Receptor-mediated endocytosis of transferrin and ferritin by guinea-pig reticulocytes. Uptake by a common endocytic pathway

Earlier studies have shown that transferrin binds to specific receptors on the reticulocyte surface, clusters in coated pits and is then internalized via endocytic vesicles. Guinea-pig reticulocytes also have specific receptors for ferritin. In this paper ferritin and transferrin endocytosis by guinea-pig reticulocytes was studied by electron microscopy using the natural electron density of ferritin and colloidal gold-transferrin (AuTf). At 4 degrees C both ligands bound to the cell surface. At 37 degrees C progressive uptake occurred by endocytosis. AuTf and ferritin clustered in the same coated pits and small intracellular vesicles. After 60 min incubations the ligands colocalized to large multivesicular endosomes (MVE), still membrane-bound. MVE subsequently fused with the plasma membrane and released AuTf, ferritin and inclusions by exocytosis. All endocytic structures labelled with AuTf contained ferritin, but 23 to 35% of ferritin-labelled endocytic structures contained no AuTf. These data suggest that ferritin and transferrin are internalized through the same pathway involving receptors, coated pits and vesicles, but that these proteins are recycled only partly in common.     

Molecules internalized by clathrin-independent endocytosis are delivered to endosomes containing transferrin receptors

We have previously demonstrated that the preendosomal compartment in addition to clathrin-coated vesicles, comprises distinct nonclathrin coated endocytic vesicles mediating clathrin-independent endocytosis (Hansen, S. H., K. Sandvig, and B. van Deurs. 1991. J. Cell Biol. 113:731-741). Using K+ depletion in HEp-2 cells to block clathrin- dependent but not clathrin-independent endocytosis, we have now traced the intracellular routing of these nonclathrin coated vesicles to see whether molecules internalized by clathrin-independent endocytosis are delivered to a unique compartment or whether they reach the same early and late endosomes as encountered by molecules internalized with high efficiency through clathrin-coated pits and vesicles. We find that Con A-gold internalized by clathrin-independent endocytosis is delivered to endosomes containing transferrin receptors. After incubation of K(+)- depleted cells with Con A-gold for 15 min, approximately 75% of Con A- gold in endosomes is colocalized with transferrin receptors. Endosomes containing only Con A-gold may be accounted for either by depletion of existing endosomes for transferrin receptors or by de novo generation of endosomes. Cationized gold and BSA-gold internalized in K(+)- depleted cells are also delivered to endosomes containing transferrin receptors. h-lamp-1-enriched compartments are only reached occasionally within 30 min in K(+)-depleted as well as in control cells. Thus, preendosomal vesicles generated by clathrin-independent endocytosis do not fuse to any marked degree with late endocytic compartments. These data show that in HEp-2 cells, molecules endocytosed without clathrin are delivered to the same endosomes as reached by transferrin receptors internalized through clathrin-coated pits.     

Endocytosis and intracellular processing of transferrin and colloidal gold-transferrin in rat reticulocytes: demonstration of a pathway for receptor shedding

Endocytosis and intracellular processing of transferrin (Tf) and Tf receptors were examined in rat reticulocytes. Subcellular fractionation revealed that Tf enters a non-lysosomal endocytic compartment with a density between those of plasma membrane and lysosomes. After 20 min of uptake at (37 degrees C) 35 to 40% of cell-associated Tf was contained in this intermediate-density compartment. To test the fidelity of colloidal gold-Tf (AuTf) as a probe for Tf processing, reticulocytes were fractionated after uptake of 131I-Tf and 125I-AuTf. The subcellular distributions of the two ligands were indistinguishable by this method, a result suggesting that AuTf is processed similarly to Tf. Electron microscopy revealed that AuTf entered multivesicular endosomes (MVEs) as well as various small vesicles and tubular structures. In addition MVE exocytosis was observed with discharge of inclusion vesicles and associated AuTf. AuTf was bound to the outside of these vesicles both before and after exocytosis. These data suggest that Tf receptors are shed from developing reticulocytes by incorporation into the limiting membrane of inclusion vesicles, followed by discharge of these vesicles by MVE exocytosis. As further evidence of this process, we isolated inclusion vesicles after their discharge and found them to contain Tf receptors. Moreover, the rate of Tf receptor shedding by inclusion vesicle discharge matches Tf receptor loss rates closely enough to suggest that this is the primary path of receptor loss during reticulocyte development.     

Transferrin and ferritin endocytosis and recycling in guinea-pig reticulocytes

Transferrin and ferritin endocytosis and exocytosis by guinea-pig reticulocytes were studied using incubation with pronase at 4 degrees C to distinguish internalized and membrane-bound protein. Internalization of both transferrin and ferritin occurred in a time- and temperature-dependent fashion. Transferrin endocytosis was more rapid than that of ferritin. Transferrin binding to receptors was not altered, but transferrin endocytosis was decreased in the presence of ferritin. Iron accumulation from transferrin was inhibited by ferritin to a greater extent than could be accounted for by the decreased rate of endocytosis. In pulse-chase experiments, almost all of the transferrin was released intact from reticulocytes, but only about 50% of the total internalized ferritin was released, of which 85% was intact. The endocytosis of transferrin by rabbit reticulocytes was 2- to 2.5-times faster than guinea-pig reticulocytes. These data suggest that ferritin and transferrin are internalized by receptor-mediated endocytosis, possibly involving the same coated pits and vesicles, but that the proteins are recycled only partly in common.     

Receptor-mediated endocytosis of transferrin by Sertoli cells of the rat

Binding of 125I-transferrin (125I-Tf) to the plasma membrane of Sertoli cells and its endocytosis were analyzed by means of light- and electron-microscope quantitative radioautography. Five minutes after 125I-Tf was injected into the interstitial space of the testis, a strong labeling of the basal aspect of the seminiferous epithelium was observed in light-microscope radioautographs. Injection of the same dose of 125I-Tf plus a 200-fold excess of cold transferrin resulted in a marked diminution of the radioautographic reaction, indicating that the initial strong labeling with radiolabeled transferrin was specific. These results were consistent with the localization of immunoreactive fluorescence of transferrin receptor at the base of the seminiferous epithelium. In electron-microscope radioautographs of tubules collected at 5 min after injection, the membrane of Sertoli cells facing the basement membrane was well labeled with 125I-Tf. At 15 and 30 min, the plasma membrane was less intensely labeled, but the silver grains were then seen overlying multivesicular bodies with an electron-lucent matrix, identified as endosomes. This population of endosomes was always seen at a short distance from the basal membrane of Sertoli cells. At 90 min, no more labeling of the plasma membrane, endosomes, or any other cytoplasmic component was observed. Isolated seminiferous tubules and Sertoli cells labeled with 125I-Tf at 4 degrees C were rinsed and reincubated in a label-free medium at 37 degrees C for various periods of time from 5 to 90 min. A radioactive protein precipitated by trichloroacetic acid, presumably intact transferrin, was released from the tubules into the incubating medium; when measured, it was found to increase rapidly from 5 to 45 min and stabilize thereafter. These results suggest that transferrin was internalized by receptor-mediated endocytosis, reached endosomes, and then was released to the extratubular space. When native ferritin (NF), a tracer for fluid-phase endocytosis, was infused within the lumen of seminiferous tubules and 125I-Tf was simultaneously injected into the interstitial space, both markers rapidly reached different populations of endosomes. Endosomes labeled with NF, scattered throughout the cytoplasm, evolved with time into dense multivesicular bodies and secondary lysosomes, whereas radiolabeled transferrin reached only the endosomes located in the basal cytoplasm of Sertoli cells. The latter thus appeared to be principally involved in the uptake and recycling of transferrin.     

Internalization and processing of transferrin and the transferrin receptor in human carcinoma A431 cells

The binding and subsequent intracellular processing of transferrin and transferrin receptors was studied in A431 cells using 125I-transferrin and a monoclonal antibody to the receptor (ATR) labeled with 125I and gold colloid. Using 125I-transferrin we have shown that, whereas at 37 degrees C uptake proceeded linearly for up to 60 min, most of the ligand that was bound was internalized and then rapidly returned to the incubation medium undegraded. At 37 degrees C, the intracellular half- life of the most rapidly recycled transferrin was 7.5 min. 125I-ATR displayed the same kinetics of uptake but following its internalization at 37 degrees C, it was partially degraded. At 22 degrees C and below, the intracellular degradation of 125I-ATR was selectively inhibited and as a result it accumulated intracellularly. Electron microscopy of conventional thin sections and of whole-cell mounts was used to follow the uptake and processing of transferrin receptors labeled with ATR- gold colloid complexes. Using a pulse-chase protocol, the intracellular pathway followed by internalized ATR gold-receptor complexes was outlined in detail. Within 5 min at 22 degrees C the internalized complexes were transferred from coated pits on the cell surface to a system of narrow, branching cisternae within the peripheral cytoplasm. By 15 min they reached larger, more dilated elements that, in thin section, appeared as irregular profiles containing small (30-50-nm diam) vesicles. By 30 min, the gold complexes were located predominantly within typical spherical multivesicular bodies lying in the peripheral cytoplasm, and by 40-60 min, they reached a system of cisternal and multivesicular body elements in the juxtanuclear area. At 22 degrees C, no other compartments became labeled but if they were warmed to 37 degrees C the gold complexes were transferred to lysosome- like elements. Extracting ATR-gold complexes with Triton X after a 30- min chase at 22 degrees C and purifying them on Sepharose-transferrin indicated that the internalized complexes remained bound to the transferrin receptor during their intracellular processing.     

Intracellular pathways of endocytosed transferrin and non-specific tracers in epithelial cells lining the rete testis of the rat

Summary The uptake and pathway of different markers and ligands for fluid-phase, adsorptive and receptor mediated endocytosis were analyzed in the epithelial cells lining the rete testis after their infusion into the lumen of these anastomotic channels. At 2 min after injection, diferric transferrin bound to colloidal gold was seen attached to the apical plasma membrane and to the membrane of endocytic coated and uncoated pits and vesicles. The injection of transferrin-gold in the presence of a 100-fold excess of unconjugated diferric transferrin revealed no binding or internalization of transferrin-gold. Similarly, apotransferrin-gold was neither bound to the apical plasma membrane nor internalized by these cells. These results thus indicate the presence of specific binding sites for diferric transferrin. At 5 min, internalized diferric transferrin-gold reached endosomes. At 15 and 30 min, the endosomes were still labeled but at these time intervals the transferrin-gold also appeared in tubular elements connected to or associated with these bodies or seen in close proximity to the apical plasma membrane. At 60 and 90 min, most of the transferrin-gold was no longer present in these organelles and was seen only exceptionally in secondary lysosomes. These results thus suggest that the tubular elements may be involved in the recycling of transferrin back to the lumen of the rete testis. The coinjection of transferrin-gold and the fluid-phase marker native ferritin revealed that both proteins were often internalized in the same endocytic pit and vesicle and shared the same endosome. However, unlike transferrin, native ferritin at the late time intervals appeared in dense multivesicular bodies and secondary lysosomes. When the adsorptive marker cationic ferritin and the fluid-phase marker albumin-gold were coinjected, again both proteins often shared the same endocytic pit and vesicle, endosome, pale and dense multivesicular body and secondary lysosomes. However, several endocytic vesicles labeled only with cationic ferritin appeared to bypass the endosomal and lysosomal compartments and to reach the lateral intercellular space and areas of the basement membrane. The rete epithelial cells, therefore, appear to be internalizing proteins and ligands by receptor-mediated and non-specific endocytosis which, after having shared the same endocytic vesicle and endosome, appear to be capable of being segregated and routed to different destinations.     

Hemopexin joins transferrin as representative members of a distinct class of receptor-mediated endocytic transport systems

Receptor-mediated transport of heme by hemopexin in vivo and in vitro results in catabolism of heme but not the protein, suggesting that intact apohemopexin recycles from cells. However, until now, the intracellular transport of hemopexin by receptor-mediated endocytosis remained to be established. Biochemical studies on cultured human HepG2 and mouse Hepa hepatoma cells demonstrate that hemopexin is transported to an intracellular location and, after endocytosis, is subsequently returned intact to the medium. During incubation at 37 degrees C, hemopexin accumulated intracellularly for ca. 15 min before reaching a plateau while surface binding was saturated by 5 min. No internalization of ligand took place during incubation at 4 degrees C. These and other data suggest that hemopexin receptors recycle, and furthermore, incubation with monensin significantly inhibits the amount of cell associated of heme-[125I]hemopexin during short-term incubation at 37 degrees C, consistent with a block in receptor recycling. Ammonium chloride and methylamine were less inhibitory. Electron microscopic autoradiography of heme-[125I]hemopexin showed the presence of hemopexin in vesicles of the classical pathway of endocytosis in human HepG2 hepatoma cells, confirming the internalization of hemopexin. Colloidal gold-conjugated hemopexin and electron microscopy showed that hemopexin bound to receptors at 4 degrees C is distributed initially over the entire cell surface, including microvilli and coated pits. After incubation at 37 degrees C, hemopexin-gold is located intracellularly in coated vesicles and then in small endosomes and multivesicular bodies. Colocalization of hemopexin and transferrin intracellularly was shown in two ways. Radioiodinated hemopexin was observed in the same subcellular compartment as horseradish peroxidase conjugates of transferrin using the diaminobenzidine-induced density shift assay. In addition, colloidal gold derivatives of heme-hemopexin and diferric transferrin were found together in coated pits, coated vesicles, endosomes and multivesicular bodies. Therefore, hemopexin and transferrin act by a similar receptor-mediated mechanism in which the transport protein recycles after endocytosis from the cell to undergo further rounds of intracellular transport.     

Morphological analysis of ligand uptake and processing: the role of multivesicular endosomes and CURL in receptor-ligand processing

The receptor-mediated endocytosis and intracellular processing of transferrin and mannose receptor ligands were investigated in bone marrow-derived macrophages, fibroblasts and reticulocytes. Mannosylated bovine serum albumin (BSA) conjugated to colloidal gold (Au-man-BSA) or colloidal gold-transferrin (AuTf) were used to trace ligand processing in these cells. These ligands appeared to be processed by mechanisms similar to those observed previously with other mannose receptor and galactose receptor ligand probes. After uptake via coated pits and coated vesicles, Au-man-BSA appeared in small uncoated vesicles and tubular structures and was transferred to large, sometimes multivesicular endosomes (MVEs), which sometimes had arm-like protrusions reminiscent of CURL (compartment of uncoupling of receptor and ligand) [10, 11]. Initially these structures became increasingly multivesicular, but during longer incubations the inclusion vesicles appeared to disintegrate to leave a denser, amorphous lumen. Inclusion vesicle disintegration may result from the introduction of lysosomal enzymes into these structures. These results suggest a model for differential receptor-ligand and ligand-ligand sorting. As suggested [10, 11] membrane constituents may be recycled to the plasma membrane from the arms of CURL. Receptor-bound ligands, such as transferrin, would also recycle. The luminal contents, including dissociated ligands, other soluble proteins and inclusion vesicles (containing some membrane proteins), would target to lysosomes. This would result in the lysosomal degradation of any membrane proteins that were incorporated in the inclusion vesicle membranes.     

Receptor-mediated endocytosis: the intracellular journey of transferrin and its receptor

A variety of ligands and macromolecules enter cells by receptor-mediated endocytosis. Ligands bind to their receptors on the cell surface and ligand-receptor complexes are localized in specialized regions of the plasma membrane called coated pits. Coated pits invaginate and give rise to intracellular coated vesicles containing ligand-receptor complexes which are thus internalized. Transferrin, a major serum glycoprotein which transports iron into cells, enters cells by this pathway. It binds to its receptor on the cell surface, transferrin-receptor complexes cluster in coated pits and are internalized in coated vesicles. Coated vesicles then lose their clathrin coat and fuse with endosomes, an organelle with an internal pH of about 5-5.5. Most ligands dissociate from their receptors in endosomes and they finally end up in lysosomes where they are degraded, while their receptors remain bound to membrane structures and recycle to the cell surface. Transferrin has a different fate: in endosomes iron dissociates from transferrin but apotransferrin remains bound to its receptor because of its high affinity for the receptor at acid pH. Apotransferrin thus recycles back to the plasma membrane still bound to its receptor. When the ligand-receptor complex reaches the plasma membrane or a compartment at neutral pH, apotransferrin dissociates from its receptor with a half-life of 18 s because of its low affinity for its receptor at neutral pH. The receptor is then ready for a new cycle of internalization, while apotransferrin enters the circulation, reloads iron in the appropriate organs and is ready for a new cycle of iron transport.     

An electron-microscope autoradiographic study of transferrin endocytosis by immature erythroid cells

The receptor-mediated endocytosis of 125I-transferrin by immature erythroid cells was studied using the technique of quantitative electron microscope autoradiography. Morphometric analysis of the grain distribution in erythroid cells from the foetal rat liver revealed that the 125I-transferrin radioactivity was localized mainly to intracellular vesicles (61%) and the cell membrane (25%) after 20 min incubation at 37 degrees C. No activity was found associated with the nucleus or mitochondria and only a small amount with the cytosol (13%). In erythroid cells which possessed a prominent Golgi complex, most of the autoradiographic grains were associated with vesicles located in this region, giving rise to a polar distribution of the 125I-transferrin. Uptake of transferrin was found to be maximal at the basophilic normoblast stage of development and then declined progressively during maturation to the reticulocyte. The kinetics of endocytosis of 125I-transferrin by rabbit reticulocytes was also studied by electron microscope autoradiography. Up to 30% of the cell-bound transferrin was internalized almost immediately upon incubation at 37 degrees C. After 30 sec incubation, 42% of the cell-bound 125I-transferrin was estimated to be internal and this rose to almost 70% at steady state between the binding and release of transferrin after 12 min incubation.     

Rapid endocytosis of the transferrin receptor in the absence of bound transferrin

The rate of endocytosis of transferrin receptors, occupied or unoccupied with transferrin, was measured on the cell line K562. At 37 degrees C, receptors, radioiodinated on the cell surface at 4 degrees C, were internalized equally rapidly in the presence or absence of transferrin. In both cases, 50% of the labeled receptors became resistant to externally added trypsin in 5 min. An antitransferrin antibody was used to show directly that the receptors had entered the cells without bound transferrin. The distribution of the receptors on the cell surface was revealed by antibody and protein A-gold staining after prolonged incubation in the presence or absence of transferrin. The receptors were concentrated in coated pits under both conditions. The data suggest that endocytosis of transferrin receptors is not "triggered" by ligand binding and raise the possibility that ligand-induced down-regulation of surface receptors may not occur by this mechanism. Instead receptors may be recognized as being ligand-occupied, not at the cell surface, but at some other site in the recycling pathway such as the endosome.     

Release of iron from endosomes is an early step in the transferrin cycle

Transferrin bound to K 562 cells at 4 degrees C was internalized quickly on temperature shift to 37 degrees C. Endosomes were isolated according to two different procedures. The endosome fraction was shown to be heterogeneous and consisted of two vesicle populations, differing in density properties and iron content. Iron was partially released from endosomes to the supernatant after 3 and 5 min endocytosis. Isolated endosomes, still capable of internal acidification, did not release iron on incubation with ATP. However, endosomes did release iron on incubation with the iron chelator pyridoxal-isonicotinoyl hydrazone. Gel-filtration of solubilized endosomes demonstrated the presence of the transferrin-transferrin receptor complexes, free transferrin and free low molecular weight iron.     

Surface interactions and intracellular localization of cationic ferritin: similarity to 125I-insulin in 3T3-L1 adipocytes

We previously reported that in 3T3-L1 adipocytes 125I-insulin associates preferentially with microvilli and coated pits at low temperatures and early times of incubation. At higher temperatures it is internalized through a series of membrane limited intracellular compartments. In the present study, we used a high resolution probe, cationic ferritin (CF), to track adsorptive endocytosis in the 3T3-L1 adipocyte. We find that CF initially associates with coated pits at 2 min of incubation at 37 degrees C. With further incubation at 37 degrees C CF is internalized and after 2 to 10 min of incubation is predominantly localized to coated and non-coated clear vesicles. Approximately 50% of the apparent coated vesicles seen near the plasma membrane on single thin sections are shown by serial sectioning to be true vesicles (i.e., without a surface connection). At later time points CF is localized predominantly to lysosomal structures and, to a much smaller extent, Golgi-related structures. The remarkable similarity between 125I-insulin and CF with respect to post-binding processing suggests that while the membrane receptor confers the initial specificity, post-binding events are common for different types of ligands after they bind to cell surfaces and are subject to adsorptive endocytosis.     

The kinetics of transferrin endocytosis and iron uptake from transferrin in rabbit reticulocytes

The endocytosis of diferric transferrin and accumulation of its iron by freshly isolated rabbit reticulocytes was studied using 59Fe-125I-transferrin. Internalized transferrin was distinguished from surface-bound transferrin by its resistance to release during treatment with Pronase at 4 degrees C. Endocytosis of diferric transferrin occurs at the same rate as exocytosis of apotransferrin, the rate constants being 0.08 min-1 at 22 degrees C, 0.19 min-1 at 30 degrees C, and 0.45 min-1 at 37 degrees C. At 37 degrees C, the maximum rate of transferrin endocytosis by reticulocytes is approximately 500 molecules/cell/s. The recycling time for transferrin bound to its receptor is about 3 min at this temperature. Neither transferrin nor its receptor is degraded during the intracellular passage. When a steady state has been reached between endocytosis and exocytosis of the ligand, about 90% of the total cell-bound transferrin is internal. Endocytosis of transferrin was found to be negligible below 10 degrees C. From 10 to 39 degrees C, the effect of temperature on the rate of endocytosis is biphasic, the rate increasing sharply above 26 degrees C. Over the temperature range 12-26 degrees C, the apparent activation energy for transferrin endocytosis is 33.0 +/- 2.7 kcal/mol, whereas from 26-39 degrees C the activation energy is considerably lower, at 12.3 +/- 1.6 kcal/mol. Reticulocytes accumulate iron atoms from diferric transferrin at twice the rate at which transferrin molecules are internalized, implying that iron enters the cell while still bound to transferrin. The activation energies for iron accumulation from transferrin are similar to those of endocytosis of transferrin. This study provides further evidence that transferrin-iron enters the cell by receptor-mediated endocytosis and that iron release occurs within the cell.     

Inhibition of coated pit formation in Hep2 cells blocks the cytotoxicity of diphtheria toxin but not that of ricin toxin

It has been recently shown (Larkin, J. M., M. S. Brown, J. L. Goldstein, and R. G. W. Anderson, 1983, Cell, 33:273-285) that after a hypotonic shock followed by incubation in a K+-free medium, human fibroblasts arrest their coated pit formation and therefore arrest receptor-mediated endocytosis of low density lipoprotein. We have used this technique to study the endocytosis of transferrin, diphtheria toxin, and ricin toxin by three cell lines (Vero, Wi38/SV40, and Hep2 cells). Only Hep2 cells totally arrested internalization of [125I]transferrin, a ligand transported by coated pits and coated vesicles, after intracellular K+ depletion. Immunofluorescence studies using anti-clathrin antibodies showed that clathrin associated with the plasma membrane disappeared in Hep2 cells when the level of intracellular K+ was low. In the absence of functional coated pits, diphtheria toxin was unable to intoxicate Hep2 cells but the activity of ricin toxin was unaffected by this treatment. By measuring the rate of internalization of [125I]ricin toxin by Hep2 cells, with and without functional coated pits, we have shown that this labeled ligand was transported in both cases inside the cells. Hep2 cells with active coated pits internalized twice as much [125I]ricin toxin as Hep2 cells without coated pits. Entry of ricin toxin inside the cells was a slow process (8% of the bound toxin per 10 min at 37 degrees C) when compared to transferrin internalization (50% of the bound transferrin per 10 min at 37 degrees C). Using the indirect immunofluorescence technique on permeabilized cells, we have shown that Hep2 cells depleted in intracellular K+ accumulated ricin toxin in compartments that were predominantly localized around the cell nucleus. Our study indicates that in addition to the pathway of coated pits and coated vesicles used by diphtheria toxin and transferrin, another system of endocytosis for receptor-bound molecules takes place at the level of the cell membrane and is used by ricin toxin to enter the cytosol.     

Lipid recycling between the plasma membrane and intracellular compartments: transport and metabolism of fluorescent sphingomyelin analogues in cultured fibroblasts

We examined the metabolism and intracellular transport of the D-erythro and L-threo stereoisomers of a fluorescent analogue of sphingomyelin, N-(N-[6-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino] caproyl])-sphingosylphosphorylcholine (C6-NBD-SM), in Chinese hamster ovary (CHO-K1) fibroblast monolayers. C6-NBD-SM was integrated into the plasma membrane bilayer by transfer of C6-NBD-SM monomers from liposomes to cells at 7 degrees C. The cells were washed, and within 10-15 min of being warmed to 37 degrees C, C6-NBD-SM was internalized from the plasma membrane to a perinuclear location that colocalized with the centriole and was distinct from the lysosomes and the Golgi apparatus. This perinuclear region was also labeled by internalized rhodamine-conjugated transferrin. C6-NBD-SM endocytosis was not inhibited when the microtubules were disrupted with nocodazole; rather, the fluorescent lipid was distributed in vesicles throughout the cell periphery instead of being internalized to the perinuclear region of the cell. The metabolism of C6-NBD-SM to other fluorescent sphingolipids at 37 degrees C and its effect on C6-NBD-SM transport was also examined. To study plasma membrane lipid recycling, C6-NBD-SM was first inserted into the plasma membrane of CHO-K1 cells and then allowed to be internalized by the cells at 37 degrees C. Any C6-NBD-SM remaining at the plasma membrane was then removed by incubation with nonfluorescent liposomes at 7 degrees C, leaving cells containing only internalized fluorescent lipid. The return of C6-NBD-SM to the plasma membrane from intracellular compartments upon further 37 degrees C incubation was then observed. The half-time for a complete round C6-NBD-SM recycling between the plasma membrane and intracellular compartments was approximately 40 min. Pretreatment of cells with either monensin or nocodazole did not inhibit C6-NBD-SM recycling.     

Azithromycin,a lysosomotropic antibiotic,has distinct effects on fluid-phase and receptor-mediated endocytosis,but does not impair phagocytosis in J774 macrophages

Pretreatment of J774 mouse macrophages by the dicationic macrolide antibiotic, azithromycin (AZ), selectively inhibited fluid-phase endocytosis of horseradish peroxidase and lucifer yellow, but not phagocytosis of latex beads. AZ delayed sequestration of receptor-bound transferrin and peroxidase-anti-peroxidase immune complexes into cell-surface endocytic pits and vesicles, but did not slow down the subsequent rate of receptor-mediated endocytosis. AZ down-regulated cell surface transferrin receptors, but not Fc gamma receptors, by causing a major delay in the accessibility of internalized transferrin receptors to the recycling route, without slowing down subsequent efflux, resulting in redistribution of the surface pool to an intracellular pool. Acidotropic accumulation of AZ was associated with an extensive vacuolation of late endosomes/lysosomes, and these compartments became inaccessible to horseradish peroxidase and immune complexes, but not to latex beads. The inhibitory profile of AZ cannot be solely accounted for by vacuolation and interference with acidification. AZ may help in dissecting various steps of the endocytic apparatus such as lateral mobility of receptors at the plasma membrane, formation of clathrin-independent endocytic vesicles, orientation of transferrin receptors into the recycling route, and fusogenicity with lysosomes.     

Microtubule-disrupting drugs blocked delivery of endocytosed transferrin to the cytocenter, but did not affect return of transferrin to plasma membrane

The fluorescence of FL cells after endocytosis of rhodamine-labeled transferrin initially appeared as a dispersed punctate pattern over the whole cell and then accumulated in the cytocenter on further incubation. In nocodazole-treated cells, the punctate fluorescence appeared along the cell edges, and stayed there on further incubation but did not accumulate in the cytocenter. The localization of transferrin was examined at the electron microscopic level with horseradish peroxidase (HRP)-labeled transferrin. Nocodazole did not affect endosome formation but affected the distribution of the endosomes. Several types of endosomes (tubular, small spherical, and microvesicular endosomes) were observed in nocodazole-treated cells, as in control cells. The endosomes were in the Golgi area of the cytocenter and also in peripheral cytoplasm in control cells. In contrast, the endosomes were only in the periplasm, along the cell edges, in nocodazole-treated cells. The uptake and release of HRP-transferrin and the release of ferric ion into the cytoplasm in nocodazole-treated cells followed in the same time-course as those in control cells. The release of transferrin was the exponential with a half-time of 12 min. The activation energy of a rate-limiting step in the recycling was 5.5 kcal.mol-1 at around 37 degrees C and increased to 29 kcal.mol-1 below 25 degrees C. These results indicated that microtubule-dependent endosome transport was faster than the overall recycling process and was independent of the return event of transferrin to the plasma membrane.     

Relations between the intracellular pathways of the receptors for transferrin, asialoglycoprotein, and mannose 6-phosphate in human hepatoma cells

We compared the intracellular pathways of the transferrin receptor (TfR) with those of the asialoglycoprotein receptor (ASGPR) and the cation-independent mannose 6-phosphate receptor (MPR)/insulin-like growth factor II receptor during endocytosis in Hep G2 cells. Cells were allowed to endocytose a conjugate of horseradish peroxidase and transferrin (Tf/HRP) via the TfR system. Postnuclear supernatants of homogenized cells were incubated with 3,3'-diaminobenzidine (DAB) and H2O2. Peroxidase-catalyzed oxidation of DAB within Tf/HRP-containing endosomes cross-linked their contents to DAB polymer. The cross-linking efficiency was dependent on the intravesicular Tf/HRP concentration. The loss of detectable receptors from samples of cell homogenates treated with DAB/H2O2 was used as a measure of colocalization with Tf/HRP. To compare the distribution of internalized plasma membrane receptors with Tf/HRP, cells were first surface-labeled with 125I at 0 degrees C. After uptake of surface 125I-labeled receptors at 37 degrees C in the presence of Tf/HRP, proteinase K was used at 0 degrees C to remove receptors remaining at the plasma membrane. Endocytosed receptors were isolated by means of immunoprecipitation. 125I-TfR and 125I-ASGPR were not sorted from endocytosed Tf/HRP. 125I-MPR initially also resided in Tf/HRP-containing compartments, however 70% was sorted from the Tf/HRP pathway between 20 and 45 min after uptake. To study the accessibility of total intracellular receptor pools to endocytosed Tf/HRP, nonlabeled cells were used, and the receptors were detected by means of Western blotting. The entire intracellular TfR population, but only 70 and 50% of ASGPR and MPR, respectively, were accessible to endocytosed Tf/HRP. These steady-state levels were reached by 10 min of continuous Tf/HRP uptake at 37 degrees C. We conclude that 30% of the intracellular ASGPR pool is not involved in endocytosis (i.e., is silent). Double-labeling immunoelectron microscopy on DAB-labeled cells showed a considerable pool of ASGPR in secretory albumin-positive, Tf/HRP-negative, trans-Golgi reticulum. We suggest that this pool represents the silent ASGPR that has been biochemically determined. A model of receptor transport routes is presented and discussed.