Transendothelial transport (transcytosis) of iron-transferrin complex in the rat liver

To determine the transport pathway of iron-transferrin complex (Fe-TF) into the hepatocyte, we labeled Fe-TF with colloidal gold and perfused rat liver through the portal vein with this probe under different conditions. The tissue was then studied by transmission electron microscopy. At a cold temperature (approximately 4 degrees C), the probe bound to the luminal surface of sinusoidal endothelium without internalization. The binding was limited to the endothelium and there was no binding to Kupffer cells or hepatocytes. The binding was inhibitable in the presence of excess soluble Fe-TF, indicating the specificity of the bindings. At 37 degrees C the probe was internalized via a system of coated pits and vesicles. Morphometric analyses of the temporal sequence of events suggested that the probe was transported, in part, across the endothelium via a system of tubules and vesicles and externalized on the abluminal side via a system of coated pits. The probe was then taken up by hepatocytes. Only minimal uptake was noted when gold-labeled bovine serum albumin was used either at the low temperature or at 37 degrees C. The findings suggest that the liver uptake of Fe-TF complex by hepatocytes is a transendothelial phenomenon (transcytosis).     

The role of liver endothelium in the binding and uptake of ceruloplasmin: studies with colloidal gold probe

To determine the mode of uptake of ceruloplasmin (CP) by liver, the protein was labeled with colloidal gold and infused into the portal vein. In cold almost all probes bound to the sinusoidal endothelium, and at 37 degrees C internalization via a system of coated pits and vesicles occurred. Only rarely did the probe appear to bypass the endothelium, moving to the abluminal side through the gaps between endothelial cells. In the endothelial cytoplasm, the probe was seen in coated vesicles, endosomes, tubules, and large vesicles which may have formed by fusion of endosomes and tubules. Moreover, externalization of the probe to the abluminal side was noted, and this also occurred via a system of coated vesicles. The findings suggest that the uptake of CP in the liver may be primarily a transendothelial phenomenon (transcytosis).     

Liver endothelium mediates the uptake of iron-transferrin complex by hepatocytes

We have previously shown that in the liver, transferrin (TF) receptors are limited to endothelial cells, and hepatocytes and Kupffer cells do not have TF receptors. To study the transport of iron into hepatocytes, we fractionated liver cell suspensions into endothelium and hepatocyte fractions. At 4 degrees C liver (but not umbilical cord) endothelium bound Fe-TF with a saturable kinetics. At 37 degrees C, the endothelial uptake was followed by its gradual release. Transendothelial transport of TF was visually demonstrated by perfusion of liver using colloidal gold-labeled TF. The released Fe-TF acquired the potential for binding to fresh target hepatocytes and binding was not inhibited by excess cold TF but was inhibitable by asialofetuin, suggesting galactosyl receptors and not TF receptors as a recognition mechanism. Isoelectrofocusing of the supernate after preincubation for 90 min at 37 degrees C with endothelial cells, demonstrated the presence of a newly generated band which co-migrated with asialotransferrin. We conclude that Fe-TF is initially removed by liver endothelium where it is modified probably by desialation to expose the galactosyl residues of the glycoproteins. The modified molecule is subsequently released and recognized by hepatocytes through a TF receptor-independent mechanism which may involve galactosyl receptors of hepatocytes. The findings indicate a key role for endothelium in the transport of Fe-TF into the liver and may suggest a physiological function for galactosyl receptors on hepatocyte surface.     

Receptor-mediated transcytosis of follicle-stimulating hormone through the rat testicular microvasculature

Accumulated data suggest that endothelial cells express specific receptors for several peptide and (glyco)protein hormones that may transport hormones across the cell to be delivered to the interstitial fluid and tissue target cells. Surprisingly, very little information is available on the actual endothelial organelles involved in this cellular process. In the present study the transfer of follicle-stimulating hormone (FSH) through the endothelial barrier of rat testes was examined by analysing the binding and transport of gold-tagged recombinant human (rh)FSH under various conditions using electron microscopy. At 4 degrees C the probe bound specifically to the luminal surface of the endothelial cells without internalization. The use of 125I-rhFSH, which allows precise quantitation of the binding, confirmed the specificity of hormone interaction with the testicular microvasculature. At 37 degrees C the hormone was internalized via coated pits and vesicles into an extensive subluminal tubulo-vesicular compartment and was transported across the endothelium via a system of tubules and vesicles. Moreover, monoclonal antibodies against the FSH receptor ectodomain coupled to colloidal gold followed the same route. In contrast, a non specific, fluid-phase uptake via caveolae was observed for a major plasma protein - rat serum albumin and a fluid-phase tracer - peroxidase. These results suggest that FSH transcytosis across the testicular endothelial barrier is receptor-mediated and involves luminal uptake via coated pits/vesicles, sorting at the level of luminal early endosomes, and transcellular transport through transcytotic tubulo-vesicular organelles. Similar receptor-mediated pathways are likely to be involved in the physiological functioning of a number of other protein and peptide hormones that must translocate specifically from blood to the target cells.     

Immunoelectron microscopic visualization of the transcytosis of low density lipoproteins in perfused rat arteries

A postembedding labeling technique was employed to visualize human native low density lipoproteins (LDL) during transcytosis in rat arterial endothelium. For this purpose human LDL was perfused through rat vasculature before fixation and processing for immunoelectron microscopy. The LDL particles were located on sections by anti-human apolipoprotein B-100 (LDL) antibodies and secondary antibodies or protein-A conjugated to 10-nm colloidal gold. LDL molecules were seen in plasmalemmal vesicles as well as in the subendothelial space. No colloidal gold was found in the intercellular junctions. Perfusion with reductively methylated LDL, which cannot bind to the LDL receptor, gave a similar labeling pattern, indicating that transcytosis of LDL via plasmalemmal vesicles is most likely receptor independent. Furthermore, the passage of LDL through intact vascular endothelium is a vesicular transport rather than an intercellular diffusion process.     

alpha 2-macroglobulin adsorbed to colloidal gold: a new probe in the study of receptor-mediated endocytosis

alpha 2-Macroglobulin (alpha 2 M) was adsorbed to colloidal gold and used as a new tool in the study of receptor-mediated endocytosis. alpha 2 M-gold is easy to prepare and is clearly visualized at the electron microscope level. When cells were incubated with alpha 2 M-gold at 0 degrees C, gold was visualized both diffusely over the cell surface and concentrated in coated pits. After cells to which alpha 2 M-gold had been bound at 0 degrees C were warmed, the gold was rapidly internalized into uncoated vesicles, previously termed receptosomes. After 30 min of incubation or longer, gold was found in small lysosomes and, later, in large lysosomes and very small vesicles in the region of the Golgi complex. This pattern of localization is similar to that previously described, using peroxidase-labeled anti-alpha 2 M antibodies. By incubating cells with both alpha 2 M-gold and vesicular stomatitis virus (VSV), we studied the internalization of these two markers simultaneously. VSV and alpha 2 M-gold rapidly clustered in the same coated pits and were internalized in the same receptosomes. Proteins and hormones adsorbed to gold may be useful in the study of receptor-mediated endocytosis.     

Glucagon receptors in endothelial and Kupffer cells of mouse liver

To determine whether hepatic sinusoidal cells contain glucagon receptors and, if so, to study the significance of the receptors in the cells, binding of [125I]-glucagon to nonparenchymal cells (mainly endothelial cells and Kupffer cells) isolated from mouse liver was examined by quantitative autoradiography and biochemical methods. Furthermore, the pathway of intracellular transport of colloidal gold-labeled glucagon (AuG) was examined in vivo. Autoradiographic and biochemical results demonstrated many glucagon receptors in both endothelial cells and Kupffer cells, and more receptors being present in endothelial cells than in Kupffer cells. In vivo, endothelial cells internalized AuG particles into coated vesicles via coated pits and transported the particles to endosomes, lysosomes, and abluminal plasma membrane. Therefore, receptor-mediated transcytosis of AuG occurs in endothelial cells. The number of particles present on the abluminal plasma membrane was constant if the amount of injected AuG increased. Therefore, the magnitude of receptor-mediated transcytosis of AuG appears to be regulated by endothelial cells. Kupffer cells internalized the ligand into cytoplasmic tubular structures via plasma membrane invaginations and transported the ligand exclusively to endosomes and lysosomes, suggesting that the ligand is degraded by Kupffer cells.     

Hepatic binding and internalization of low density lipoprotein-gold conjugates in rats treated with 17 alpha-ethinylestradiol

Receptor-mediated hepatic uptake of low density lipoproteins (LDL) conjugated to colloidal gold was studied by perfusion of livers from rats treated for 5 d with 17 alpha-ethinylestradiol. Estrogen treatment resulted in a marked decrease in serum lipid and lipoprotein concentrations. After 15 min of perfusion the conjugate was bound to the hepatic microvilli of both control and estrogen-treated rats; the estrogen-treated rats showed an 8- to 11-fold greater number of membrane-bound conjugates. The conjugates were bound to the membrane receptor by the LDL particle because the gold granules were regularly displaced from the membrane by 20 +/- 3.2 nm, the diameter of LDL. Internalization of the conjugate, evident by gold particles in multivesicular bodies, occurred at coated pits at the base of the microvillus where coated vesicles containing a single gold-LDL conjugate were released. After 1 h of perfusion, the livers from the estrogen-treated rats showed all phases of endocytosis and incorporation into multivesicular bodies of the conjugate. After 2 h of perfusion, there was congregation of gold-labeled lysosomes near the bile canaliculi. Gold-LDL conjugates were also observed to bind and be internalized by Kupffer cells and sinusoidal endothelium. These findings indicate that estrogen treatment induces hepatic receptors for LDL. The catabolic pathway of binding and endocytosis of the conjugate is similar to that seen in fibroblasts, although slower. Because gold-LDL conjugates were also present in the Kupffer and endothelial cells, the uptake of LDL by the liver involves the participation of more than a single cell type.     

Histochemical evidence for the differential surface labeling, uptake, and intracellular transport of a colloidal gold-labeled insulin complex by normal human blood cells

A colloidal gold-labeled insulin-bovine serum albumin (GIA) reagent has been developed for the ultrastructural visualization of insulin binding sites on the cell surface and for tracing the pathway of intracellular insulin translocation. When applied to normal human blood cells, it was demonstrated by both visual inspection and quantitative analysis that the extent of surface labeling, as well as the rate and degree of internalization of the insulin complex, was directly related to cell type. Further, the pathway of insulin (GIA) transport via round vesicles and by tubulo-vesicles and saccules and its subsequent fate in the hemic cells was also related to cell variety. Monocytes followed by neutrophils bound the greatest amount of labeled insulin. The majority of lymphocytes bound and internalized little GIA, however, between 5-10% of the lymphocytes were found to bind considerable quantities of GIA. Erythrocytes rarely bound the labeled insulin complex, while platelets were noted to sequester large quantities of the GIA within their extracellular canalicular system. GIA uptake by the various types of leukocytic cells appeared to occur primarily by micropinocytosis and by the direct opening of cytoplasmic tubulo-vesicles and saccules onto the cell surface in regions directly underlying surface-bound GIA. Control procedures, viz., competitive inhibition of GIA labeling using an excess of unlabeled insulin in the incubation medium, preincubation of the GIA reagent with an antibody directed toward porcine insulin, and the incorporation of 125I-insulin into the GIA reagent, indicated the specificity and selectivity of the GIA histochemical procedure for the localization of insulin binding sites.     

Receptor-mediated endocytosis of transferrin and recycling of the transferrin receptor in rat reticulocytes

At 4 degrees C transferrin bound to receptors on the reticulocyte plasma membrane, and at 37 degrees C receptor-mediated endocytosis of transferrin occurred. Uptake at 37 degrees C exceeded binding at 4 degrees C by 2.5-fold and saturated after 20-30 min. During uptake at 37 degrees C, bound transferrin was internalized into a trypsin- resistant space. Trypsinization at 4 degrees C destroyed surface receptors, but with subsequent incubation at 37 degrees C, surface receptors rapidly appeared (albeit in reduced numbers), and uptake occurred at a decreased level. After endocytosis, transferrin was released, apparently intact, into the extracellular space. At 37 degrees C colloidal gold-transferrin (AuTf) clustered in coated pits and then appeared inside various intracellular membrane-bounded compartments. Small vesicles and tubules were labeled after short (5-10 min) incubations at 37 degrees C. Larger multivesicular endosomes became heavily labeled after longer (20-35 min) incubations. Multivesicular endosomes apparently fused with the plasma membrane and released their contents by exocytosis. None of these organelles appeared to be lysosomal in nature, and 98% of intracellular AuTf was localized in acid phosphatase-negative compartments. AuTf, like transferrin, was released with subsequent incubation at 37 degrees C. Freeze-dried and freeze-fractured reticulocytes confirmed the distribution of AuTf in reticulocytes and revealed the presence of clathrin-coated patches amidst the spectrin coating the inner surface of the plasma membrane. These data suggest that transferrin is internalized via coated pits and vesicles and demonstrate that transferrin and its receptor are recycled back to the plasma membrane after endocytosis.     

Receptor-mediated endocytosis of a ricin-colloidal gold conjugate in vero cells. Intracellular routing to vacuolar and tubulo-vesicular portions of the endosomal system

We have prepared a conjugate (Ri-Au) of the toxic plant protein ricin and colloidal gold (particle size 5 nm) and used it for internalization studies in monolayer cultures of Vero cells. The Ri-Au conjugate was very stable, with only little release of ricin ([125I]Ri) from the gold particles within a pH range of 4.5-8.0. Within 2 h at 37 degrees C, only very little intracellular degradation of the ricin preparation ([125I]Ri-Au) occurred. The cells bound the same proportion of native ricin ([125I]Ri) and Ri-Au from the medium, and the kinetics of toxicity (decrease in cellular incorporation of [3H]leucine) of [125I]Ri and [125I]Ri-Au were also comparable. At 4 degrees C, the cell-surface binding of Ri-Au was continuous and distinct, as revealed by electron microscopy. This binding was specific, since almost no Ri-Au surface binding occurred at 4 degrees C in the presence of 0.1 M lactose or 1 mg/ml native (unlabelled) ricin. Within the first 30 min of warming prelabelled cells to 37 degrees C, the amount of surface-associated Ri-Au decreased considerably (from 150 to 60 gold particles per micron cell surface in 40 nm sections). Coated pits and vesicles were involved in the internalization of Ri-Au, and within 5-30 min at 37 degrees C Ri-Au had been delivered to vacuolar and tubulo-vesicular portions of the endosomal system, and later also to lysosomes. Analysis of very thin (ca 20 nm) serial sections revealed that most of the tubulo-vesicular elements were separate structures not connected to the membrane of the vacuolar portion. Data here presented indicate that our ricin conjugate, like many "physiological' ligands and viruses, is internalized by receptor-mediated endocytosis via the coated pit-endosomal pathway.     

Transport of membrane-bound macromolecules by M cells in follicle-associated epithelium of rabbit Peyer's patch

Summary M cells in Peyer's patch epithelium conduct transepithelial transport of luminal antigens to cells of the mucosal immune system. To determine the distribution of specific lectin-binding sites on luminal membranes of living M cells and to follow the transport route of membranebound molecules, lectin-ferritin conjugates and cationized ferritin were applied to rabbit Peyer's patch mucosa in vivo and in vitro. The degree to which binding enhances transport was estimated by comparing quantitatively the transport of an adherent probe, wheat germ agglutinin-ferritin, to that of a nonadherent BSA-colloidal gold probe. When applied to fixed tissue, the lectins tested bound equally well to M cells and columnar absorptive cells. On living mucosa, however, ferritin conjugates of wheat germ agglutinin and Ricinus communis agglutinins I and II bound more avidly to M cells. Absorptive cells conducted little uptake and no detectable transepithelial transport. Lectins on M cell membranes were endocytosed from coated pits, rapidly transported in a complex system of tubulocisternae and vesicles, and remained adherent to M cell basolateral membranes. Cationized ferritin adhered to anionic sites and was similarly transported, but was released as free clusters at M cell basolateral surfaces. When applied simultaneously to Peyer's patch mucosa, wheat germ agglutinin-ferritin was transported about 50 times more efficiently than was bovine serum albumin-colloidal gold.     

Binding and endocytosis of heparin-gold conjugates by the fenestrated endothelium of the rat choriocapillaris

Summary Heparin-gold probes were used to localize regions of heparin binding on the luminal surface of the diaphragmed-fenestrated endothelium of the rat choriocapillaris. Structures of endothelial cells were unlabeled when rats were kept on ice and perfused with solutions at 4° C. When the heparin-gold quantity was doubled, only a few heparin-gold particles marked some diaphragms spanning fenestrae, vesicles and channels, parajunctional regions of the plasmalemma, and coated pits. With solutions at 4° C, but the animals kept at room temperature, all of these structures in the endothelial cells were labeled. This binding was not altered by the perfusion of low levels of unlabeled heparin, but was eliminated following high levels of unlabeled heparin, and by digestion with trypsin and pronase. At 37° C, heparin was localized to the above structures and, in addition, was internalized into coated vesicles, endosomes, and multivesicular bodies, but not other types of lysosomes. Some particles were found in tubules adjacent to the Golgi stacks. Heparin-gold was also transported to the abluminal front of the endothelium by vesicles. A desulfated, non-anticoagulant, fraction of heparin bound to gold was localized to the endothelium in the same manner. These results demonstrate receptors for heparin on the surface of a fenestrated endothelium. Furthermore, they show the pathway of endocytosis and transport of heparin. The possible roles of heparin in the function of the endothelium is discussed.     

G-protein ligands inhibit in vitro reactions of vacuole inheritance

In many organs the vascular endothelium forms a barrier which impedes the free diffusion of large molecules. The mechanism by which protein hormones are transported through the endothelial cells to reach their target cells is unknown. We have examined the transport of human chorionic gonadotropin (hCG) in rat testicular microvasculature by electron microscopy and by analysing the transfer of radiolabeled hormone and antibodies. Surprisingly, we have observed that the same receptor molecule which is present in target Leydig cells is also involved in transcytosis through the endothelial cells. The hormone was internalized by coated pits and vesicles on the luminal side of the endothelium. It was then localized in the endosomal compartment and subsequently appeared to be delivered by smooth vesicles into the subendothelial space. Moreover, anti-LH/hCG receptor antibodies were efficiently transported via the same system and delivered into the interstitial space. If generalized, these observations may define a new level of modulation of hormone action and may be of importance for drug targeting into the numerous organs which are responsive to the various protein hormones.     

The role of sialated glycoproteins in endocytosis, permeability and transmural passage in the myeloid endothelium.

Changes in the anionic charge distribution at the luminal face of the endothelium of the sinusoids of the bone marrow have been studied at sites of endocytosis by large bristle coated vesicles and at the sites of molecular permeability through diaphragmed fenestrae. The anionic charge distribution has also been studied at the abluminal aspect of these vessels at sites of transmural blood cell passage. Cationic surface markers such as colloidal iron, native ferritin and polycationic ferritin used at low pH, 1.8, and the use of neuraminidase show that the nonmodified endothelial cell surface has exposed sialic acid groups, which are absent at the sites of these functional specializations. Polycationic ferritin binding over a range of pH levels indicates the prsence of another species of anionic materials present at both the nonmodified cell surface and at the sites of the cell surface modifications. This second group of anionic compounds is neuraminidase resistant and has a pKa higher than that of sialic acid (pKa:2.6).     

Receptor-mediated endocytosis of immunoglobulin-coated colloidal gold particles in cultured mouse peritoneal macrophages. Chloroquine and monensin inhibit transfer of the ligand from endocytic vesicles to lysosomes

Earlier studies have shown that immunoglobulin G (IgG)-coated colloidal gold particles bind to specific receptors on the macrophage surface and accumulate in coated pits. They are then internalized via endocytic vesicles and transferred to lysosomes. During this process the plasma membrane is depleted of binding sites for IgG, suggesting that both the receptor and the ligand end up in lysosomes. Here, we have examined the effects of the weak base chloroquine and the Na+-H+ ionophore monensin on endocytosis and intracellular transport of IgG-coated colloidal gold particles in cultured macrophages. The results indicate that chloroquine and monensin do not arrest uptake of IgG-coated particles bound to the cell surface. On the other hand, the drugs strongly inhibit transfer of the particles from endocytic vesicles to lysosomes, the latter marked by prior pulse-chase labeling of the cells with horseradish peroxidase. Since the main effect shared by chloroquine and monensin is to raise pH in acid compartments such as endocytic vesicles and lysosomes, the findings suggest that the transfer of IgG-coated particles into the lysosomes is a pH-dependent process. It remains to be shown whether it is the membrane fusion as such that is controlled by pH or, more specifically, the transfer of receptor-bound ligands into the lysosomes.     

Receptor-mediated endocytosis of insulin by cultured endothelial cells.

Label-fracture immunochemistry and pre-embedding indirect immunocytochemistry were applied to investigate insulin uptake by endothelial cells. Freeze fracture replicas showed that a small percentage of native insulin receptors are associated with non-coated pits (4%) and coated pits (2%). After warming, receptor bound insulin became increasingly associated with such endocytotic vesicles. After 2 min the percentage of detectable insulin associated with non-coated and coated pits increased to 16% and 8%, respectively. Pre-embedding immunocytochemical localization of insulin gave results consistent with those obtained from the label-fracture studies. Both non-coated and coated vesicles appeared labelled after 5 min of warming. Non-coated vesicles contained 25% of the cell associated insulin while 9% was associated with coated pits and vesicles. After 10 min of warming, 9% of label was located in non-coated vesicles and 7% in coated vesicles. A large proportion (29%) of the label was found in tubular-vesicular endosomes at this time. After 15 min of warming, 30% of the remaining cell-associated gold label was found in multivesicular bodies. These experiments demonstrate that insulin uptake by endothelium is mediated by both coated and non-coated vesicles and that, once internalized, insulin is routed through endosomal pathways that primarily result in transcytosis.     

In vitro clustering and multiple fusion among macrophage endosomes

Early steps of receptor-mediated endocytosis appear to require the fusion of endosomes with each other. Recently, these fusion events have been reconstituted in vitro using vesicle preparations from J774 macrophages which have internalized ligands via the mannose receptor (Diaz, R., Mayorga, L., and Stahl, P. (1988) J. Biol. Chem. 263, 6093-6100). The present studies indicate that endosomes first form clusters when incubated under fusogenic conditions. Aggregation state was determined by electron microscopy using vesicles containing ligand-coated colloidal gold of different sizes previously internalized via the mannose receptor. Aggregation required cytosol and ATP. Afterwards, the limiting membranes of the vesicles composing these aggregates undergo multiple fusion and bring about the formation of large diameter vesicles that maintained the same density as endosomes when analyzed by Percoll gradient sedimentation. These large diameter vesicles were no longer fusogenic in the fusion assay. Multiple fusion was determined morphologically by the co-localization of three different size colloidal gold vesicles inside endocytic vesicles and biochemically by the fusion-dependent formation of triple immune complexes between three endocytic ligands internalized by receptor-mediated endocytosis: anti-dinitrophenol mouse IgG and dinitrophenol-derivatized beta-glucuronidase, ligands for the mannose receptor, and aggregated rabbit anti-mouse IgG, a ligand for the macrophage Fc receptor.     

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.     

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.