Study on membrane recycling in the rat visceral yolk-sac endoderm using concanavalin-A conjugates

Summary The internalization and intracellular movements of apical-cell-membrane material were investigated in the endodermal cells of cultured visceral yolk-sacs of rats (whole-embryo culture; explanted at 10.5 days of gestation and cultured for 24h) using horseradish peroxidase- and ferritin-labelled concanavalin A (Con-A HRP, Con-A Fer). When visceral yolk-sac endoderm was exposed to Con-A HRP or Con-A Fer for 5 min at 4°C, the apical cell membranes containing a well-developed fuzzy coat were heavily labelled, whereas apical vacuoles, lysosomes and apical canaliculi were not. Incubation of Con-A-labelled endoderm for 5 60 min at 20° and 37°C in Con-A-free serum resulted in a temperature-dependent internalization of membranebound lectin into coated vesicles, apical vacuoles and lysosomes, and the apical cell membranes were cleared of the heavy labelling. With increasing incubation time, the number of labelled vacuolar structures and the intensity of their labelling decreased gradually, whereas the number of labelled apical canaliculi increased. Thus, after 30 and 60 min at 37°C, most of the apical canaliculi contained high concentrations of the markers. It was possible to observe labelled apical canaliculi that were in continuity with labelled apical vacuoles and lysosomes as well as with the apical cell membrane. These findings in rat endodermal cells indicate that constitutents of the apical cell membrane are internalized in apical vacuoles and lysosomes, and are then brought back to the apical cell membrane by the apical canaliculi, which concentrate and store this membrane material.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

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.     

Study on membrane recycling in the rat visceral yolk-sac endoderm using concanavalin-A conjugates

The internalization and intracellular movements of apical-cell-membrane material were investigated in the endodermal cells of cultured visceral yolk-sacs of rats (whole-embryo culture; explanted at 10.5 days of gestation and cultured for 24 h) using horseradish peroxidase- and ferritin-labelled concanavalin A (Con-A HRP, Con-A Fer). When visceral yolk-sac endoderm was exposed to Con-A HRP or Con-A Fer for 5 min at 4 degrees C, the apical cell membranes containing a well-developed fuzzy coat were heavily labelled, whereas apical vacuoles, lysosomes and apical canaliculi were not. Incubation of Con-A-labelled endoderm for 5-60 min at 20 degrees and 37 degrees C in Con-A-free serum resulted in a temperature-dependent internalization of membrane-bound lectin into coated vesicles, apical vacuoles and lysosomes, and the apical cell membranes were cleared of the heavy labelling. With increasing incubation time, the number of labelled vacuolar structures and the intensity of their labelling decreased gradually, whereas the number of labelled apical canaliculi increased. Thus, after 30 and 60 min at 37 degrees C, most of the apical canaliculi contained high concentrations of the markers. It was possible to observe labelled apical canaliculi that were in continuity with labelled apical vacuoles and lysosomes as well as with the apical cell membrane. These findings in rat endodermal cells indicate that constituents of the apical cell membrane are internalized in apical vacuoles and lysosomes, and are then brought back to the apical cell membrane by the apical canaliculi, which concentrate and store this membrane material.     

Effects of leupeptin on endocytosis and membrane recycling in rat visceral yolk-sac endoderm

The effect of exposure to leupeptin (25 micrograms/ml for 24 h) on the endocytotic activity and the membrane flow of apical cell membranes was studied in endodermal cells of cultured rat visceral yolk sacs by applying a double-labelling method using concanavalin-A ferritin (Con-A Fer) and horseradish peroxidase (HRP). Control and leupeptin-treated yolk sacs were labelled with Con-A Fer at 4 degrees C and then incubated with HRP for 5, 15 or 60 min at 37 degrees C. In controls, HRP reaction product was detected after 5 min in many of the apical vacuoles as well as a few lysosomes; after 15 min, reaction product was observed in all apical vacuoles and in lysosomes of various sizes. These HRP-positive structures usually contained a variable amount of membrane-bound Fer. After 60 min, all apical vacuoles and almost all lysosomes exhibited HRP reactions, but only some of these structures contained Fer particles. At this time, many apical canaliculi (which are involved in membrane recycling) exhibited positive HRP reactions and sometimes also contained Fer particles. In leupeptin-treated cells, HRP reaction product and variable amounts of membrane-bound Fer particles were found in apical vacuoles after 5 min; after 15 min, both labels were also observed in some small lysosomes, and after 60 min, they were found in all apical vacuoles as well as some small and middle-sized lysosomes. Significantly fewer labelled apical vacuoles, lysosomes and apical canaliculi were present after leupeptin treatment than in controls at corresponding times. At all times examined, the giant lysosomes found in leupeptin-treated cells did not exhibit any labeling.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of leupeptin on endocytosis and membrane recycling in rat visceral yolk-sac endoderm

Summary The effect of exposure to leupeptin (25 g/ml for 24 h) on the endocytotic activity and the membrane flow of apical cell membranes was studied in endodermal cells of cultured rat visceral yolk sacs by applying a doublelabelling method using concanavalin-A ferritin (Con-A Fer) and horseradish peroxidase (HRP). Control and leupeptintreated yolk sacs were labelled with Con-A Fer at 4°C and then incubated with HRP for 5, 15 or 60 min at 37°C. In controls, HRP reaction product was detected after 5 min in many of the apical vacuoles as well as a few lysosomes; after 15 min, reaction product was observed in all apical vacuoles and in lysosomes of various sizes. These HRP-positive structures usually contained a variable amount of membrane-bound Fer. After 60 min, all apical vacuoles and almost all lysosomes exhibited HRP reactions, but only some of these structures contained Fer particles. At this time, many apical canaliculi (which are involved in membrane recycling) exhibited positive HRP reactions and sometimes also contained Fer particles. In leupeptin-treated cells, HRP reaction product and variable amounts of membrane-bound Fer particles were found in apical vacuoles after 5 min; after 15 min, both labels were also observed in some small lysosomes, and after 60 min, they were found in all apical vacuoles as well as some small and middle-sized lysosomes. Significantly fewer labelled apical vacuoles, lysosomes and apical canaliculi were present after leupeptin treatment than in controls at corresponding times. At all times examined, the giant lysosomes found in leupeptintreated cells did not exhibit any labelling. These findings indicate that, after leupeptin treatment, both endocytotic activity and membrane recycling decrease, and that fusions of the apical vacuolar system with giant lysosomes are retarded or inhibited.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Intracellular transport of horseradish peroxidase in the absorptive cells of goldfish hindgut in vitro,with special reference to the cytoplasmic tubules

Summary This study was undertaken to determine whether the numerous cytoplasmic tubules (CT) in the apical cytoplasm of goldfish hindgut absorptive cells are directly involved in the endocytotic transport of macromolecules into the cells, or whether they are derived from the intracellular membrane components. The absorptive cells were exposed to horseradish peroxidase (HRP)-containing medium in organ culture and subsequently fixed and prepared for electron microscopy. Analysis revealed that 5 sec after exposure, many vesicular structures, including coated vesicles, were labelled with reaction product whereas almost all CT were negative. After a 1-min exposure, reaction product was detected in about 11 % of the CT, and thereafter, the percentage increased to about 95% after 15 min exposure. As labelled CT increased in number, the number of densely labelled vacuoles with attached CT also increased. CT connected to vacuoles with a peripheral margin of dense reaction product were always HRP-positive, whereas those connected to vacuoles which were not distinctly labelled were themselves also devoid of HRP reaction product. This indicated that the labelling of CT was closely associated with the labelling of the inner surface of the vacuolar membrane. These results indicate that CT are probably formed by a budding off from these vacuoles, rather than being directly involved in endocytosis.     

Endocytosis at 0° C, 5° C,and 10° C in trophotaenial absorptive cells of goodeid embryos (Teleostei)

Summary The absorptive epithelium of the trophotaeniae of goodeid embryos is involved in the micropinocytotic uptake of protein macromolecules from the ovarian embryotrophe. Incubations of viable Xenoophorus captivus embryos in vitro with horseradish peroxidase (HRP) and/or cationized ferritin (CF) allows the tracing of the fluid-phase and receptor-mediated pathways, respectively. Effects of lowered temperature on both these endocytotic mechanisms have been investigated. At 10° C, trophotaenial absorptive cells (TACs) have a strong capacity to ingest marker proteins from double tracer media. Surface-bound ligands (CF) and solutes (HRP), taken up in primary pinocytic vesicles, are rapidly channelled to the endosomal compartment. Part of the ingested CF is segregated into dense apical tubules and small vesicles indicating that membrane recycling and transcytosis continue at 10° C. Adsorptive endocytosis of CF at 5° C proceeds at a decreased rate. After incubation periods of 30 min and 1 h, tracer molecules can be found in vesicular, tubular and vacuolar compartments of the apical endocytic zone. At 0° C, no uptake of ligand worth mentioning could be ascertained. Fluid-phase endocytosis, on the other hand, is observable at this temperature. Enzyme reaction product accumulates in flattened vacuoles rather than typical voluminous endosomes. After prolonged exposure to HRP, the epithelial junctional complex becomes leaky and the marker protein penetrates the intercellular space and the lateral lamellar membrane invaginations of TACs.Supported by the Deutsche Forschungsgemeinschaft     

Protein transport to the epididymal lumen

Summary Experiments were performed to clarify the debate over the entry of circulating proteins into the epididymal lumen by use of the marker horseradish peroxidase (HRP). Epididymal tubules from the caput epididymidis of the rat were immersed in medium TC 199 containing HRP (3.5 mg/ ml) for 5 min to 3 h at 33° C. Sections were examined for the presence of tracer within the epithelial cells by electron microscopy. From 5 min to 3 h, vesicles containing peroxidase reaction products were found throughout the cytoplasm of the principal cells. Vesicles occurred close to both the basal and apical membranes, and many were found opening into the interstitial space and lumen, depending on the length of incubation. By 5 min labelled vesicles were infrequently found in the apical part of the cells. Reaction product was observed in the epididymal lumen adhering to the microvilli from 30 min of incubation onwards. At all periods of incubation peroxidase was present at the base of the epithelium and between the cells, but it was never found within the tight junctional complexes, and no reaction deposits were found within epithelial cells of tubules incubated in the absence of peroxidase. It is concluded that large molecules leaving the capillaries may enter the epididymal lumen in the caput by means of fluid-phase endocytosis.     

Morphological studies of the goldfish hindgut mucosa in organ culture

Summary The morphology of the absorptive cells of the goldfish hindgut mucosa, and their capability for horseradish peroxidase (HRP) uptake, were investigated by electron microscopy after a 24-h organ culture. The columnar appearance and the fine structure of the absorptive cells were well preserved for 24 h at room temperature and 37° C with 5% CO₂ in air, in all the media used in this study. Mitoses were frequently observed in the epithelium at the bottom of cultured mucosal folds, and re-epithelization was also observed in many explants.Some structural changes were, however, noted in the cultured absorptive cells, as compared with the non-cultured absorptive cells; the deep invaginations of the surface membrane between the microvilli decreased in number; supranuclear giant vacuoles were reduced in size or almost disappeared; the distributional pattern of mitochondria in the absorptive cells was altered.The HRP uptake experiments showed that the absorptive cells cultured for 24 h could still take up HRP by endocytosis and transport it, indicating that the absorptive cells maintained their capability of macromolecule uptake and transport after 24 h of culture. In addition, HRP experiments, in which reaction product was detected within numerous cytoplasmic tubules (CT), various vacuoles and CT-vacuole complexes, suggested a close relationship between CT and vacuolar system in the apical cytoplasm during endocytotic events in the absorptive cells.     

Maternal-embryonic relationships in the goodeid teleost,Xenoophorus captivus

Summary The endodermal trophotaenial epithelium in goodeid embryos acts as a placental exchange site. Fine structural and cytochemical data indicate that the trophotaenial absorptive cells are endocytotically highly active. To test their micropinocytotic capacity and characterize the cellular mechanisms involved in membrane, solute and ligand movements, living embryos of Xenoophorus captivus were incubated in saline media containing horseradish peroxidase (HRP) and/or cationized ferritin (CF) in vitro, and the uptake of these tracer proteins examined by both time sequence analysis and pulse-chase procedures. In some embryos, the effects of prolonged exposure to CF injected into the ovarian cavity, was also investigated.Labelling of the free cell surface was detectable with CF only, but interiorization of both probes was quick from all incubation media. Adsorptive pinocytosis of CF and fluid-phase uptake of HRP sequentially labelled pinocytic vesicles, endosomes, and lysosome-like bodies. In addition, CF-molecules were sequestered within apical tubules and small vesicles. HRP was largely excluded from both organelles and ended up in the lysosomal compartment. For CF, two alternative pathways were indicated by the pulse-chase experiments; transcellular passage and regurgitation of tracer molecules to the apical cell surface. The latter procedure involves membrane and receptor recycling, in which apical tubules are thought to mediate.In double-tracer experiments, using an 81 excess of HRP, external labelling with CF was light or lacking after 1–3 min, and the initial uptake-phase produced pinocytic vesicles and endosomes that mainly contained HRP-reaction product. Prolonged incubation, however, resulted in densely CF-labelled plasmalemmal invaginations and pinocytic vesicles that predominantly carried ferritin granules. After 60 min, the vacuoles of the endosomal compartment contained either high concentrations of HRP-reaction product, both tracers side by side, or virtually exclusively CF.     

Uptake and fate of luminally administered horseradish peroxidase in resting and isoproterenol-stimulated rat parotid acinar cells

The formation and fate of apical endocytic vesicles in resting and isoproterenol-stimulated rat parotid acinar cells were studied using luminally administered horseradish peroxidase (HRP) to mark the vesicles. The tracer was taken up from the lumen by endocytosis in small, smooth-surfaces "c"- or ring-shaped vesicles. About 1 h after HRP administration the vesicles could be found adjacent to the Golgi apparatus. At later times HRP reaction product was localized in multivesicular bodies and lysosomes; in isoproterenol-stimulated cells it was also present in autophagic vacuoles. HRP reaction product was never localized in any structure associated with secretory granule formation. These results suggest that the apical endocytic vesicles play a role in membrane recovery, but that they are degraded and not reutilized directly in secretory granule formation. Additionally, it was found that when isoproterenol was injected before HRP administration, the apical junctional complexes became permeable to the tracer, allowing it to gain access to the lateral and basal intercellular spaces. This permeability may provide an additional route whereby substances in the extracellular fluid could reach the saliva.     

Transtubular transport of proteins in rabbit proximal tubules

The purpose of the present experiments was to study possible different pathways of intracellular transport of proteins after luminal and basolateral uptake in isolated rabbit proximal tubules. Tubules were exposed to cationized ferritin (CF) in the perfusion fluid and horseradish peroxidase (HRP) in the bath simultaneously or to HRP in the bath alone for 30 min. The peritubular fluid (bath) and perfusion fluid were then exchanged and the tubules either fixed immediately or allowed to function during chase-periods for 10, 20, 30, or 60 min before fixation to follow the migration of the proteins through the cells. The proteins were to a large extent found separated in different vacuoles and lysosomes at all time periods studied, indicating separate pathways after uptake via the luminal and basolateral membranes respectively. About 0.5% of the CF taken up by the cells was transported through the cells and became located in the intercellular spaces. HRP was transported from the peritubular fluid to the apical cytoplasm of the tubules indicated by a gradual accumulation of small HRP-containing vesicles, first in the basal part of the cells and then in the apical cytoplasm. In tubules perfused with both CF and HRP in the perfusate, the CF and HRP were found together in apical vacuoles and lysosomes. After perfusion with HRP alone, this tracer was found in similar large vacuoles and lysosomes in the apical cytoplasm, in contrast to the small HRP-filled vacuoles seen after uptake from the bath.     

Protamine induced intracellular uptake of horseradish peroxidase and vacuolation in mouse skeletal muscle in vitro

Summary The uptake in vitro of horseradish peroxidase (HRP) in mouse skeletal muscle was examined by electron microscopy and chemical determination.In muscles exposed to an HRP solution for 60 min at +37°C, HRP infiltrated the basal lamina of muscle fibres and caused an intense labelling of their sarcolemma. In addition HRP was found within the transverse tubules. Exposure to HRP for 30 min at +37°C followed by HRP together with a polycationic protein (protamine) for 30 min at +37°C caused an intracellular vesicular uptake of HRP. Intracellular HRP was found in numerous vesicles, membrane limited bodies and vacuoles. Protamine also induced focal autophagic vacuolation with progressive muscle fibre degeneration. An intracellular HRP uptake or muscle cell vacuolation could not be detected in the absence of protamine or when the incubation temperature was + 4°C. Chemical determination of HRP uptake was in general agreement with the morphological results. The uptake of HRP in the presence of protamine was stimulated at +31°C and blocked at +4°C.The results suggest that in skeletal muscle in vitro intracellular uptake of macromolecules occurs by endocytosis.     

An electron microscopic study of absorption of peroxidase-conjugated immunoglobulin G by guinea pig visceral yolk sac in vitro.

In the guinea pig and some other animals, passive immunity is conferred on the developing fetus by passage of immunoglobulin from mother to fetus across the yolk sac. In order to examine the cytological pathway involved in immunoglobulin transport, guinea pig visceral yolk sacs from late in gestation were exposed in vitro to peroxidase-conjugated guinea pig immunoglobulin G (IgG-HRP). Tissue was then fixed, incubated to show the site of localization of peroxidase reaction product and prepared for electron microscopy. The results suggested that the first step in the uptake of IgG-HRP by yolk sac is attachment of the protein to the surface coats of endocytic invaginations at the apical surfaces of the endodermal cells. The endocytic vesicles then appear to pinch off from the surface and move deeper into the cytoplasm. Some of the small endocytic vesicles fuse with large apical vacuoles, which often contain large amounts of reaction product. Other small endocytic vesicles pinch off from the surface, move deeper into the cytoplasm and fuse with the lateral plasmalemma; their protein content is emptied into the intercellular space by exocytosis. From the intercellular spaces the protein presumably diffuses across the basement membrane and connective tissue spaces and enters the vitelline capillary bed. It is postulated that the latter cellular pathway, involving small vesicles and the intercellular spaces, is utilized by those immunoglobulins which are transferred intact across the yolk sac endoderm.     

Membrane-bound and fluid-phase macromolecules enter separate prelysosomal compartments in absorptive cells of suckling rat ileum

The absorptive cell of the suckling rat ileum is specialized for the uptake and digestion of milk macromolecules from the intestinal lumen. The apical cytoplasm contains an extensive tubulocisternal system, a variety of vesicles and multivesicular bodies (MVB), and a giant phagolysosomal vacuole where digestion is completed. To determine if sorting of membrane-bound and fluid-phase macromolecules occurs in this elaborate endocytic system, we infused adsorptive and soluble tracers into ligated intestinal loops in vivo and examined their fates. Lysosomal compartments were identified by acid phosphatase histochemistry. Native ferritin and two ferritin-lectin conjugates that do not bind to ileal membranes (Con A, UEAI) served as soluble tracers. Horseradish peroxidase binds to ileal membranes and thus was not useful as a fluid-phase tracer in this system. Cationized ferritin and a lectin that binds to terminal B-D-galactosyl sites on ileal membranes (Ricinus communis agglutinin [RCAI]-ferritin) were used as tracer ligands. All tracers entered the wide apical invaginations of the luminal cell surface and were transported intracellularly. Membrane-bound tracers were found in coated pits and vesicles, and throughout the tubulocisternal system (where cationized ferritin is released from the membrane) and later, in large clear vesicles and MVB. In contrast, fluid-phase tracers appeared within 5 min in vesicles of various sizes and were not transported through the tubulocisternae, rather, they were concentrated in a separate population of vesicles of increasing size that contained amorphous dense material. Large clear vesicles, large dense vesicles, and MVB eventually fused with the giant supranuclear vacuole. Acid phosphatase activity was present in MVB and in the giant vacuole but was not present in most large vesicles or in the tubulocisternae. These results demonstrate that membrane-bound and soluble protein are transported to a common lysosomal destination via separate intracellular routes involving several distinct prelysosomal compartments.     

Absorption of intraarticularly injected horseradish peroxidase in synoviocytes of rat synovial membrane: an ultrastructural-cytochemical study

The ability of type A and type S synoviocytes to absorb horseradish peroxidase (HRP) and the intracellular fate of this tracer were studied by electron microscopic cytochemistry. Different concentrations of HRP (0.1-5 mg/ml) were injected into the left knee joint of rats and at intervals ranging from 1 min to 24 hr after injection the synovial membrane was fixed and incubated for HRP. Type A synoviocytes showed a striking ability to absorb HRP at low concentrations. At 1 and 5 min after injection reaction product was localized in coated pits and coated vesicles (110 nm) as well as in smooth-walled vesicles, vacuoles, and tubules. At 15 min to 4 hr postinjection the lysosomal system became increasingly loaded with reaction product. At 24 hr after injection reaction product had disappeared. At higher concentrations of HRP similar observations were made in the A cells, but reaction product was still apparent in lysosomes at 24 hr postinjection. With respect to type S synoviocytes no reaction product was detected within these cells at any time interval after injection of low concentrations of HRP. However, at 5 min after injection of higher concentrations of HRP reaction product was localized in smooth vesicles and vacuoles mainly restricted to the large cytoplasmic processes facing the joint cavity. At 30 min to 4 hr postinjection the lysosomal system became progressively more loaded with HRP reaction product. At 24 hr after injection reaction product still remained in the lysosomal system. The present findings that type A and type S synoviocytes showed major differences with respect to endocytic capacity and cellular structures involved in absorption of HRP support the interpretation that the A and S cells represent two distinct types of cells and further suggest that endocytosis in these two types of cells serve different functions.     

Redistribution of cell surface transferrin receptors prior to their concentration in coated pits as revealed by immunoferritin labels

Summary Immunocytochemistry has been used to study distribution of cell surface transferrin receptors in erythroid, leukemic (K562) cells. The cells were fixed and labelled with monoclonal (OKT-9) anti-transferrin receptor antibodies; the antibody-labelled receptors were then detected by either immunofluoresceinor immunoferritin-antimouse-antibody conjugates. Typically, the immunoferritin labels were distributed diffusely at the non-coated regions of the cell surface as well as concentrated in the clathrincoated pits. To examine further this pattern of distribution, cells were labelled at 0° C and then warmed to 37° C for zero to 30 min prior to fixation. The majority of the immunoferritin labels were initially dispersed in small groups at the non-coated regions of the cell surface (mean = 6 immunoferritin labels/cluster), but larger groups were common subsequent to incubation at 37° C (mean = 13 immunoferritin labels/cluster). However, the size of immunoferritin labels in the coated pits was unchanged (mean = 12 immunoferritin labels/pit). Immunoferritin labels were typical in coated and uncoated vesicles l min after warming to 37° C, but common in endosomes, multivesicular bodies and lysosomes by 30 min. It appears that single cell-surface receptors form large aggregates prior to their concentration in coated pits. Coated vesicles, uncoated vesicles, and endosomal vacuoles may together form the non-lysosomal compartment where the internalized receptors might be dissociated from the ligands (antibodies).     

Endocytotic pathways in the melanotroph of the rat pituitary

Summary The internalization of the extracellular markers horseradish peroxidase (HRP) and cationized ferritin (CF) by the melanotrophs of the intermediate lobe of the rat pituitary was studied during short-time incubation of mechanically dissociated cells or in cell culture after 5 days. After a 30 min exposure, the tracers were found in electron-lucent granules or vacuoles of approximately the same size as the secretory granules, situated 200–500 nm from the cell membrane. In the cultured cells, which showed a higher rate of tracer uptake, internalization was followed for 1, 2 and 5 min after labelling and during 2 h of exposure. Initially, the label was seen only in coated pits and coated vesicles at the cell membrane. Larger vacuoles were first seen after 2–5 min of incubation. After 2 h of exposure the labelling pattern was distinctly different for the two tracers. CF was found in larger vacuoles of varying morphology, in dilatations at the base of cilia, within Golgi saccules and at the edge of the electron-dense core of forming secretory granules. HRP was found in an extensive array of tubulovesicular structures extending throughout the cytoplasm. The Golgi complex and forming granules were, however, not labelled with HRP. The study identifies part of the electron-lucent granules or vacuoles in the melanotroph as endosomes, and shows that the melanotrophs sort CF and HRP via diverting pathways after internalization, suggesting that granule membrane, and possibly its functional components, can be recycled in these cells.     

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.     

Colchicine-induced tubular, vesicular and cisternal organelle aggregates in absorptive cells of the small intestine of the rat. II.--Endocytosis studies

Administration of the antimicrotubular agent colchicine to adult rats (0.5 mg/100 g of body weight for 6 hr) induces formation of extended aggregates of tubular, vesicular, and cisternal organelles in the absorptive cells of the small intestine. The phosphatase reaction pattern (thiamine pyrophosphatase, acid phosphatase, acid trimetaphosphatase) suggests that the majority of them belongs to the lysosomal system (Ellinger and Pavelka, 1984). The present study extends these findings and examines the uptake and fate of intravenously injected horseradish peroxidase (HRP) at the basal and lateral cell surfaces and of intraluminally applied HRP at the apical cell surface. HRP, applied to control animals and animals pretreated with colchicine, was internalized at both apical and basolateral cell surfaces of the absorptive cells, and delivered into endosome-like vesicles, multivesiculated bodies (mvbs), dense bodies (dbs), and in several instances into Golgi cisternae. Following intraluminal application, evidence was obtained for the transport of HRP across the cell; in contrast, intravenously applied HRP was never detected at the apical cell surface. Colchicine pretreatment did not stop the uptake of HRP, which was rapidly sequestered to the clustered tubules, vesicles, and cisternae, as well as to the mvbs and dbs. After longer intervals, the portion of HRP-reactive tubules, vesicles, and cisternae within the clusters increased: 60 min after HRP-administration all of them contained HRP-activity. These results indicate that the colchicine-induced clustered organelles are recipients of endocytic materials internalized at the apical as well as at the basolateral cell surface.