Endocytosis of the C3b receptor of complement within coated pits in human polymorphonuclear leukocytes and monocytes

The distribution and endocytosis of the C3b receptor by human polymorphonuclear leukocytes and monocytes were visualized by both fluorescent and electron microscopic examination of cells that had been labeled with monospecific F(ab')2 anti-C3b receptor and anti-F(ab')2 conjugated with rhodamine or ferritin. When prefixed or unfixed cells that were labeled at 0 to 4 degrees C were examined, the receptor was distributed within clusters on the plasma membrane. After the cells had been warmed to room temperature or to 37 degrees C for 5 minutes, the fluorescently labeled receptors appeared to enter the cells, and the ferritin-tagged receptors often occurred within coated endocytic pits and coated vesicles within the cytoplasm. After incubation at 37 degrees C for 20 minutes, the C3b receptor-antibody complexes were largely cleared from the cell surface, and much of the label was found within lysosomes. These results indicate that C3b receptors may directly mediate endocytosis within coated pits, thus utilizing a mechanism shared by a variety of other receptors for the rapid, efficient, and selective internalization of extracellular ligands.     

Ultrastructural localization of interferon receptors on the surfaces of cultured cells and erythrocytes.

The bindings sites for interferon (IFN) on the limiting cell membranes of human and mouse fibroblasts and erythrocytes were revealed by an indirect immunoferritin technique. Mouse IFN-beta and human IFN-beta of high specific activity were used with the corresponding purified antibodies. Species-specific IFN binding was demonstrated by ferritin deposition on human erythrocytes and fibroblast membranes treated with human IFN and on mouse erythrocytes and fibroblast membranes treated with mouse IFN, but not on human erythrocytes or fibroblast membranes treated with mouse IFN. IFN binding sites on fibroblasts were located on regions of membranes between microvilli, whereas diphtheria toxin receptors were demonstrated mainly on microvilli. IFN binding altered the diphtheria toxin after IFN treatment. This reduced toxicity correlated with a decrease in the quantity of receptors for diphtheria toxin on the cell membrane. Thus, the species-specific binding of IFN appears to depend on membrane receptors in discrete regions of the limiting membrane which are present not only on functionally responsive fibroblasts but also on erythrocytes.     

Membrane activity and topography of F-Met-Leu-Phe-Treated polymorphonuclear leukocytes. Acute and sustained responses to chemotactic peptide.

The chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (f-Met-Leu-Phe) causes a dramatic stimulation of membrane ruffling and a fluid pinocytosis in polymorphonuclear leukocytes (PMNs). These responses are maximal by 1 minute and subside within 5-10 minutes. The same immediate responses characterize cells exposed to several peptide hormones and may thus represent an essential component of target cell activation by peptides. The stimulation of the whole membrane following f-Met-Leu-Phe binding is succeeded by the development of a polarized cell shape with a posterior uropod and a broad anterior lamellipodium, both subtended by microfilaments. Membrane components and functions segregate into distinct domains on polarized PMNs. Thus, succinyl concanavalin A-receptor complexes are capped and internalized by receptor-mediated endocytosis at the uropod; the uptake by fluid pinocytosis of fluorescein-dextran is restricted to the uropod; and coated pits and coated vesicles are concentrated at the uropod. The lamellipodium excludes coated pits and lacks pinocytic activity but shows preferential binding of immunoglobulin aggregates, presumably to Fc receptors. The origin and physiologic implications of these asymmetries of membrane molecular and functional topography on polarized cells are discussed.     

Morphological characterization of beta-VLDL and acetylated-LDL binding and internalization by cultured pigeon monocytes

The ultrastructure of binding, internalization, and translocation of beta-migrating very low density lipoprotein (beta-VLDL) and acetylated low density lipoprotein (Ac-LDL) by cultured pigeon monocytes was examined using lipoprotein-gold conjugates. Through morphometry, differences in the binding and uptake of beta-VLDL-gold and Ac-LDL-gold were documented. Cells exposed to either beta-VLDL-gold or Ac-LDL-gold for 2 hr at 4 degrees C had the label over noncoated regions of the plasma membrane. Upon warming the cells to 37 degrees C for 2 min, 35% of the surface-bound beta-VLDL-gold was within coated pits on the cell surface. Although coated pits occupied less than 2% of the surface, binding of beta-VLDL-gold was 53 times more concentrated in coated pits as compared to noncoated membrane regions. In contrast, Ac-LDL-gold neither bound to coated pits nor relocated into coated regions of the membrane upon warming to 37 degrees C. Both the beta-VLDL-gold and the Ac-LDL-gold were internalized when the cells were rewarmed at 37 degrees C. Most of the internalized gold particles for both lipoproteins were located in electron-lucent vesicles; however, 9% of the intracellular beta-VLDL-gold was observed within coated vesicles at early times. Upon prolonged rewarming (30-90 min), both lipoprotein-gold conjugates were within acid phosphatase-positive lysosomes. Ultimately 83% of the Ac-LDL-gold and 90% of the beta-VLDL-gold were within electron-dense and electron-lucent lysosomes. These results suggested that the receptor-mediated binding and internalization of beta-VLDL and Ac-LDL by pigeon monocyte macrophages proceeded by separate, distinct routes; beta-VLDL by both coated and noncoated pathways while Ac-LDL was internalized exclusively by noncoated mechanisms. Regardless of these internalization differences, both lipoproteins were delivered to lysosomes for degradation.     

Cationized ferritin and phosvitin uptake by coated vesicles of the early chick embryo

Summary The endocytosis of cationized ferritin and of a phosvitinferritin conjugate by cells of the chick embryo area pellucida has been examined. Cationized ferritin was bound mainly to the free surface of the epiblast but was absent from the region of the primitive streak. The binding was patchy and experiments suggest that the anionic sites which bind cationized ferritin are themselves naturally clustered. Uptake of cationized ferritin was exclusively by coated pits. The resulting coated vesicles delivered the cationized ferritin to membrane-bound sites of accumulation in the cytoplasm and to the close vicinity of Golgi bodies. The cationized ferritin was frequently found to share intracellular vacuoles with yolk granules. The uptake was not affected by the presence of microfilament or microtubule-inhibiting agents. Native ferritin, even at concentrations forty times that of cationized ferritin, was not bound or endocytosed.Coated pits in the epiblast were often associated with overlying extracellular yolk granules. This suggested that the yolk might be inducing the formation of the coated pits. The yolk protein phosvitin was coupled to ferritin and this conjugate was found to be endocytosed by coated pits. This uptake was inhibited in the presence of an excess of free phosvitin but not by albumin, indicating some selectivity for phosvitin over other proteins. The phosvitin conjugate was also found sharing intracellular vacuoles with yolk. We conclude that the cells of the area pellucida, and in particular those of the epiblast, have an active coated vesicle uptake system which may be able to selectively endocytose yolk or yolk protein.     

Receptor utilization by reovirus type 3: distinct binding sites on thymoma and fibroblast cell lines result in differential compartmentalization of virions.

Reovirus type 3/D infects cells following binding to specific cell-surface receptors. The characteristics of these receptors may play an important role in determining post-binding events critical to the viral life cycle. Some cell lines, i.e. L-cells, appear to bind reovirus type 3/D utilizing sialylated proteins as specific receptors for viral adsorption. Such binding results in productive infection. Other cell lines, i.e. R1.1 thymoma cells, bind reovirus type 3/D in a sialic acid independent manner which does not result in productive infection. Yet, a peptide analogue of the viral binding site is capable of inhibiting binding of reovirus type 3 to both cell types, suggesting the same viral epitope interacts with both cellular receptors. When binding of reovirus is studied by electron microscopy, the virus particles enter the L cells via coated pits, and are later seen in large accumulations in endocytic vesicles near the transGolgi network. In contrast, R1.1 cells appear to divert the reovirus particles to a cell membrane elaboration, with reovirus remaining bound to the cell membrane. At later time points with R1.1 cells, there are no apparent intracellular accumulations. These studies demonstrate that viruses can attach to different cells utilizing distinct receptors, and this may play a role in the ability of the virions to productively infect the cells. The capacity of virus to be adsorbed to cellular receptors which do not lead to internalization may be an important mechanism for the sequestration and clearance of virus. These observations have implications for the tissue tropism demonstrated by reovirus type 3/D and other viruses.     

Binding of Mannosylated Ferritin to Chicken Bone Marrow Macrophages

Ferritin conjugated to diazotated p-aminophenylmannoside and mannan was used for ultrastructural visualization of binding and endocytosis via the mannose receptor. Conjugates were bound by live macrophages but not by glutaraldehyde-fixed cells. Binding was inhibited by 0.1 mM mannan and 0.2 M α-methylmannoside, and strongly reduced, but not abolished, after trypsin degradation of surface receptors. Binding sites were rapidly assembled on coated pits, which often showed a polar distribution on the cell surface and entered the cell following the direction of microtubuli. Some coated vesicles occasionally kept in touch with the extracellular space. 10-min exposure to the conjugate resulted in changes in the surface morphology, such as a loss of infoldings and increased membrane tension. Ferritin accumulated in large smooth vesicles with coated membrane regions. It was mostly detached from the membrane and agglutinated into big clumps, together with fibrillar material and small vesicles which may derive from the vesicle membrane.     

Interferon induces an antiviral state in ganglioside-deficient transformed mouse fibroblasts

The antiviral activity of mouse fibroblast interferon against vesicular stomatitis virus was investigated in L-929 mouse fibroblasts and the ganglioside-deficient L-929 mutant cells (ATCC clone NCTC 2071). Although it has been widely reported that gangliosides serve as primary receptors for interferon at the cellular membrane, only a small difference in interferon sensitivity was observed between the wild-type L-929 and the ganglioside-deficient NCTC 2071 cells. It was not possible, however, to overcome this difference by administration of exogenous gangliosides.     

Incomplete replication of human parainfluenza virus type 4 in LLC-MK2 cells and in L929 cells

Human parainfluenza virus type 4A (hPIV-4A) and type 4B (hPIV-4B) were tested for their ability to replicate in the monkey kidney LLC-MK2 cell line (MK2 cells) and the murine L929 cell line (L929 cells). These cells are normally non-permissive for replication of hPIV-4; however, treatment with acetylated trypsin led to virus replication in MK2 cells, but was less effective for L929 cells. Endogenously produced interferon (IFN) played no role in virus replication in L929 cells. Synthesis of virus-specific polypeptides was suppressed in L929 cells. WhereasNP-mRNA and HN-mRNA were detected in MK2 cells, no HN-mRNA was detected in L929 cells. These results indicate that hPIV-4 can infect both MK2 cells and L929 cells. In MK2 cells, when protease exists in the extracellular medium, hPIV-4 exhibits multistep growth. In L929 cells, however, the cause of incomplete replication might be lack of other unknown factors. Received: 10 January 2000     

Lens culinaris lectin receptors in the plasma membrane of rat liver cells: comparative electron microscopic studies on normal cells, on cells in vivo transformed by diethylnitrosamine and on Zajdela ascites hepatoma cells.

The distribution of the Lens culinaris lectin receptors on normal rat liver cells, on rat liver cells in vivo transformed by diethylnitrosamine and on Zajdela ascites hepatoma cells of the rat is investigated by means of the Lens culinaris lectin-peroxidase method and by ferritin conjugated Lens culinaris lectin. The normal rat liver cells show a continuous labeling at the outer membrane surface by the lectin complexes, whereas the transformed rat liver cells exhibit a strong tendency for patchy distribution of the cell surface label. The discontinuous cell surface label in the transformed rat liver cells is obviously caused by an internalization of plasma membrane areas. The importance of these morphological findings in their relationship to Lens culinaris lectin mediated agglutination of rat liver cells and the membrane fluidity in general are discussed. In the experiments no hints to a rotation of the Lens culinaris lectin receptors from the outer membrane surface to the inner membrane surface of rat liver cells can be found.     

Endocytic appartus and transcytosis in epithelial cells of the vas deferens in the rat

The apex of the principal epithelial cells lining the vas deferens of the rat contains coated pits in continuity with the apical plasma membrane and large subsurface-coated vesicles (100–125 um). In the apical cytoplasm, large, pale, uncoated vesicles (150–300 nm), small coated and uncoated vesicles (50–60 nm), uncoated vesicles about 75–90 nm, and membranous apical tubules are present, in addition to large, vacuolar, pale, multivesicular bodies, dense multivesicular bodies, and secondary lysosomes seen deeper in the cytoplasm amongst numerous ER cisternae, saccules of the Golgi apparatus, and mitochondria. The endocytic activity of these cells was investigated by using cationic ferritin (CF) as a marker of adsorptive endocytosis and native ferritin (NF) for demonstrating fluid-phase endocytosis. These tracers were injected separately into the lumen of the vas deferens, and the animals were killed at various time intervals thereafter from 2 to 90 minutes. At 2 minutes CF was seen bound predominantly to microvilli and to areas of the apical plasma membrane delimiting coated pits as well as in large, coated vesicles. At 5 and 15 minutes the tracers were seen in apical tubules and pale multivesicular bodies; at 30 minutes moderately dense multivesicular bodies were labeled. At 1 hour and longer time intervals dense multivesicular bodies and secondary lysosomes were labeled. NF followed the same pathway as CF; however, no binding to microvilli or areas delimiting coated pits was observed. The numerous other vesicular structures, i.e., the large uncoated vesicles (150–300 nm) and the small coated and uncoated vesicles (50–60 nm), never became labeled with the tracers and therefore were not involved in the endocytic process. There was, however, and exception in the case of several small (75–90 nm) uncoated vesicles seen deeper in the apical cytoplasm of these cells which were labeled exclusively with CF. With time such vesicles appeared along the lateral and basal surfaces of these cells and discharged their content of CF into the lateral intercellular space or the connective tissue space at the base of these cells. Thus the principal epithelial cells in addition to sequestering the endocytosed tracers within secondary lysosomes where they are presumably degraded also appear to be involved in the transcytosis of material from the lumen of the vas deferens to the underlying lamina propria.     

Endocytic apparatus and transcytosis in epithelial cells of the vas deferens in the rat

The apex of the principal epithelial cells lining the vas deferens of the rat contains coated pits in continuity with the apical plasma membrane and large subsurface-coated vesicles (100-125 nm). In the apical cytoplasm, large, pale, uncoated vesicles (150-300 nm), small coated and uncoated vesicles (50-60 nm), uncoated vesicles about 75-90 nm, and membranous apical tubules are present, in addition to large, vacuolar, pale, multivesicular bodies, dense multivesicular bodies, and secondary lysosomes seen deeper in the cytoplasm amongst numerous ER cisternae, saccules of the Golgi apparatus, and mitochondria. The endocytic activity of these cells was investigated by using cationic ferritin (CF) as a marker of adsorptive endocytosis and native ferritin (NF) for demonstrating fluid-phase endocytosis. These tracers were injected separately into the lumen of the vas deferens, and the animals were killed at various time intervals thereafter from 2 to 90 minutes. At 2 minutes CF was seen bound predominantly to microvilli and to areas of the apical plasma membrane delimiting coated pits as well as in large, coated vesicles. At 5 and 15 minutes the tracers were seen in apical tubules and pale multivesicular bodies; at 30 minutes moderately dense multivesicular bodies were labeled. At 1 hour and longer time intervals dense multivesicular bodies and secondary lysosomes were labeled. NF followed the same pathway as CF; however, no binding to microvilli or areas delimiting coated pits was observed. The numerous other vesicular structures, i.e., the large uncoated vesicles (150-300 nm) and the small coated and uncoated vesicles (50-60 nm), never became labeled with the tracers and therefore were not involved in the endocytic process. There was, however, an exception in the case of several small (75-90 nm) uncoated vesicles seen deeper in the apical cytoplasm of these cells which were labeled exclusively with CF. With time such vesicles appeared along the lateral and basal surfaces of these cells and discharged their content of CF into the lateral intercellular space or the connective tissue space at the base of these cells. Thus the principal epithelial cells in addition to sequestering the endocytosed tracers within secondary lysosomes where they are presumably degraded also appear to be involved in the transcytosis of material from the lumen of the vas deferens to the underlying lamina propria.     

Concanavalin A receptor sites on lymph node cells in vivo and in vitro

The distribution and density of receptors for concanavalin A (Con A) on the surfaces of cells of intact and isolated popliteal and axillary lymph nodes were investigated in the rabbit. Intact lymph nodes were perfused via the subcapsular (marginal) sinus with either Con A peroxidase or Con A ferritin, fixed with glutaraldehyde, and processed for electron microscopy. Both Con A peroxidase and Con A ferritin were distributed on the plasmalemma of lymphocytes, macrophages, neutrophils, plasma cells, reticular endothelial cells, and the vascular endothelium. Counts of Con A-conjugated ferritin particles indicated that the density of Con A receptors was generally similar for lymphocytes, macrophages, and neutrophils but lower on plasma cells. When lymph node cells were isolated by mechanical methods and exposed to Con A ferritin, the label was homogenously distributed on the cell surfaces of most cells. However, Con A binding was significantly higher on the surface of isolated cells than in the intact node. It is suggested that the increase in density of Con A binding sites on isolated cells may possibly be due to an unmasking of cell surface moieties in which additional Con A receptor sites become available as a result of the isolation procedure. The density of Con A ferritin binding sites was also significantly lower on the surface of isolated plasma cells than the lymphocyte and macrophage, suggesting that the density distribution of cell surface saccharides is different for various lymphoid cells.     

Interferon-induced antiviral actions in Friend leukemia cells: role of membrane gangliosides

The interactions between interferon (IFN) and membrane gangliosides have been analyzed in Friend leukemia cells (FLC). The G_M1-cholera toxin (CT) model has been selected as a reference system for a high-affinity ganglioside-ligand binding. Two gangliosides have been detected in FLC extracts: a disialoganglioside (～ 95%) and a monosialoganglioside (～ 5%) which migrate respectively as G_D1a and G_M1. Unilammellar liposomes containing either mixed commercial gangliosides or G_D1a or G_M1 bind three to eight fold more IFN (both partially purified and pure) than gangliosides-free liposomes. Only 3–4% of the input IFN was bound, whereas G_M1-containing liposomes bound almost 50% of [³H]acetyl-CT. Neuraminidase treatment of FLC, which converts G_D1a-like into the G_M1-like ganglioside, does not significantly modify the IFN-induced antiviral effects. CT binding to FLC as well as CT-induced elevation of endocellular cyclic AMP levels in FLC were instead markedly increased by the same neuraminidase treatment. Further, pretreatment of FLC with either G_D1a or G_M1 or gangliosides extracted from FLC did not change the IFN-induced establishment of the antiviral state. These data provide convincing evidence that FLC gangliosides do not represent the IFN high-affinity receptors nor specifically mediate IFN effects, even though they bind IFN to some extent.     

Receptor-mediated endocytosis of glucagon in isolated mouse hepatocytes

The binding of glucagon to the cell surface and the pathway of intracellular transport of the hormone in isolated mouse hepatocytes were studied by autoradiography, colloidal gold-labeled glucagon (Au-glucagon), and biochemical methods. In cells incubated with 1251-glucagon at 4 degrees C, the label was mainly localized to the plasma membrane even after 60 min of incubation. At 20 degrees C, the labeled ligand was internalized by the cells and the amount of internalized ligand increased with time of incubation. At 37 degrees C, the ligand was rapidly internalized and found to be associated with coated or uncoated vesicles. Au-glucagon experiments revealed clearly the process of internalization of glucagon. Au-glucagon bound to the plasma membrane was transported to coated regions and then internalized into vesicles via coated pits. Biochemical results supported these findings from autoradiography and Au-glucagon experiments. Thus, glucagon is internalized by hepatocytes via receptor-mediated endocytosis.     

Studies on reovirus receptors of L cells: virus binding characteristics and comparison with reovirus receptors of erythrocytes

"Saturation binding experiments" were carried out to characterize the attachment of reovirus to mouse L fibroblasts. Scatchard analysis of data obtained from such experiments suggests that one homogeneous set of noncooperative, high affinity binding sites are involved in reovirus attachment. It is estimated that L cells possess between 3 and 5 X 10(5) reovirus binding sites per cell and that the equilibrium dissociation constant (KD) is approximately 3 X 10(-9) M. Scatchard analysis of data from similar experiments, carried out in the presence of anti-sigma 1 and anti-sigma 3 antibodies, revealed that although both antibodies prevent viral attachment, they exhibit distinct binding inhibition characteristics: anti-sigma 1 effectively abolishes high-affinity, specific binding, whereas anti-sigma 3 apparently blocks low-affinity, nonspecific interactions. The nature of the L-cell receptor was then probed using various enzymes and reagents, and compared with that of the reovirus receptor on human type O erythrocytes. It was found that whereas reovirus hemagglutination (HA) is inhibited by pretreatment of erythrocytes with various proteases or neuraminidase, virus binding to L cells is unaffected by such treatments. Neither HA nor cell binding is inhibited by the various sugars tested, including N-acetyl-D-glucosamine, which was previously reported to inhibit reovirus HA (L. D. Gelb and A. M. Lerner, 1965, Science 147, 404-405). Both L cells and erythrocyte reovirus receptors are nevertheless highly sensitive to periodate treatment, which presumably destroys the high-affinity reovirus binding sites since protein sigma 1, which is capable of attaching to L cells by itself, does not bind to cells pretreated with periodate. It is therefore concluded that sugar residues on the receptor may be involved in this specific interaction. The possibility that gangliosides may serve as reovirus receptors was also probed. It was found that bovine brain gangliosides, but not cerebrosides, readily aggregate reovirus, inhibit HA, and block viral attachment to L cells. However, binding of protein sigma 1 to L cells is unaffected by gangliosides. Inhibition of reovirus HA and L-cell binding by these gangliosides is therefore most likely due to a steric hindrance effect brought about by interactions between the gangliosides and other components of the outer viral capsid.     

Coated pits and vesicles in the osteoclast

The osteoclast is a cell with a phagocytic ability not dissimilar to the macrophage. Nevertheless, the mechanisms by which it resorbs bone are poorly understood. The aim of this study was to examine the distribution of coated membrane structures in the osteoclast in order to gain further information about endocytosis in this cell. Osteoclasts around the developing tooth germs of young rats were examined using transmission electron microscopy, and immunocytochemistry. Results showed that previously described coated membrane structures within the ruffled border do not appear to be associated with coated pits or vesicles. Coated pits were, however, evident on the dorsal and lateral surfaces of the cell, particularly opposite the clear zone areas. Immunogold staining for clathrin confirmed that coated pits and vesicles are absent within both the clear zone and ruffled border areas, but present on the lateral and dorsal surfaces of the actively resorbing cell. It is suggested that clathrin-associated receptor-mediated endocytosis occurs along the lateral and dorsal surfaces of the osteoclast for the uptake of nutrients and macromolecules, while endocytosis of bone mineral by the ruffled border is mediated by a non-clathrin associated coated membrane structure.     

小鼠枯否细胞的抗肿瘤活性

检测了小鼠Ｋｕｐｆｆｅｒ细胞（ＫＣ）膜受体、吞噬功能和抗肿瘤活性。结果证明ＫＣ的Ｆｃ受体和Ｃ３ｂ受体活性都在９０％以上，对披复有Ｃ３ｂ的酵母多糖的吞噬作用明显强于腹腔巨噬细胞。正常昆明和Ｃ５７ＢＬ／６小鼠的ＫＣ具有对ＹＡＣ－１，Ｐ８１５和Ｌ９２９瘤细胞介导低水平的天然细胞毒作用和产生ＴＮＦ。用ＢＣＧ或双岐杆菌在原位激活的小鼠ＫＣ对上述三株瘤细胞介导的细胞毒作用和产生ＴＮＦ活性都明显增强。     

Transitional cells at the junction of seminiferous tubules with the rete testis of the rat: Their fine structure,endocytic activity,and basement membrane

Transitional cells line the intermediate region of rat seminiferous tubules situated between the rete testis and the seminiferous epithelium proper. These tall elongated cells orient themselves in a downstream direction and converge on one another distally in the lumen of the rete testis where they form a distinct papillalike structure through which a narrow patent lumen is apparent. In addition to widely dispersed Golgi apparatus and mitochondria, these cells contain an abundance of microtubules, cisternae of endoplasmic reticulum, and a distinct lobulated nucleus showing clumps of chromatin and a prominent nucleolus. The endocytic activity of these cells was examined by employing adsorptive (cationic ferritin, concanavalin A ferritin) and fluid-phase tracers (native ferritin, horseradish peroxidase-colloidal gold complex, and concanavalin A ferritin in presence of a methyl-D-mannoside). Such tracers were injected separately into the lumen of the rete testis, and the animals were killed at 2, 5, 15, and 30 min and 1, 2, and 6 hr after injection. At 2 min, both adsorptive and fluid-phase tracers were found within coated and uncoated pits of the apical plasma membrane of these cells as well as in large, subsurface, uncoated spherical, C-shaped, and tubular membranous elements. At 5 min the tracers were seen in endosomes of different sizes; while at 15 min and 30 min, pale and dense multivesicular bodies of small and large sizes, respectively, were labeled. At 1-hr and longer time intervals secondary lysosomes became labeled. While both fluid-phase and adsorptive tracers followed the same pathway and fate, binding to the apical and lateral plasma membranes of the transitional cells and to the membrane delimiting coated and uncoated pits was observed only with the adsorptive tracers. These results demonstrate that the transitional cells are actively involved in both fluid-phase and adsorptive endocytosis, which may play an important role in modifying the composition of the luminal fluid. The transitional cells of the distal zone of the intermediate region rest on an elaborate basement membrane (BM) complex which includes a thin BM immediately underlying these cells, a thick distal layer of BM, and strands of BM spanning the distance between the two in the form of a loose anastomotic network. Use of antisera against heparan sulfate proteoglycan, laminin, and type IV collagen revealed the presence of all three components within all areas of the BM complex. In the meshes of the anastomotic BM network, extracellular vesicles were observed. Vesicles of similar size and appearance were also noted in the basal region of the transitional cells as well as in their basal-foot-like processes which extended deep into the meshes of the anastomotic network. While such extracellular vesicles may be derived from the transitional cells, their functional significance is as yet unclear.     

Endocytic pathway of high density lipoprotein via trans-Golgi system in rat resident peritoneal macrophages

Interaction of high density lipoprotein (HDL) with rat resident peritoneal macrophages was investigated by morphological and biochemical approaches. Binding studies at 0 degree C demonstrated saturable binding sites for 125I-HDL on the surface membrane. When cells were incubated with 125I-HDL at 37 degrees C, the cell-associated radioactivity increased with time, but intracellular degradation of HDL did not occur. Rather, the cell-associated 125I-HDL was released intact into the medium. Two morphological probes were employed to visualize the post-binding fate of HDL. Horseradish peroxidase and ferritin conjugates of HDL showed specific bindings to the coated pits of the plasma membranes and were internalized and delivered through receptosomes to a trans-Golgi system. They were then resecreted as secretory vesicles from the cells. Parallel experiments with transferrin-horseradish peroxidase conjugates revealed an endocytic pathway identical or very similar to that observed with HDL. These results provide evidence that internalization and subsequent nonlysosomal pathway via the trans-Golgi system are involved in receptor-mediated endocytosis of HDL by macrophages.