Biomedical subjects

B van Deurs

Publications and source records attributed to B van Deurs.

At least 91 records · Page 5Linked to original sources

PubMed1988

Human proteins IEF 58 and 57a are associated with the Golgi apparatus.

A mouse monoclonal antibody (mAB 22-II-D8B) raised against lysed transformed human amnion cells (AMA) has been characterized. The mAB decorated the Golgi apparatus in growing and quiescent cultured monolayer cells (fibroblasts and epithelial cells) of various species as determined by double immunofluorescence labeling and colocalization with galactosyltransferase antibodies. It reacted with the acidic human proteins IEF 58 (Mr = 29,000) and 57a, respectively (Mr = 30,000) (HeLa protein catalogue number; [(1982) Clin. Chem. 28, 766]), Golgi staining was also observed in BS-C-1 cells microinjected with mAB 22-II-D8B suggesting that the epitopes recognized by the antibody are most likely located on the cytoplasmic face of the membranes. The precise localization of the antigens to the various cisternae of the Golgi apparatus could not be demonstrated by immunogold cytochemistry on ultrathin cryosections due to either weak reactivity of the antibody or low concentration of the antigens. Immunofluorescence staining with mAB 22-II-D8B of lymphoid human Molt-4 cells and some human tissues failed to reveal any significant staining even though these expressed high levels of both IEF 58 and 57a. These results are taken to imply that the epitopes recognized by mAB 22-II-D8B may be masked in some cell types.

Animals↗

PubMed1988

Inhibition of endocytosis from coated pits by acidification of the cytosol.

Binding and endocytosis of the ligands transferrin, epidermal growth factor (EGF), and ricin were measured in a number of different cell lines after treatment of cells with compounds that react with SH-groups and under conditions where the cytosolic pH was lowered. N-ethylmalemide and diamide irreversibly inhibited endocytosis of all ligands tested, whereas low pH in the cytosol strongly inhibited endocytosis of transferrin and EGF. Data obtained by electron microscopy indicated that the formation of coated vesicles from coated pits is inhibited in acidified cells. Entry of ricin was much less affected, and ricin endocytosed under these conditions was able to intoxicate the cells. At low pH in the cytosol there was a calcium-dependent increase in the number of transferrin receptors at the cell surface. The increase was even larger in the presence of the calcium ionophore A23187, whereas it was completely blocked by the calmodulin antagonists trifluoperazine and W7. The results show that endocytosis from coated pits can be inhibited in a reversible way by acidification of the cytosol and they suggest that a second pathway of endocytosis exists, possibly involving formation of vesicles from uncoated areas of the membrane.

Ammonium Chloride↗

PubMed1988

Estimation of the amount of internalized ricin that reaches the trans-Golgi network.

We have used a protocol for internalization of ricin, a ligand binding to plasma membrane glycoproteins and glycolipids with terminal galactosyl residues, and infection with the vesicular stomatitis virus ts 045 mutant in BHK-21 cells to determine whether internalized plasma membrane molecules tagged by ricin reach distinct compartments of the biosynthetic-exocytic pathway. At 39.5 degrees C newly synthesized G protein of ts 045 was largely prevented from leaving the endoplasmic reticulum. At the same temperature ricin was endocytosed and reached, in addition to endosomes and lysosomes, elements of the Golgi complex. When the temperature was lowered to 19.5 degrees C, no more ricin was delivered to the Golgi complex, but now G protein accumulated in the Golgi stacks and the trans-Golgi network (TGN). Double-labeling immunogold cytochemistry on ultracryosections was used to detect G protein and ricin simultaneously. These data, combined with stereological and biochemical methods, showed that approximately 5% of the total amount of ricin within the cells, corresponding to 6-8 X 10(4) molecules per cell, colocalized with G protein in the Golgi complex after 60 min at 39.5 degrees C. Of this amount approximately 70-80% was present in the TGN. Since most of the ricin molecules remain bound to their binding sites at the low pH prevailing in compartments of the endocytic pathway, the results indicate that a fraction of the internalized plasma membrane molecules with terminal galactose are not recycled directly from endosomes or delivered to lysosomes, but are routed to the Golgi complex. Also, the results presented here, in combination with other recent studies on ricin internalization, suggest that translocation of the toxic ricin A-chain to the cytosol occurs in the TGN.

Animals↗

PubMed1988

Growth factor control of myoepithelial-cell differentiation in cultures of human mammary gland.

The relationship between growth and cytodifferentiation was studied in cultured human mammary myoepithelial cells under serum-free culture conditions. Myoepithelial-cell differentiation was monitored by quantifying cells showing immunoreactivity to the muscle isoform of actin; to the membrane glycoprotein common acute lymphoblastic leukemia antigen (CALLA); and to type IV collagen. Growth was quantified either by measuring the actual increase in cell number, or in a more-sensitive assay using immunoreactivity to the cell-proliferation-associated nuclear antigen Ki-67 as a measurement of the number of cells leaving the G0-phase of the cell cycle. The results showed that: (a) Primary cultures of myoepithelial cells on DME-F12 supplemented with cholera toxin (CT) alone resulted in the formation of quiescent cell islets (in the G0-phase of the cell cycle) showing phenotypic traits preserved from the in vivo situation (actin- and CALLA-positive cells with little or no type-IV-collagen immunoreactivity). (b) After addition of epidermal growth factor (EGF), with an ED50 of 1-10 ng/ml, in the presence of CT, the cells entered the G1-phase of the cell cycle, without further increase in cell number. At the same ED50 of EGF, the frequency of CALLA-positive cells decreased, while the number of cells immunoreactive for type IV collagen increased with a maximal effect of EGF seen after 7-11 days. During the same period, the cells remained fully differentiated with respect to actin immunoreactivity. (c) Further addition of insulin (I) to the medium in the presence of EGF and CT resulted in the cells entering an exponential growth phase associated with simultaneous decrease in actin immunoreactivity with a maximal effect of I after 11 days of exposure. The dose-response curve to I was virtually identical for stimulating cell proliferation and for reducing the frequency of actin-immunoreactive cells (ED50 in the range of 30 ng/ml), suggesting that the two processes were controlled by the same initial I-receptor interaction. (d) Some reduction in the number of actin-positive cells was exerted by I-EGF-CT independently of the mitogenic response, but this reduction was further augmented if the cells were allowed to proliferate. (e) Time-course studies of quiescent (G0-phase) cells stimulated to exponential growth revealed that entrance of cells into the G1-phase of the cell cycle preceded the loss of muscle actin filaments. (f) Exponentially growing actin-negative epithelial cells did not resume a myoepithelial phenotype in density-arrested postconfluent cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1987

Frequency and distribution of estrogen receptor-positive cells in normal, nonlactating human breast tissue.

We have analyzed the frequency and distribution of cells expressing estrogen receptor (ER) in cryosections of normal human breast tissue using quantitative microspectrophotometry and the estrogen receptor immunocytochemical assay. We found that the human mammary gland contained a small but distinct population of ER-positive cells, comprising approximately 7% of the total epithelial cell population from all biopsies. Stromal cells were found to be ER negative. The ER-positive cells were distributed as scattered single cells, with the highest frequency and intensity of measured staining in the lobules as compared to the interlobular ducts. Moreover, on the average, 87% of the ER-positive cells were luminal epithelial cells or occupied an intermediate position in the duct wall. The intermediate cells were found not to express basal cell phenotype as determined by combined immunocytochemistry to ER and "common acute lymphoblastic leukemia antigen" selectively decorating myoepithelial cells (B.A. Gusterson et al., J. Natl. Cancer Inst., 77: 343-349, 1986).

Adolescent↗

PubMed1987

Preservation of defined phenotypic traits in short-term cultured human breast carcinoma derived epithelial cells.

Interpretation of primary monolayer culture of organs and tissues with different epithelial cell types demands well-defined criteria for distinguishing between such cells. Epithelial components in breast carcinomas comprise, in addition to carcinoma epithelial cells (CEP), at least two epithelial cell types organized in mammary ductules of normal appearance as an inner layer of luminal epithelial cells (LEP) and an outer layer of basal or myoepithelial cells (MEP) resting on a basement membrane. In a previous study (Petersen and van Deurs, Cancer Res., 46: 2013-2020, 1986) we have defined a population of CEP in vivo and in vitro, appearing in about 50% of primary carcinomas, by a cytochemical reaction for reduced nicotinamide adenine dinucleotide phosphate neotetrazolium reductase. Carcinoma derived epithelial cells not showing this cytochemical reaction in culture were believed to originate from either carcinoma cells or from mammary ductules of normal or benign appearance. In the present study we show that carcinoma derived NADPH-NT reductase negative cell islets often exhibit phenotypic traits of apparently normal mammary ductules as defined in vivo. Moreover, it is shown that the reductase positive CEP, apart from the reductase reaction, has preserved several other features in culture distinguishing them from cells of apparently normal origin. Thus, whereas reductase positive CEP often consisted of only one cell type, as revealed by phase contrast microscopy, some reductase negative cell islets showed a distinct two-cell-type composition. One cell type exhibited cobblestone-like appearance and remained in the center of the islets whereas the other was more loosely arranged and rapidly left the central area by migration below the cobblestone-like cells to the periphery of the islets. Cobblestone-like cells and loosely arranged cells were found by immunocytochemistry to express elements of LEP and MEP phenotype, respectively. LEP phenotype was defined in vivo by expression of milk fat globule membrane antigen and cytokeratins, whereas MEP expressed basement membrane-associated type IV collagen. Computerized image analysis revealed mean population doubling times for cells with MEP phenotype of 1 day and for those with LEP phenotype of 2 days. Both cell types showed a diploid DNA pattern as revealed by fluorimetry. Reduced nicotinamide adenine dinucleotide phosphate neotetrazolium reductase positive CEP expressed milk fat globule membrane antigen and cytokeratins, thus resembling the reductase negative LEP.(ABSTRACT TRUNCATED AT 400 WORDS)

Antigens, Neoplasm↗

PubMed1987

Effect of reduced endocytosis induced by hypotonic shock and potassium depletion on the infection of Hep 2 cells by picornaviruses.

Potassium depletion after a brief exposure of the cells to hypotonic medium was used to inhibit endocytosis from coated pits in Hep 2 cells. After such treatment the endocytic uptake of transferrin was arrested, and electron microscopy revealed that virtually no coated pits were present at the cell surface, while smooth (uncoated) pits were abundant. Under the same conditions the cells were strongly protected against poliovirus, while the cytopathogenic effect of human rhinovirus type 2, HRV 2, was increased. The cytopathogenic effect of encephalomyocarditis (EMC) virus was only slightly affected. Potassium depletion without hypotonic shock reduced the endocytic uptake of transferrin 2-3-fold and the number of coated pits at the cell surface about 3-fold. Furthermore, the cells were not protected against poliovirus after such treatment. The data indicate that the productive uptake of poliovirus occurs by receptor-mediated endocytosis from coated pits, while the productive uptake of the other two picornaviruses may occur by another endocytic pathway. In order to efficiently arrest endocytosis from coated pits in these cells, hypotonic shock seems to be a critical component of the potassium depletion protocol.

Cell Line↗

PubMed1987

Delivery of internalized ricin from endosomes to cisternal Golgi elements is a discontinuous, temperature-sensitive process.

Galactose-terminating membrane glycoproteins and glycolipids on two established human breast carcinoma cell lines were tagged at 4 degrees C with a ricin-horseradish peroxidase conjugate (Ri-HRP). The cells were then incubated for various periods of time at 37 or 18 degrees C. After fixation and diaminobenzidine cytochemistry, the compartments reached by Ri-HRP were studied by analyzing thin serial sections. In both cell types a highly pleomorphic endosomal system comprising vacuolar elements as well as smaller, sometimes branched, tubular elements (tubular endosomes) was revealed at both 37 and 18 degrees C. At 37 degrees C Ri-HRP was consistently observed in flattened cisterns of the Golgi region in 30-40% of the Golgi complexes examined after 30-60 min of incubation. However, no Ri-HRP reached such Golgi elements at 18 degrees C, even after incubation for 180 min. Moreover, at 18 degrees C the ability of ricin to inhibit protein synthesis was virtually abolished, whereas the effect of diphtheria toxin was reduced much less. Following incubation with a monovalent transferrin-HRP conjugate or with unconjugated HRP, no labeling of cisternal Golgi elements was detected. These data indicate that delivery of galactose-terminating membrane molecules from endosomes to the Golgi complex is a discontinuous, temperature-sensitive process and that this process may be required for optimal ricin A-chain translocation.

Breast Neoplasms↗

PubMed1987

Increased plasma membrane traffic in daunorubicin resistant P388 leukaemic cells. Effect of daunorubicin and verapamil.

Numerous studies have indicated that the plasma membrane plays an important role in the development of resistance to anthracycline and vinca alkaloid drugs (pleiotropic resistance). We have previously shown that pleiotropically resistant Ehrlich ascites tumour cells, which are of epithelial origin, have a significantly increased plasma membrane traffic (endo/exocytosis) to the endosomal compartment compared to sensitive cells. The present study, using the same ultrastructural morphometric technique, has demonstrated a similar significant difference in plasma membrane traffic between daunorubicin resistant and sensitive P388 cell lines (which are of lymphoid origin). Furthermore, we have shown that this difference between the P388 sublines is accompanied by an approximately 4 fold increase in the plasma membrane area participating in recycling together with an increased endosomal volume, number and membrane area in resistant cells. Plasma membrane traffic in resistant cells was significantly inhibited by the calcium channel blocker verapamil, a well known modulator of anthracycline resistance, but unaffected by daunorubicin itself. The confirmation of this phenotype in an additional pleiotropically resistant cell type with a different histogenesis further supports a hypothesis of endosomal drug trapping and vesicular extrusion as a possible resistance mechanism.

Animals↗

PubMed1987

Acidification of the cytosol inhibits endocytosis from coated pits.

Acidification of the cytosol of a number of different cell lines strongly reduced the endocytic uptake of transferrin and epidermal growth factor. The number of transferrin binding sites at the cell surface was increased in acidified cells. Electron microscopic studies showed that the number of coated pits at the cell surface was not reduced in cells with acidified cytosol. Experiments with transferrin-horseradish peroxidase conjugates and a monoclonal anti-transferrin receptor antibody demonstrated that transferrin receptors were present in approximately 75% of the coated pits both in control cells and in cells with acidified cytosol. The data therefore indicate that the reason for the reduced endocytic uptake of transferrin at internal pH less than 6.5 is an inhibition of the pinching off of coated vesicles. In contrast, acidification of the cytosol had only little effect on the uptake of ricin and the fluid phase marker lucifer yellow. Ricin endocytosed by cells with acidified cytosol exhibited full toxic effect on the cells. Although the pathway of this uptake in acidified cells remains uncertain, some coated pits may still be involved. However, the data are also consistent with the possibility that an alternative endocytic pathway involving smooth (uncoated) pits exists.

Cell Line↗

PubMed1987

Increase in nonspecific adsorptive endocytosis in anthracycline- and vinca alkaloid-resistant Ehrlich ascites tumor cell lines.

Numerous biochemical and drug transport studies have focused on the role of the plasma membrane or membrane-associated processes in anthracycline and vinca alkaloid resistance. Cationized ferritin, which binds electrostatically to anionic sites on the plasma membrane, was used as a membrane marker in a morphometrical ultrastructural study. The nonspecific adsorptive endocytosis was significantly increased in Ehrlich ascites tumor cell lines resistant to daunorubicin (DNR), doxorubicin, vincristine, and vinblastine compared to that in sensitive cells. Thus cells resistant to DNR internalized 2.04% of their plasma membrane per minute, in contrast to 1.17% in sensitive cells. Because the measured cell surface areas in sensitive and resistant cells were unchanged during the experiments, increased membrane traffic (recycling) from intracytoplasmic compartments to the plasma membrane must have occurred in resistant cells. Possible implications of these findings include a resistance-linked phenotype without significance for resistance per se, an increase in membrane repair, and a connection between increased membrane traffic and increased active drug extrusion in resistant cells.

Adsorption↗

PubMed1987

Epithelial membrane polarity: a stable, differentiated feature of an established human breast carcinoma cell line MCF-7.

Carcinoma cells typically show little or no polarity as compared to normal, differentiated epithelial cells. We have studied polarity in two established human breast carcinoma cell lines, T47D and MCF-7, by various techniques (electron microscopic enzyme- and immunocytochemistry, freeze-fracture) and show that one of them (MCF-7) is characterized by a high degree of polarity. Thus, in contrast to T47D cells, MCF-7 cells in monolayer culture form apical tight junctions, do not allow a ricin-horseradish peroxidase conjugate, which binds to terminal galactose residues on the apical surface, to stain the basolateral membrane domain, and express a surface antigen (MFGM-A) only in the apical surface membrane domain, as do normal mammary epithelial cells in vivo. This polarization is independent of a basement membrane, since it is maintained when MCF-7 cells, which do not deposit type IV collagen themselves, are grown directly on plastic. Moreover, even though MCF-7 cells express estrogen receptors rather homogeneously, estrogen has no effect on this polarity, neither in vitro nor after transplantation to nude mice. We conclude that polarity is a stable, differentiated feature of MCF-7 cells.

Animals↗

PubMed1986

Routing of internalized ricin and ricin conjugates to the Golgi complex.

Receptor-mediated endocytosis and intracellular routing of native ricin, and of ricin conjugated to colloidal gold (Ri-Au) and to horseradish peroxidase (Ri-HRP), have been studied in cultured MCF-7 and Vero cells by electron microscopical techniques including serial section analysis. Both native ricin, as demonstrated by immunoperoxidase cytochemistry, and the ricin conjugates were internalized via a common coated pit-coated vesicle pathway to reach vacuolar and tubulo-vesicular portions of the endosomal system. In addition, native ricin and a purified monovalent fraction of Ri-HRP reached distinct Golgi cisterns, whereas Ri-Au and polyvalent Ri-HRP did not. The results delineate intracellular routing of native ricin and compare it with the routing of different ricin conjugates. Moreover, our study shows that conjugates of a particular ligand (ricin) and various probes (e.g., gold and peroxidase), may be handled differently by cells. Sorting apparently takes place in the endosomal system, allowing some but not other molecules to reach Golgi elements. This sorting seems to depend on the valency of the ricin conjugate.

Biological Transport↗

PubMed1986

Characterization of epithelial membrane antigen expression in human mammary epithelium by ultrastructural immunoperoxidase cytochemistry.

Ultrastructural immunocytochemistry was used to analyze cell surface distribution and intracellular localization of milk fat globule membrane antigen (MFGM-A) in cryosections from human breast carcinomas and benign breast biopsy specimens. The specimens were fixed in formaldehyde and frozen. Cryostat sections were cut at 15 micron, incubated with mouse monoclonal antibody to MFGM-A, and then with a peroxidase-conjugated goat anti-mouse antibody. After glutaraldehyde fixation, the sections were incubated with diaminobenzidine-H2O2 and further processed for electron microscopy. MFGM-A was specific for epithelial cells. MFGM-A staining was strictly confined to the apical surface membrane of normal ductal epithelium, never involving basolateral membranes below the tight junctions. In normal epithelial cells, MFGM-A was readily detected in cisternae of the endoplasmic reticulum (ER), but only to a lesser extent in Golgi complexes and presumptive secretory vesicles. In carcinoma cells, surface staining for MFGM-A was either distributed in a non-polarized manner on the entire cell surface or else was totally absent. In some carcinoma cells without surface-associated MFGM-A, very pronounced intracellular MFGM-A staining was seen in the ER, in the nuclear envelope, and in annulate lamellae. The observations on MFGM-A expression were supported by studies on a cell culture model system.

Antigens, Neoplasm↗

PubMed1986

Demonstration of human breast carcinoma cells in cryosections and primary monolayer cultures of surgical biopsies by neotetrazolium reductase cytochemistry.

The present study describes a cytochemical approach to demonstrate human breast carcinoma cells in cryosections and in primary monolayer cultures from surgical biopsies. The material consisted of biopsies from 52 carcinomas and 29 benign lesions. Cryosections and cultures were incubated to demonstrate NADPH-neotetrazolium reductase in an atmosphere of 99.5% oxygen. Incubation time and section thickness were adjusted to accomplish the same level of reaction in cells of cryosections and corresponding cultures. Positive reaction was thus confined to epithelial elements and to the wall of some smaller blood vessels. More than one-half of the carcinoma cells showed moderate to strong reaction in cryosections from 29 of 52 carcinomas whereas no reaction was seen in ductules of normal appearance adjacent to these carcinoma cells. Positive reaction was seen in epithelial cell islets in primary cultures of 16 of the 40 carcinomas cultured. In cryosections from fibroadenomas and fibrocystic disease specimens only apocrine metaplasia consistently showed positive reactions compared with less than 10% of other ductular profiles in a given cryosection. This pattern of reaction was reflected in the derived primary cultures in which positive reaction was found in epithelial cell islets in only one of 19 biopsies cultured. The presence of human milk fat globule membrane antigen was used to demonstrate the epithelial nature of the cell islets seen in cultures of biopsies from both benign lesions and carcinomas. NADPH-neotetrazolium reductase positive islets from carcinoma biopsies were frequently aneuploid whereas most negative islets from carcinoma biopsies were diploid as were all islets from benign tissues.

Adenofibroma↗

PubMed1985

Effect of oxygen on the tetrazolium reaction for glucose 6-phosphate dehydrogenase in cryosections of human breast carcinoma, fibrocystic disease and normal breast tissue.

We here report a method for the characterization of carcinoma cells in cryosections of human breast tissue. Cryosections from 37 biopsies including carcinomas, fibrocystic disease specimens and reduction mammoplasties were incubated in an atmosphere of either pure nitrogen or 99.5% oxygen. The incubation medium contained neotetrazolium, phenazine methosulphate and the colloid stabilizer Polypep 5115. A marked overlap in formation of reaction product was found between carcinomas, benign tissue and normal breast tissue when sections were incubated in pure nitrogen. However, following 5 min of oxygen incubation no reaction was seen in normal breast tissue, whereas carcinoma cells consistently showed reaction. Moreover, a statistically significant difference was seen between the level of reaction in carcinoma cells and that of ductal epithelial cells in fibrocystic disease not containing apocrine metaplasia. Apocrine metaplasia, on the other hand, showed the highest activity recorded at all.

Breast↗

PubMed1985

Characterization of epithelial cell islets in primary monolayer cultures of human breast carcinomas by the tetrazolium reaction for glucose 6-phosphate dehydrogenase.

Epithelial cell islets in primary monolayer cultures of human breast biopsies were characterized by combined immuno-, enzyme- and DNA cytochemistry as well as by analysis of attachment-, spread- and growth patterns. For cultivation we used explants from reduction mammoplasties, benign lesions, primary carcinomas and metastases. Milk fat globule membrane antigen (MFGM-A) was detected with a monoclonal antibody, and the tetrazolium reaction for glucose 6-phosphate dehydrogenase (G6PDH) as well as DNA content of the cultured cells were quantified. Spreading and growth of individual islets were studied by image analysis. Fibroblast-like cells did not express MFGM-A, and whereas epithelial (MFGM-A positive) cell islets of normal and benign origin showed cells with no or low G6PDH reaction, respectively, the majority of epithelial cell islets from 11 out of 21 carcinomas showed strong reaction. Cell islets with strong G6PDH reaction were sometimes hyperdiploid. Moreover, whereas cell islets with no or low reaction from both benign lesions and carcinomas readily attached and spread in a serum-free medium and showed population doubling times of 30 to 110 h, cell islets with strong reaction from carcinomas and metastatic lesions required serum for attachment and their growth rate was too low to be determined.

Antigens, Surface↗

PubMed1985

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.

Animals↗