Endocytosis of wheat germ agglutinin binding sites from the cell surface into a tubular endosomal network

By using fluorescence and electron microscopy, the endocytic pathway encountered by cell surface components after they had bound wheat germ agglutinin (WGA) was visualized. The majority of these components are thought to consist of sialylated glycoproteins (HMWAG) that represent a subpopulation of the total cell surface proteins but most of the externally disposed plasma membrane proteins of the cell. Examination of semi-thin sections by medium- and high-voltage electron microscopy revealed the three-dimensional organization of vesicular and tubular endosomes. Binding of either fluorescein isothiocyanate-, horseradish peroxidase-, or ferritin-conjugated WGA to cells at 4 degrees C showed that the HMWAG were distributed uniformly over the cell surface. Warming of surface-labeled cells to 37 degrees C resulted in the endocytosis of WGA into peripheral endosomes via invagination of regions of both coated and uncoated membrane. The peripheral endosome appeared as isolated complexes comprising a vesicular element (300-400 nm diam.) surrounded by and continuous with tubular cisternae (45-60 nm diam.), which did not interconnect the endosomes. After 30 min or more label also became localized in a network of anastomosing tubules (45-60 nm diam.) that were located in the centrosomal region of the cell. Endocytosed WGA-HMWAG complexes did not become associated with cisternae of the Golgi apparatus, although tubular and vesicular endosomes were noted in the vicinity of the trans-Golgi region. The accumulation of WGA-HMWAG in the endosomes within the centrosomal region was inhibited when cells were incubated at 18 degrees C. None of these compartments contained acid phosphatase activity, a result that is consistent with other data that the HMWAG do not pass through lysosomes initially. The kinetics of labeling were consistent with the interpretation that recycling of most of the WGA binding surface glycoproteins occurred rapidly from early peripheral endosomes followed by the late trans-Golgi compartment. In conclusion, a portion of cell surface glycoproteins are routed to a complex arrangement of tubular and vesicular compartments following endocytosis that includes a putative post-endosomal, tubular reticulum that appears to be separate from the trans-most Golgi saccule.     

Cell surface glycoproteins of CHO cells. I. Internalization and rapid recycling

The major cell surface proteins of Chinese hamster ovary (CHO) cells have been investigated after reacting cells at 4 degrees C with the membrane-impermeant reagent, trinitrobenzenesulfonate (TNBS). Immunoprecipitation and subsequent two-dimensional, sodium-dodecyl sulfate, polyacrylamide gel electrophoresis (SDS-PAGE) of proteins from derivatized cells that had been labelled previously with [3H]D-glucosamine or [3H]L-leucine showed that TNBS reacted with most of the high molecular weight (HMW) acidic glycoproteins that became labelled with iodine by the lactoperoxidase technique and that bind the lectin, wheat germ agglutinin (WGA). After warming the cells to allow endocytosis to proceed, molecules haptenized with trinitrophenol (TNP) groups were followed radiochemically by means of [125I]anti-DNP antibodies. The half-life for internalization of proteins tagged with either [125I]anti-DNP IgG or Fab averaged about 5 min. A similar result was obtained when a monoclonal antibody directed against a single plasma membrane glycoprotein was used, or when the rate of surface loss of TNP groups unoccupied by antibodies was measured. Within 15 min at 37 degrees C, a steady-state between surface and cytoplasmic label was reached, with about 65% of the hapten located internally. Recycling of internalized TNP groups back to the cell surface also occurred rapidly (t 1/2 approximately 5 min). Most of the intracellular radioactivity was associated with a membrane fraction of density similar to that of the plasma membrane. Over a 4-h period, there was no significant entry of labeled molecules into lysosomes. By contrast, the fluid-phase marker, horseradish peroxidase, became associated with the lysosomes within 1 h. Our results are consistent with the view that the majority of plasma membrane glycoproteins are continuously being internalized and recycled at a high rate.     

Receptor-mediated endocytosis in cultured fibroblasts: cryptic coated pits and the formation of receptosomes

Concentrative receptor-mediated endocytosis of many specific ligands by cultured fibroblasts occurs through the coated pit-receptosome pathway. The formation of receptosomes was studied using two impermeant electron-dense labels for the cell surface, ruthenium red and concanavalin A-horseradish peroxidase. These studies show that at 4 degrees C, virtually all coated structures near the plasma membrane are in communication with the cell surface, and are not isolated coated vesicles. On warming cells to 37 degrees C for only 1 minute, a major portion of these structures become cryptic, that is, not labeled by these surface markers. However, on cooling cells immediately back to 4 degrees C, virtually all of these structures are again in communication with the surface. Many images showed that membrane of these cryptic pits to be continuous with the cell surface when caught in the appropriate plane of section; often there was a very narrow entrance that excluded extracellular label. At 37 degrees C, receptosomes could be occasionally seen forming as an invagination of membrane adjacent to the coated region. Mechanisms by which receptosomes may form and other evidence demonstrating the failure of coated pits to pinch off to form isolated coated vesicles during endocytosis are discussed.     

Receptor-mediated endocytosis of immunoglobulin-coated colloidal gold particles in cultured mouse peritoneal macrophages

Endocytosis of immunoglobulin G (IgG)-coated colloidal gold particles in cultured mouse peritoneal macrophages was studied by scanning and transmission electron microscopy. At 4 degrees C, the tracers adhered to the plasma membrane and accumulated in coated pits located in the bottom of furrows or deep invaginations on the cell surface. In the presence of an excess of unlabeled mouse IgG, cellular binding of the tracer was reduced by 80 to 90%. After warming to 37 degrees C, surface-bound tracer particles were rapidly ingested and transported to small and large vesicles lacking membrane coat. From here, they were then passed over to multivesicular bodies and lysosomes characterized by their content of myelin-like figures and other inclusions. Double-labeling experiments with IgG-coated colloidal gold particles of two different sizes (20 and 5 nm diameter) indicated that the plasma membrane was depleted of binding sites after uptake of a polyvalent ligand. The restoration of the binding capacity was a slow process requiring ongoing protein synthesis. On the basis of these observations, a model for endocytosis of immune complexes in macrophages is presented. It includes the following four steps: IgG-containing macromolecular aggregates bind to specific receptors in the plasma membrane. These appear to be preclustered in coated pits or able to move laterally within the membrane even at 4 degrees C. The receptor-ligand complexes are internalized and transferred sequentially to larger uncoated vesicles or endosomes, multivesicular bodies, and lysosomes with inclusions of varying appearance. Receptors and ligands are degraded within the lysosomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluid-phase endocytosis by intrahepatic bile duct epithelial cells isolated from normal rat liver

Although recent data from our laboratory have established the occurrence of receptor-mediated endocytosis in intrahepatic bile duct epithelial cells (IBDEC) isolated from normal rat liver, no studies have assessed the role of isolated IBDEC in fluid-phase endocytosis. Therefore, to determine if IBDEC participate in fluid-phase endocytosis, we incubated morphologically polar doublets of IBDEC isolated from normal rat liver with horseradish peroxidase (HRP, 5 mg/ml), a protein internalized by fluid-phase endocytosis, and determined its intracellular distribution by electron microscopic cytochemistry. Pulse-chase studies using quantitative morphometry were also performed to assess the fate of HRP after internalization. After incubation at 37 degrees C, IBDEC internalized HRP exclusively at the apical (i.e., luminal) domain of their plasma membrane; internalization was completely blocked at 4 degrees C. After internalization, HRP was seen in acid phosphatase-negative vesicles and in acid phosphatase-positive multivesicular bodies (i.e., secondary lysosomes). Small acid phosphatase-negative vesicles containing HRP moved progressively from the apical to the basal domain of IBDEC. Pulse-chase studies showed that HRP was then discharged by exocytosis at the basolateral cell surface. These results demonstrate that IBDEC prepared from normal rat liver participate in fluid-phase endocytosis. After internalization, HRP either is routed to secondary lysosomes or undergoes exocytosis after transcytosis from the luminal to the basolateral cell surface. Our results suggest that IBDEC modify the composition of bile by internalizing both biliary proteins and fluid via endocytic mechanisms.     

Pathways of endocytosis in cultured macrophages. Electron microscopic autoradiographic tracing of iodinated plasma membrane proteins

Lactoperoxidase-mediated iodination at 4 degrees C--an established method for covalent labelling of plasma membrane proteins--and quantitative electron microscopic autoradiography were used to follow the pathways of endocytosis in mouse macrophages in vitro. Directly after the labelling, the autoradiographic grains were concentrated to the cell surface. After warming to 37 degrees C, radioactive material was rapidly internalized into cytoplasmic vesicles and subsequently transferred to lysosomes as well as to the Golgi complex. Maximum grain density (% grains/% volume) over the vesicles was observed after 15 min, over the lysosomes after 30 to 45 min and over the Golgi complex after 30 and 90 min. Throughout the experimental period (120 min), the vesicles showed the largest fraction of intracellular grains, but higher grain densities occurred in lysosomes as well as in stacked Golgi cisternae and Golgi-associated vesicles. In spite of the internalization process, the labelling of the cell surface came to a steady state already after 30 min and at all intervals more than 50% of the autoradiographic grains were localized to this compartment. About 25% of the cell-associated radioactivity was lost rapidly with a half-life of 20 to 25 min and the remaining 75% slowly with a half-life of 7 to 9 h. The results indicate that membrane internalized by endocytosis partly follows a route to the lysosomes and that, additionally, there exists a route to and through the Golgi complex. They further support earlier notions of a bidirectional traffic between the surface and interior of the cell and suggest that recycling of membrane components may take place from endocytic vesicles, lysosomes, as well as the Golgi complex.     

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.     

The preendosomal compartment comprises distinct coated and noncoated endocytic vesicle populations

The transfer of molecules from the cell surface to the early endosomes is mediated by preendosomal vesicles. These vesicles, which have pinched off completely from the plasma membrane but not yet fused with endosomes, form the earliest compartment along the endocytic route. Using a new assay to distinguish between free and cell surface connected vesicle profiles, we have characterized the preedosomal compartment ultrastructurally. Our basic experimental setup was labeling of the entire cell surface at 4 degrees C with Con A-gold, warming of the cells to 37 degrees C to allow endocytosis, followed by replacing incubation medium with fixative, all within either 30 or 60 s. Then the fixed cells were incubated with anti-Con A-HRP to distinguish truly free (gold labeled) endocytic vesicles from surface-connected structures. Finally, analysis of thin (20-30 nm) serial sections and quantification of vesicle diameters were carried out. Based on this approach it is shown that the preendosomal compartment comprises both clathrin-coated and non-coated endocytic vesicles with approximately the same frequency but with distinct diameter distributions, the average noncoated vesicle being smaller (95 nm) than the average coated one (110 nm). In parallel experiments, using an anti-transferrin receptor gold-conjugate as a specific marker for clathrin-dependent endocytosis it is also shown that uncoating of coated vesicles plays only a minor role for the total frequency of noncoated vesicles. Furthermore, after perturbation of clathrin-dependent endocytosis by potassium depletion where uptake of transferrin is blocked, noncoated endocytic vesicles with Con A-gold, but not coated vesicles, exist already after 30 and 60 s. Finally, it is shown that the existence of small, free vesicles in the short-time experiments cannot be ascribed to recycling from the early endosomes.     

Internalization and delivery to lysosomes of hydrazide horseradish peroxidase, a covalent membrane probe

Hydrazide horseradish peroxidase, (hydHRP), a hydrazide derivative of the common cytochemical tracer HRP, was covalently coupled to the surface of periodate-treated Chinese hamster ovary (CHO) cells and used to study the distribution and internalization of plasma membrane glycoconjugates. The Schiff-base coupling of hydHRP to the cell surface at 4 degrees C had little effect on cell viability. After coupling, cells were washed at 4 degrees C and the subcellular distribution of hydHRP was determined immediately or after incubation at 37 degrees C. Within 1 hr, hydHRP was observed to cap over pseudopodal-like extensions and then accumulate over a 2.5 h period in a punctate to perinuclear staining pattern over the cell body. By electron microscopy, the pseudopodal-like regions were found to be areas of extensive cell surface invaginations, rich in microfilaments. HydHRP internalized over a 2.5 to 18 hr period was observed in smooth vesicles resembling pinosomes/endosomes, multivesicular bodies (lysosomes), and small perinuclear vesicles. Little, if any, hydHRP activity was detected in association with elements of Golgi apparatus. By cell fractionation in 10% Percoll gradients, hydHRP was found to have accumulated in prelysosomal endocytic vesicles and lysosomes. For cells that were first surface labeled with 125I at 4 degrees C and then conjugated with hydHRP, little, if any, cotransport of the 125I label with hydHRP was observed. Over the entire capping and internalization period, most hydHRP activity remained membrane associated. Overall, these results indicate that the dominant intracellular transport route for a covalent membrane probe, hydHRP glycoconjugate, is similar if not identical to that previously reported for the solute probe native HRP (16) in CHO cells. HydHRP internalization provides further evidence for the independent sorting of proteins in endocytic transport.     

Endocytosis by African trypanosomes. I. Three-dimensional structure of the endocytic organelles in Trypanosoma brucei and T. congolense

African trypanosomes multiply rapidly during the course of infection obtaining nutrients from the host blood and other body fluids. The organelles involved in endocytosis were revealed ultrastructurally using horseradish peroxidase (HRP) and colloidal gold coupled to bovine transferrin (Au-Tf) or bovine serum albumin (Au-BSA). At 0 degree C the markers bound to the cell surface and neither entered the flagellar pocket nor were internalized. Upon warming to 37 degrees C, the markers were found in the flagellar pocket and appeared to enter all the intracellular endocytic organelles within 5 min. Serial sectioning of resin-embedded cells was employed to obtain pseudo three-dimensional views of these organelles. The organelles involved were of three types: (1) small vesicles and cisternae (20-25 nm in diameter), (2) large tubular networks (200 nm diameter) similar to endosomes of mammalian cells, and (3) large lysosome-like vesicles. These organelles were located between the flagellar pocket and the nucleus and were also associated with one face of the Golgi apparatus. In pulse-chase experiments HRP was not detected in intracellular organelles after 410 min but Au-Tf was seen in residual bodies. No exocytosis of Au-Tf from the flagellar pocket was observed. The data suggests that the processes of endocytosis in these parasitic protozoa may be similar to the endocytic processes found in mammalian cells.     

Quantitative ultrastructural, autoradiographic evidence for the magnitude and early involvement of the Golgi apparatus complex in the endocytosis of wheat germ agglutinin by cultured neuroblastoma

Several ligands undergo endocytosis into the Golgi apparatus. We have examined with a quantitative ultrastructural, autoradiographic method the sequential endocytosis of tritiated wheat germ agglutinin (3H-WGA) by cultured murine neuroblastoma cells. Cells were incubated with 3H-WGA for 1 hour at 4 degrees C, washed, and incubated in complete medium without ligand at 37 degrees C for 5, 15, 30, and 120 minutes. At 5 minutes, the optimized sources/micron 2 of neuroblastoma cell area, which represented the grain density of each compartment, were as follows: smooth vesicles and tubules, 1.03 +/- 0.88; Golgi-associated vesicles, i.e., clusters of vesicles within a 1 micron radius of the Golgi cisterns, 1.03 +/- 0.31; Golgi cisterns, less than 0.01; and lysosomes, 0.26 +/- 0.16. At 15 minutes grain densities were: smooth vesicles and tubules, 0.9 +/- 0.34; Golgi-associated vesicles, 1.41 +/- 0.28; Golgi cisterns, 0.73 +/- 0.41; and lysosomes, 0.1 +/- 0.09. At 30 minutes grain densities were: smooth vesicles and tubules, 0.46 +/- 0.46; Golgi-associated vesicles, 1.78 +/- 0.34; Golgi cisterns, 0.89 +/- 0.78; and lysosomes, 0.39 +/- 0.14. At 2 hours, smooth vesicles, tubules, and Golgi cisterns were not labeled, Golgi-associated vesicles were still labeled (0.71 +/- 0.1), and lysosomes were heavily labeled (2.17 +/- 0.22). These results are consistent with the hypotheses that either the Golgi complex (cisterns and associated vesicles) is an early and intermediate step of the endocytosis of 3H-WGA into lysosomes or that it constitutes part of a separate and quantitatively significant pathway of endocytosis of this ligand.     

Prolactin internalisation by the epithelial mammary cell: effects of lysosomotropic agents and transglutaminase inhibitors

Prolactin endocytosis was studied by electron microscopy with 125I-prolactin 125I-hGH (human growth hormone) and prolactin-ferritin. Endocytosis and intracellular transit of the labelled hormone proceeded identically in epithelial cells isolated from the mammary glands of pseudopregnant rabbits and in surviving fragments from mammary glands of lactating rabbits. After binding of the hormone to its receptor, the labelled material was rapidly detectable in vesicles showing an homogeneous aspect; 15 min later part of the labelled material was still localized within the same kind of vesicles, but in addition it appeared to have migrated into microvesicles of the Golgi region and into vesicles of heterogeneous aspect tentatively identified with lysosomes. Endocytosis of bovine serum albumin, labelled with ferritin followed the same intracellular pathway. Native ferritin accumulated in vesicles of various sizes, but seemed excluded from the microvesicles of the Golgi zone. In the presence of lysosomotropic agents labelled prolactin accumulated in cytoplasmic vesicles. In the presence of dansylcadaverine, endocytosis of the labelled material proceeded unimpaired. Conversely, in the presence of bacitracin, the internalisation of labelled prolactin seemed to be reduced. These observations show that the endocytosis of the hormone/receptor complex is linked to membrane movements, which eventually lead to its location within both the Golgi apparatus and the lysosomes.     

Binding and degradation of proteoglycans by cultured arterial smooth muscle cells. I. Endocytosis and intracellular translocation of proteoglycan-gold conjugates

Proteoglycans (Mr approximately 200 000) isolated from bovine arterial tissue were decorated with 17 nm diameter gold particles for tracing in electron microscopic thin sections and surface replicas. Lysine and arginine residues of their proteoglycan protein core are assumed to be essential for gold conjugation. The resulting proteoglycan-gold conjugates, which appear as pearl string-like gold strands of about 170 nm in length were used to visualize binding, endocytosis and intracellular translocation of proteoglycans by homologous cultured arterial smooth muscle cells. The proteoglycan-gold conjugates bind to coated as well as to non-coated cell surface membrane areas at 4 degrees C. This is followed by the formation of membrane invaginations. Postincubation at 37 degrees C leads to a time-dependent uptake of proteoglycan-gold conjugates via non-coated and coated vesicles which after fusion are translocated to multivesicular bodies and to large sized vesicles within 1 h. After conversion of these vesicles to lysosomal compartments the gold particles are uncoupled from the proteoglycans and are concentrated within residual vacuoles. From these vacuoles the gold particles are extruded. In contrast to the surface-bound proteoglycan-gold conjugates the released gold particles are condensed to bulky aggregates. The results, which include competition, inhibition and pulse chase experiments, extend biochemical data on endocytosis and degradation of proteoglycans.     

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.     

Redistribution and recycling of internalized membrane in seminal vesicle secretory cells

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

Platelet-derived growth factor: morphologic and biochemical studies of binding, internalization, and degradation

Kinetic studies of binding and internalization of 125I-platelet-derived growth factor (PDGF) demonstrate that up to 15% of membrane-associated radioactivity is internalized within 2 minutes after warming to 37 degrees C in a variety of cell types. The T 1/2 for internalization is approximately 20 minutes. The T 1/2 for the subsequent appearance of degradation products in the culture medium is between 60-90 minutes following initiation of internalization. Internalization and lysosomal association of 125I-PDGF were confirmed by EM autoradiography. Quantitative studies using PDGF adsorbed to colloidal gold (gold-PDGF) demonstrate that 17% of the cell-associated sites are along coated regions of the plasma membrane (1.0 sites/micron), while 82% are associated with noncoated membrane (0.2 sites/micron). There is a significant redistribution of the gold-PDGF complexes upon warming. Within 1-2 minutes at 37 degrees C, gold particles are found within endocytic vesicles, endosomes (0.09-0.3 micron diameter), and lysosomes (greater than 0.2 micron diameter). At this time the vesicle/endosome compartment comprises 15% of the total sites and contains 0.9 sites per micron2 of surface area. The lysosomes account for 8% of the total sites and contain 0.8 sites per micron2 of surface area. Simultaneously, there is an increase in the number of gold-PDGF binding sites within coated-pits (1.6 sites/micron, 18% of the total sites) and a decrease along noncoated regions of the membrane (0.11 sites/micron, 58% of the total sites). After 15 minutes at 37 degrees C, 26% of the total sites (1.4 sites/micron2) are highly concentrated within lysosomes, while sites in the vesicle/endosome compartment remain constant. At the same time, binding sites within coated pits decrease substantially (0.5 sites/micron, 4% of the total sites), while the number of sites along noncoated regions of the membrane remain constant. Gold-PDGF was not observed associated with the Golgi complex at any time up to 120 minutes following warming. We conclude that gold-PDGF is processed via both receptor-mediated and nonspecific endocytosis and follows an intracellular pathway comparable to that followed by some other protein ligands.     

The organelles of the trans domain of the cell. Ultrastructural localization of sialoglycoconjugates using Limax flavus agglutinin

The subcellular distribution of sialic acid was determined at the ultrastructural level using Limax flavus agglutinin (LFA). This lectin, which is specific for N-acetylneuraminic acid and N-glycolylneuraminic acid, was covalently conjugated to horseradish peroxidase (HRP). The conjugates (LFA-HRP) were applied to aldehyde-fixed, saponin-permeabilized 3T3 cells in pre-embedding labeling electron microscopy. Peroxidase label was detected in a patchy distribution at the cell surface, and in plasma-membrane-coated pits, endocytic vesicles (receptosomes), multivesicular bodies, and lysosomes. Smooth-surfaced tubular and vesicular structures, similar to those that participate in membrane recycling, were labeled. In the Golgi complex, more than half of the cisternae contained label--typically only one cisterna on the cis side was unlabeled. Heavily labeled structures of the trans Golgi included a reticular membranous system with coated regions--50-80 nm diameter vesicular or pit-like profiles and larger coated vacuoles. Smooth 200-300 nm vacuoles were labeled on the trans side of the Golgi stack. Similar structures have been previously shown to participate in the exocytosis of plasma membrane and secretory glycoproteins from the Golgi stacks. These findings identify those intracellular organelles that are functionally at the level of, or distal to, the sialyltransferase-containing membranes of the Golgi, and distinguish them from the pre-Golgi membranous structures. The LFA-HRP conjugate is an indicator for this functional trans domain of the cell, and should be applicable for ultrastructural double-label experiments as a cis versus trans marker of the exocytic pathway.     

Role of the coated endocytic vesicle in the uptake of receptor-bound low density lipoprotein in human fibroblasts.

125I-labeled and ferritin-labeled low density lipoprotein (LDL) were used as visual probes to study the surface distribution of LDL receptors and to examine the mechanism of the endocytosis of this lipoprotein in cultured human fibrobasts. Light microscopic autoradiograms of whole cells incubated with 125I-LDL at 4 degrees C showed that LDL receptors were widely but unevenly distributed over the cell surface. With the electron microscope, we determined that 60-70% of the ferritin-labeled LDL that bound to cells at 4 degrees C was localized over short coated segments of the plasma membrane that accounted for no more than 2% of the total surface area. To study the internalization process, cells were first allowed to bind ferritin-labeled LDL at 4 degrees C and were then warmed to 37 degrees C. Within 10 min, nearly all the surface-bound LDL-ferritin was incorporated into coated endocytic vesicles that were formed by the invagination and pinching-off of the coated membrane regions that contained the receptor-bound LDL. With increasing time at 37 degrees C, these coated vesicles were observed sequentially to migrate through the cytoplasm (1 min), to lose their cytoplasmic coat (2 min), and to fuse with either primary or secondary lysosomes (6 min). The current data indicate that the coated regions of plasma membrane are specialized structures of rapid turnover that function to carry receptor-bound LDL, and perhaps other receptor-bound molecules, into the cell.     

Receptor-mediated endocytosis of alpha 2-macroglobulin in cultured fibroblasts

Alpha 2-macroglobulin is internalized into cultured fibroblasts by receptor-mediated endocytosis. This ligand binds initially to diffusely distributed receptors on the cell surface which cluster rapidly into bristle-coated pits. Within a few minutes at 37 degrees C, these complexes are internalized into uncoated cytoplasmic vesicles, called receptosomes, which move about in the cell by saltatory motion. These vesicles interact with the Golgi-endoplasmic reticulum-lysosome system in the cell to deliver the ligand to newly formed lysosomes within 30--60 min.     

Movements and other distinguishing features of small vesicles identified by darkfield microscopy in living macrophages

Perinuclear vesicles (estimated diameter less than 0.15 micron), too small to be seen in living mouse macrophages by direct phase-contrast microscopy, could be detected by darkfield microscopy thanks to their rapid non-saltatory movements at 37 degrees C, contrasting with the slower saltations of accompanying phase-visible larger vesicles (0.25-0.5 micron, presumed secondary lysosomes). The movements of these 'small visicles' also differed from those of the 'larger visicles' in their responses to changes in temperature, and to chemical agents known to inhibit both the saltations of secondary lysosomes and the latter's fusion with phagosomes. Thus the 'larger vesicles' stopped moving at 25 degrees C, the small ones did not; both stopped at 18 degrees C. The 'small vesicles' continued to move actively after cell uptake of the polyanion poly-D-glutamic acid, while the saltations of the 'larger vesicles' were markedly slowed; both sets of vesicles stopped after uptake of ammonium chloride. Degranulation of the small vesicles paralleled that of the larger, while simultaneously observed preformed pinosomes (labelled with fluorescent wheat germ agglutinin (WGA) appeared to be unaffected. On the basis also of refractivity, location and speed the 'small vesicles' are considered not to be pinosomes, but probably to be lysosomes. The question of whether they are a subgroup of small immature secondary lysosomes or primary lysosomes (0.05-0.08 micron) is discussed. The broad spectrum of movement inhibited by ammonia in macrophages raises the possibility that this weak base inhibits movements of all lysosomes. Further characterization of these 'small vesicles' requires their relation to be defined to the small particles in other cell types (especially in axoplasm) which have been detected by video-enhanced microscopy.