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H J Geuze

Publications and source records attributed to H J Geuze.

156 records · Page 9Linked to original sources
PubMed1983

The pathway of the asialoglycoprotein-ligand during receptor-mediated endocytosis: a morphological study with colloidal gold/ligand in the human hepatoma cell line, Hep G2.

The human hepatoma cell line, Hep G2, was used as a model system to delineate the morphological aspects of receptor-mediated endocytosis of asialoglycoproteins (ASGP). These hepatoma cells, which have several structural features in common with liver parenchymal cells, contain a well-differentiated endocytotic apparatus which includes coated vesicles and CURL (compartment of uncoupling receptor and ligand) similar to that found in rat liver cells in vivo (Geuze et al., 1983). In order to precisely identify the route of ligand from the cell surface to the CURL sorting vesicles, Hep G2 cells were allowed to endocytose in synchrony ASGP bound to 12 nm colloidal gold for 5 to 60 min. Thereafter the subcellular distribution of the gold particles was examined in ultrathin cryosections. This study demonstrates that, once internalized, ASGP ligand enters a system of CURL tubules prior to delivery to the lumenal contents of attached endocytotic vesicles. These vesicles then in turn transform into multivesicular bodies and secondary lysosomes. Thus, the tubular portions of CURL provide the entry for ASGP ligand into the sorting compartment.

Asialoglycoprotein Receptor↗
PubMed1983

Albumin localization in rat liver parenchymal cells.

In this study, we re-investigated the localization of albumin in hepatocytes using immunocytochemistry (ICC) on ultrathin cryo-sections of rat livers, which were immersion or perfusion-fixed with paraformaldehyde or a mixture of paraformaldehyde and glutaraldehyde. The sections were indirectly labelled with rhodamine-conjugated IgG or with 8 nm protein A-colloidal gold particles. We demonstrate that perfusion-fixation is obligatory for a proper localization of albumin. After perfusion fixation, all hepatocytes of fed and fasted rats were uniformly labelled. At the subcellular level albumin was localized in hepatocytes in the membrane-bound compartments of the entire secretory pathway, i.e. the rough and smooth ER, peripheral Golgi vesicles, all Golgi cisternae and the secretory granules. Concentration of albumin appeared mainly to occur at the cis-Golgi side.

Albumins↗
PubMed1982

Recycling of the asialoglycoprotein receptor: biochemical and immunocytochemical evidence.

One of the best documented systems of receptor-mediated endocytosis is the clearance of asialoglycoproteins (ASGP) from the blood plasma by liver parenchymal cells. There are 200 000-500 000 ligand binding sites per cell, which makes this system favourable for molecular studies of receptor function. By using both biochemical and immunocytochemical approaches, we have obtained evidence for receptor recycling. We have also localized the intracellular site at which the endocytosed receptor and ligand dissociate. The human hepatoma cell Hep G2 contains abundant ASGP receptors (approximately 225 000 per cell). In growing cells approximately 85% of the functional receptors are on the cell surface and the remaining 15% are internal. The maximal rate of ligand uptake in this cell system at 37 degrees C is approximately 30 000 molecules per cell per minute. Each functional receptor can therefore bind and internalize more than 50 ligand molecules during a 6 h period (in the absence of new receptor synthesis), or one ligand each 8 min. To follow both ligand and receptor during their common endocytosis and to visualize the compartment in which the dissociation of ligand from receptor occurs, we have used our recently developed double-labelling immunocytochemical electron microscopic techniques with purified antibodies against ASGP ligand and ASGP receptor. In normal rat hepatocytes, both ligand and receptor are taken up from the sinusoidal cell surface in clathrin-coated vesicles. Both receptor and ligand are associated with the membrane of small clathrin-coated vesicles close to the cell surface. Larger vesicles, farther removed from the surface, contain ligand accumulated within the lumen. The membranes of these larger vesicles contain little receptor, but receptor was concentrated in detached vesiculotubular extensions, which were largely free of ligand. These vesicles represent the compartment of uncoupling of receptor and ligand (CURL) during their common endocytosis. Ligand contained within the vesicle lumen is then transferred to multivesicular bodies and lysosomes; the tubular extensions may carry receptor back to the cell surface.

Animals↗
PubMed1982

Immunocytochemical localization of the receptor for asialoglycoprotein in rat liver cells.

We used high-resolution immunocytochemistry on ultrathin frozen sections labeled with colloidal gold to study the subcellular distribution of the asialoglycoprotein receptor in rat liver. The receptor was localized along the entire hepatocyte plasma membrane, including the bile capillary membrane, but was scarce intracellularly. Sinusoidal lining (Kupffer) cells and blood cells showed no immunoreactivity. In liver cells of rats injected with 1 to 100 micrograms of asialoorosomucoid (ASOR) 2-15 min before tissue fixation, endocytotic internalization of receptors at the blood front was conspicuous. At all times in this interval, receptor was present in approximately 100-nm vesicles and larger vacuoles adjacent to the sinusoidal plasma membrane. No other significant intracellular receptor was noted during the 15-min exposure to ASOR; in particular, lysosomes and Golgi complex were not labeled. Our observations, in combination with data from the literature which demonstrate that, under these conditions, the ligand is transferred further to the Golgi complex-lysosome region, suggest that the receptor and ligand are dissociated in the vicinity of the plasma membrane, after which the receptor rapidly returns to the cell surface.

Animals↗
PubMed1982

Immunocytochemical localization of beta-glucuronidase in the male rat preputial gland.

Present knowledge of the in situ intracellular localization of acid hydrolases is mainly based on enzyme-cytochemical observations. In the preputial gland cells beta-glucuronidase and acid phosphatase were thus demonstrated in lysosome-like secretory granules and the GERL system. We applied immunocytochemistry to localize beta-glucuronidase at the earliest sites of biosynthesis. Lysosomal beta-glucuronidase was purified from preputial gland by column chromatography and SDS gel electrophoresis. Antibodies were raised in rabbit and affinity-purified preparations were used for immunocytochemistry on thin frozen sections of perfusion fixed preputial glands. Indirect procedures were applied with a second antibody labelled with rhodamine for fluorescence, and 5 or 8 nm protein A-gold probes for electron microscopy. beta-Glucuronidase occurred in all cells, except for the precursor cells, and was localized throughout the endoplasmic reticulum and perinuclear space, in all Golgi cisternae and storage granules, and in autophagic vacuoles. Thus in the preputial gland cell, beta-glucuronidase is present in both the lysosomal and the secretory system.

Animals↗
PubMed1981

Use of colloidal gold particles in double-labeling immunoelectron microscopy of ultrathin frozen tissue sections.

Complexes of protein-A with 5 and 16 nm colloidal gold particles (PA/Au5 and PA/Au16) are presented as sensitive and clean immunoprobes for ultrathin frozen sections of slightly fixed tissue. The probes are suitable for indirect labeling and offer the opportunity to mark multiple sites. The best procedure for double labeling was to use the smaller probe first, i.e., antibody 1 - PA/Au5 - antibody 2 - PA/Au16. When this was done, no significant interference between PA/Au5 and PA/Au16 occurred. Using this double-labeling procedure we made an accurate comparison between the subcellular distributions of amylase as a typical secretory protein and of GP-2 a glycoprotein, characteristic for zymogen granule membrane (ZGM) preparations. We prepared two rabbit antibodies against GP-2. One antibody (R x ZGM) was obtained by immunizing with native membrane material. The specificity of R x ZGM was achieved by adsorption with the zymogen granule content subfraction. The other, R x GP-2, was raised against the GP-2 band of the SDS polyacrylamide profile of ZGM. We found that the carbohydrate moiety of GP-2 was involved in the antigenic determinant for R x ZGM, while R x GP-2 was most likely directed against GP-2 polypeptide backbone. THe immunocytochemical observations showed that GP-2, on the one hand, exhibited the characteristics of a membrane protein by its occurrence in the cell membrane, the Golgi membranes, and its association with the membranes of the zymogen granules. On the other hand, GP-2 was present in the contents of the zymogen granules and in the acinar and ductal lumina. Also, a GP-2-like glycoprotein was found in the cannulated pancreatic secretion (Scheffer et al., 1980, Eur. J. Cell Biol. 23:122-128). Hence, GP-2 should be considered as a membrane-associated secretory protein of the rat pancreas.

Amylases↗
PubMed1981

Sizing of protein A-colloidal gold probes for immunoelectron microscopy.

Gold particles in colloidal solutions often vary considerably in size. The finest sols (diameter less than 15 nm), especially, are very heterogeneous, as is indicated by coefficients of variance (CV) of 25-35%. We have complexed staphylococcal protein A with gold particles (PA/Au) and then fractionated the preparations by glycerol or sucrose gradient centrifugation into very homogeneous subfractions. In this way, PA/Au probes of almost any size between 4.5 and 15 nm could be prepared. The variation of the gold particles in these fractions resulted in CV's between 9 and 16%. The reactivity of the PA/Au complex was not affected by the gradient procedure, as was shown by single- and double-labeling immunocytochemistry of ultrathin cryosections of rat pancreatic tissue.

Animals↗
PubMed1980

The subcellular localization of immunoglobulin in mouse plasma cells, as studied with immunoferritin cytochemistry on ultrathin frozen sections.

Cells of mineral oil-induced plasmacytomas (MOPC) and normal plasma cells from mouse lymph nodes were used to study the intracellular localization of IgG by means of immunoferritin cytochemistry on ultrathin frozen sections IgG was demonstrated in the rough endoplasmic reticulum, in virus-containing smooth ER of the tumor cells, in peripheral elements at the cis-side of the Golgi complex, and in all Golgi cisternae. It is suggested that the peripheral elements transfer IgG molecules from the RER to the Golgi complex. Vacuoles showing a strong immunoreaction occurred at the trans-side of the Golgi complex. These vacuoles were normal in lymph node plasma cells and were occasionally seen in the tumor plasma cells. It is proposed that these vacuoles carry IgG from the Golgi complex to the cell membrane and, hence, can be considered as secretory vacuoles.

Animals↗
PubMed1980

Disproportional immunostaining patterns of two secretory proteins in guinea pig and rat exocrine pancreatic cells. An immunoferritin and fluorescence study.

Amylase (Am) and chymotrypsinogen (Chtg) were demonstrated in rat and guinea pig exocrine pancreatic cells by immunofluorescence and immunoferritin cytochemistry on thin and ultrathin frozen sections. We describe two observations indicating that Am and Chtg may behave differently in the pre-Golgi phase of their intracellular transport. Firstly, aggregates of material within the RER cisternae of the guinea pig (so-called intracisternal granules) reacted strongly with anti-Chtg, but showed no affinity for anti-Am. Secondly, in both rat and guinea pig, the increase in labeling intensity from cytoplasm (RER) to secretory granules was larger for Chtg than for Am. We hypothesize that the two proteins do not travel in-parallel towards the Golgi complex. Compared with Chtg, Am would lag behind in the RER cisternae.

Amylases↗
PubMedDate not supplied

Quantitative aspects of immunogold labeling in embedded and in nonembedded sections.

We tried to control immunolabeling conditions so that information about antigen concentration could be achieved by quantifying labeling patterns. Working with immunogold labeling procedures in ultrathin cryosections, we observed that differential penetration of immunoreagents causes considerable differences in labeling efficiency between various cell structures. Therefore, in these nonembedded sections, labeling densities can only be used to measure variations in antigen concentration within one cell structure. After embedding the tissue in 30% polyacrylamide (PAA), differences in penetration were negated. The equalizing effect of PAA on the labeling efficiency enabled us to design a simple immunocytochemical method by which concentrations of a protein can be measured in situ at subcellular levels, provided that no variations in the protein's structural conformation occur that would affect its immunoreactivity. In spite of a higher sensitivity observed for Ig-gold, we preferred to use protein A-gold in our system because of the low nonspecific labeling and the more precise antigen detection by the latter immunomarker.

Amylases↗
PubMedDate not supplied

Overall major histocompatibility complex class I expression is not downregulated in cervix cancer, as detected by immunoelectron microscopy.

Downregulation of major histocompatibility complex (MHC) class I molecules in cervix cancer has been proposed as a mechanism for cancer cells to escape immunodetection. By means of light microscopic immunohistochemistry, it has been shown that in 20-70% of cervix cancers MHC class I is downregulated. We have reinvestigated this phenomenon by quantitative immunogold analysis of MHC class I labeling on the plasma membrane of cervix epithelial cells in ten human squamous cancers and ten normal human cervices. We have not found a statistically significant difference in MHC class I expression between normal and cancer cells. The difference with published light microscopic data probably reflects the higher morphologic resolution and quantifiable immunoreactivity of the immunoelectron microscopy.

Adult↗