB), lysosomes (LYS), and the cell membrane basolateral domain (BLD). About 58% of non-internalized radioactivity was removed by hydrolysis. During the 2nd incubation at 37 degrees C the concentration of label increased in BLD and LYS, decreased in SV and MVB, and fluctuated in GC, GV and AD. The distribution of grains observed at 15 min, as compared using the chi-square test, was highly significantly different from that expected without recycling. The results show that cell membrane glycoproteins internalized at the cell apex recycle back to the membrane apical domain and are consistent with the involvement of GC and SV in the recycling pathway. Membrane shuttle between the apical and basolateral domains of the cell membrane is also suggested by these observations.     

Vesicular transport of cationized ferritin by the epithelium of the rat choroid plexus

We have studied the transport of ferritin that was internalized by coated micropinocytic vesicles at the apical surface of the choroid plexus epithelium in situ. After ventriculocisternal perfusion of native ferritin (NF) or cationized ferritin (CF), three routes followed by the tracers are revealed: (a) to lysosomes, (b) to cisternal compartments, and (c) to the basolateral cell surface. (a) NF is micropinocytosed to a very limited degree and appears in a few lysosomal elements whereas CF is taken up in large amounts and can be followed, via endocytic vacuoles and light multivesicular bodies, to dark multivesicular bodies and dense bodies. (b) Occasionally, CF particles are found in cisterns that may represent GERL or trans-Golgi elements, whereas stacked Golgi cisterns never contain CF. (c) Transepithelial vesicular transport of CF is distinctly revealed. The intercellular spaces of the epithelium, below the apical tight junctions, contain numerous clusters of CF particles, often associated with surface-connected, coated vesicles. Vesicles in the process of exocytosis of CF are also present at the basal epithelial surface, whereas connective tissue elements below the epithelium are unlabeled. Our conclusion is that fluid and solutes removed from the cerebrospinal fluid by endocytosis either become sequestered in the lysosomal apparatus of the choroidal epithelium or are transported to the basolateral surface. However, our results do not indicate any significant recycling via Golgi complexes of internalized apical cell membrane.     

The Pathway of Golgi Cluster Formation in Okadaic Acid-Treated Cells

Using stereology and immunoelectron microscopy we examined the pathway of Golgi duster formation during treatment with the phosphatase inhibitor okadaic acid. During the first hour the Golgi stack of suspension HeLa cells lost 90% of its membrane without appreciable reduction in the number of cisternae. During this time clusters of tubules and vesicles (Golgi clusters) appeared and these contained only a fraction of the Golgi membrane present in untreated cells. Despite the overall reduction in membrane the total amount of immunolabeling for galactosyltransferase over the Golgi clusters of a typical cell was maintained, indicating that galactosyltransferase had been retained in Golgi membranes. The observation that, after 40 min okadaic acid treatment, labeling density for galactosyltransferase within trans Golgi cisternae increased 1.6-fold (n = 3, CE 10%) suggests that membrane loss from trans cisternae was selective. Careful evaluation of immunolabeled clusters showed that most of the galactosyltransferase labeling was located over complex tubular profiles and not vesicular profiles. Tubular structures were also observed during disassembly and these were found both connected to disassembling cisternae and within forming Golgi clusters, indicating that they were intermediates in cluster formation. We also investigated the role of vesicular transport in cluster formation. During disassembly we found no accumulation of COP-coated buds and vesicles over Golgi membrane. However, aluminium fluoride, previously found to arrest transport in the Golgi stack, completely inhibited membrane depletion and stack disassembly. Taken together, our results indicate that during Golgi cluster formation, membrane leaves the Golgi but galactosyltransferase is retained within a tubular reticulum which is a direct descendant of trans-Golgi cisternae. Membrane depletion may require ongoing vesicular transport and we postulate that it arises because of an imbalance in membrane traffic into and out of the Golgi apparatus.     

Redistribution and recycling of internalized membrane in seminal vesicle secretory cells

This study was performed to clarify the fate of membrane constituents internalized from the apical domain in secretory cells, in particular their possible recycling and the compartments involved in it. Glycoproteins of the apical membrane of seminal vesicle secretory cells from guinea-pig were covalently labeled in vitro (0°C, 20 min) with ³H-galactose and the epithelium incubated for 15 min (37°C, first incubation) to allow endocytosis. The label which was not internalized was then exposed to enzymatic hydrolysis (0°C, 30 min) and the epithelium re-incubated to allow membrane movement for 15 and 30 min (37°C, 2nd incubation). After each step of the protocol, tissue pieces were fixed and processed for electron microscope autoradiography and the results studied by morphometric analysis. Following labeling, 99% of the silver grains were associated with the apical domain of the cell membrane (AD). After the 1st incubation at 37°C, 30° of the grains were inside the cells in association with the cytoplasmic vesicles (Cyt ves), secretory vacuoles (SV), Golgi vesicles (GV), Golgi cisternae (GC), multivesicular bodies (MVB), lysosomes (LYS), and the cell membrane basolateral domain (BLD). About 58% of non-internalized radioactivity was removed by hydrolysis. During the 2nd incubation at 37°C the concentration of label increased in BLD and LYS, decreased in SV and MVB, and fluctuated in GC, GV and AD. The distribution of grains observed at 15 min, as compared using the χ-square test, was highly significantly different from that expected without recycling. The results show that cell membrane glycoproteins internalized at the cell apex recycle back to the membrane apical domain and are consistent with the involvement of GC and SV in the recycling pathway. Membrane shuttle between the apical and basolateral domains of the cell membrane is also suggested by these observations.     

Immunoelectron microscopic studies of the intracellular transport of the membrane glycoprotein (G) of vesicular stomatitis virus in infected Chinese hamster ovary cells

An immunoelectron microscopic study was undertaken to survey the intracellular pathway taken by the integral membrane protein (G-protein) of vesicular stomatitis virus from its site of synthesis in the rough endoplasmic reticulum to the plasma membrane of virus-infected Chinese hamster ovary cells. Intracellular transport of the G-protein was synchronized by using a temperature-sensitive mutant of the virus (0-45). At the nonpermissive temperature (39.8 degrees C), the G-protein is synthesized in the cell infected with 0-45, but does not leave the rough endoplasmic reticulum. Upon shifting the temperature to 32 degrees C, the G-protein moves by stages to the plasma membrane. Ultrathin frozen sections of 0-45-infected cells were prepared and indirectly immunolabeled for the G-protein at different times after the temperature shift. By 3 min, the G-protein was seen at high density in saccules at one face of the Golgi apparatus. No large accumulation of G-protein-containing vesicles were observed near this entry face, but a few 50-70-mm electron-dense vesicular structures labeled for G-protein were observed that might be transfer vesicles between the rough endoplasmic reticulum and the Golgi complex. At blebbed sites on the nuclear envelope at these early times there was a suggestion that the G-protein was concentrated, these sites perhaps serving as some of the transitional elements for subsequent transfer of the G-protein from the rough endoplasmic reticulum to the Golgi complex. By 3 min after its initial asymmetric entry into the Golgi complex, the G-protein was uniformly distributed throughout all the saccules of the complex. At later times, after the G-protein left the Golgi complex and was on its way to the plasma membrane, a new class of G-protein-containing vesicles of approximately 200-nm diameter was observed that are probably involved in this stage of the transport process. These data are discussed, and the further prospects of this experimental approach are assessed.