Biomedical subjects

H J Geuze

Publications and source records attributed to H J Geuze.

At least 145 records · Page 8Linked to original sources

PubMed1986

Intracellular localization of the copper-zinc and manganese superoxide dismutases in rat liver parenchymal cells.

Both the copper-zinc (CuZn) and the manganese (Mn) containing superoxide dismutases (SOD) have been immunolocalized in rat liver sections using protein A-gold labeling on ultrathin cryosections. The CuZnSOD was found to distribute uniformly throughout the nuclear and cytoplasmic matrix. The CuZn enzyme was excluded from membrane-bound compartments such as the nuclear envelope, endoplasmic reticulum, Golgi elements, secretory vesicles, and mitochondria. The primary exception was that lysosomes frequently labeled heavily for CuZnSOD. The lysosomal nature of these membrane-bound bodies was confirmed by double immunolabeling with a lysosomal enzyme, cathepsin-D. The MnSOD was located in mitochondria, particularly the matrix between the cristae. Light but distinct labeling for the MnSOD occurred in the cytoplasmic matrix.

Animals↗

PubMed1985

Immunoelectron microscopic localization of acidic intracellular compartments in hepatoma cells.

Using protein A-colloidal gold immunoelectron microscopy and monospecific antibodies to the weak base primaquine, we have delineated acidic intracellular compartments in the human hepatoma cell line, HepG2. Primaquine specifically accumulated within endocytotic compartments (including CURL vesicles, multivesicular bodies and lysosomes). In addition, the Golgi cisternae were positive. However, the CURL tubules, which contain recycling asialoglycoprotein receptor, did not accumulate primaquine. Thus, there may be a gradient of acidification within the endocytotic pathway.

Antibodies↗

PubMed1985

Cell biology of the asialoglycoprotein receptor system: a model of receptor-mediated endocytosis.

Substantial information about the ASGP-R has accumulated in the 10 years following the initial studies of this receptor by Ashwell and Morell. Many of its biochemical properties, its structure, and its orientation within the plasma membrane are now known. The pathways of ASGP ligand and receptor, with the CURL organelle being a central component, are summarized in Fig. 18. The major pathway of the ligand through the cell, beginning with binding at the cell surface and ending with degradation in lysosomes, has been investigated in detail. Recently, alternate routes of the ligand such as the ligand recycling pathway have been observed. With regard to the itinerary of the receptor, there is now biochemical, kinetic, and morphological evidence to support receptor recycling. The new concept of CURL as an important intracellular organelle has originated from studies of ASGP-R recycling. Its importance in the dissociation and segregation of ligand and receptor as well as in receptor recycling is now evident. In addition, there has been a concurrent investigation of other receptor systems that participate in receptor-mediated endocytosis, providing parallels and contrasts to the ASGP-R of hepatocytes. Many critical issues still exist in the cell biology of the ASGP-R. What are the structural requirements of the receptor for ligand binding and subsequent endocytosis of the receptor-ligand complex? Very little is known about the interactions between the receptor and the lipid bilayer in which it resides. How does the receptor move laterally in the plasma membrane? Are there proteins or glycolipids closely associated with the ASGP-R and, if so, what is their function? What is the mechanism that causes receptor clustering into coated pits? Although the existence of a pathway for ligand recycling has been demonstrated, there are still many issues to be addressed. What signals a particular ligand molecule for recycling? Is it a stochastic process? What is the function of this route of ligand movement? How are the various ligand pathways coordinated and regulated? In addition, there are many unanswered questions regarding the receptor pathway. How does CURL mediate the sorting of ASGP-R from ligand? How are receptors with different destinations (e.g., ASGP-R and IgA receptor) sorted in CURL? What is the mechanism of ASGP-R degradation and how is it regulated? Finally, how does the Golgi function in the ASGP system and what is the relationship between the Golgi and CURL? Future investigation of these issues will require further observations with existing techniques as well as new approaches.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1985

Possible pathways for lysosomal enzyme delivery.

Immunogold double-labeling and ultrathin cryosections were used to compare the subcellular distribution of albumin, mannose 6-phosphate receptor (MPR), galactosyltransferase, and the lysosomal enzymes cathepsin D, beta-hexosaminidase, and alpha-glucosidase in Hep G2 cells. MPR and lysosomal enzymes were found throughout the stack of Golgi cisternae and in a trans-Golgi reticulum (TGR) of smooth-surfaced tubules with coated buds and vesicles. The trans-Golgi orientation of TGR was ascertained by the co-localization with galactosyltransferase. MPR was particularly abundant in TGR and CURL, the compartment of uncoupling receptors and ligands. Both TGR and CURL also contained lysosomal enzymes, but endogenous albumin was detected in TGR only. The coated buds on TGR tubules contained MPR, lysosomal enzymes, as well as albumin. MPR and lysosomal enzymes were also found in coated pits of the plasma membrane. CURL tubules seemed to give rise to smooth vesicles, often of the multivesicular body type. In CURL, the enzymes were found in the lumina of the smooth vesicles while MPR prevailed in the tubules. These observations suggest a role of CURL in transport of lysosomal enzymes to lysosomes. When the cells were treated with the lysosomotropic amine primaquine, binding of anti-MPR to the cells in culture was reduced by half. Immunocytochemistry showed that MPR accumulated in TGR, especially in coated buds. Since these buds contain endogenous albumin and lysosomal enzymes also, these data suggest that coated vesicles originating from TGR provide for a secretory route in Hep G2 cells and that this pathway is followed by the MPR system as well.

Albumins↗

PubMed1985

Effect of monensin on the metabolism, localization, and biosynthesis of N- and O-linked oligosaccharides of galactosyltransferase.

The effect of monensin on the metabolism and glycosylation of the trans-Golgi enzyme galactosyltransferase (GT) was studied in HeLa cells. The synthesis of GT was not affected by monensin; however, in the presence of the drug GT precursor molecules (Mr 54 000 and 47 000) were transported to the Golgi system at a slower rate than in control cells. the half-life of GT as an intrinsic membrane protein of the trans-Golgi system was prolonged in the presence of monensin. Mature GT (Mr 54 000) contained in addition to one N-linked oligosaccharide several O-linked oligosaccharides; the majority of them consisted of the disaccharide galactose-N-acetylgalactosamine containing one or two sialic acid residues. We conclude that GT resides at an intracellular location where significant sialylation occurs. The sialylation of O-linked oligosaccharides was not hampered by the presence of monensin. Two-dimensional gel electrophoresis combined with neuraminidase treatment revealed that GT consists of a number of species in a pI range between 4 and 7. Removal of sialic acid residues resulted in a less acidic pI range of GT but the charge heterogeneity persisted. Immunoelectron microscopy with antibodies against GT showed that monensin affects primarily the GT-containing cisternae.

Biological Transport, Active↗

PubMed1985

Immunocytochemical localization of acrosin and hyaluronidase in epididymal and ejaculated porcine spermatozoa.

Acrosin and hyaluronidase demonstrated different release patterns following treatment of living spermatozoa with the Ca2+-ionophore A 23187. One hour after the acrosomal reaction about 50% of the acrosin was still associated with the spermatozoal membranes, while hyaluronidase could no longer be detected in the spermatozoal remnants. Strong fixation conditions with acrolein and glutaraldehyde were used to prevent redistribution and leakage of these sperm proteins. Lost antigenicity was restored with sodium-borohydride and pronase E treatment. Immunofluorescent localization showed hyaluronidase to be confined to the anterior portion of the acrosome. Acrosin was localized throughout the entire acrosome including the equatorial segment. By immunoelectron microscopy, hyaluronidase was exclusively found in the acrosomal matrix. The equatorial segment was devoid of hyaluronidase. Acrosin was found in the acrosomal matrix as well as on the outer acrosomal membrane. Furthermore, labeling for acrosin in the equatorial segment was clearly demonstrated. Localization of hyaluronidase was the same for epididymal and ejaculated sperm cells but in approximately 70% of the epididymal spermatozoa acrosin could not be detected in the equatorial segment. In most of these epididymal cells acrosin was confined to the anterior part of the acrosome comparable with hyaluronidase. These observations support the motion that the appearance of acrosin in the equatorial segment is part of the maturation process during passage through the epididymis.

Acrosin↗

PubMed1985

Immuno-electron microscopical demonstration of lysosomes in human blood platelets and megakaryocytes using anti-cathepsin D.

Immunocytochemistry with affinity-purified anti-human cathepsin D was applied to ultrathin frozen sections of human bone marrow megakaryocytes and of blood platelets from peripheral blood. The fixative used was paraformaldehyde (concentration gradient 2----8%). Protein A/colloidal gold (5 and 8) particles were used as second label. Cathepsin D was localized in primary and secondary lysosomes in blood platelets and in primary and secondary lysosomes in megakaryocytes. Primary lysosomes in megakaryocytes were identified by their localization on the trans-side of the Golgi complex and secondary lysosomes by the presence of inclusions. The lysosomes in platelets differed from alpha-granules by being smaller, lacking an electron dense core, and by the presence of a transparent submembrane halo. Platelets undergoing a release reaction after stimulation with thrombin showed cathepsin-D staining in the surface-connecting tubules.

Blood Platelets↗

PubMed1985

A new method of preparing gold probes for multiple-labeling cytochemistry.

A new method is described for preparing colloidal gold particles in any size between approximately 3 and 17 nm for electron microscopy. The gold particles are homogeneous in size (homodisperse). When bound to various proteins (e.g. IgG and protein A), the complexes were stable for long periods and suitable for affinity cytochemistry. We demonstrated the usefulness of the new gold probes bound to protein A for multiple labeling in a current immunocytochemical study on receptor mediated transport of IgA in human duodenal crypt cells. Other gold-protein complexes were useful for studying macromolecular arrangements at high resolution.

Duodenum↗

PubMed1984

Immunocytochemical localization of albumin and factor XIII in thin cryo sections of human blood platelets.

Albumin and factor XIII alpha 2 were localized in human blood platelets by immunofluorescence and protein A-colloidal gold immuno-electron microscopy using thin and ultrathin cryosections of mildly fixed human blood platelets. Albumin was found in alpha-granules, co-distributing with beta-thromboglobulin in double label experiments. In platelets directly fixed in plasma, without previous washing, albumin was also localized on the plasma membrane and in the surface connecting system. Factor XIII alpha 2 was localized diffusely through the entire cytoplasmic matrix.

Blood Platelets↗

PubMed1984

A cycloheximide-resistant pool of receptors for asialoglycoproteins and mannose 6-phosphate residues in the Golgi complex of hepatocytes.

Following in vivo administration of cycloheximide (20 mg/kg body weight i.p.) protein synthesis was completely inhibited (99%) in rat liver. No newly synthesized asialoglycoprotein receptor (ASGP-R) could be detected by metabolic labeling. Fluorescence immunocytochemistry of several secretory proteins and plasma membrane proteins, including the receptors for polymeric IgA (IgA-R), demonstrated a rapid loss from the Golgi complex following cycloheximide administration. On the other hand, two membrane proteins, the receptors for ASGP-R and mannose 6-phosphate (MP-R), were not altered in their cellular localization including the Golgi. Using quantitative immunoelectron microscopy with colloidal gold, we found that 2 h and 4 h after cycloheximide administration, the densities of ASGP-R and MP-R in the membranes of the Golgi complex were unaltered compared with control liver. Similarly, there was no significant effect of cycloheximide on the receptor labeling in coated vesicles and compartment of uncoupling receptors and ligands (CURL). These observations are consistent with an involvement of the Golgi and CURL pools of the receptors in intracellular trafficking, endocytosis and receptor recycling.

Animals↗

PubMed1984

Immunocytochemistry of lysosomal hydrolases and their precursor forms in normal and mutant human cells.

The acid hydrolases alpha-glucosidase, beta-galactosidase, N-acetyl-beta-D-hexosaminidase, beta-glucocerebrosidase and cathepsin D were studied immunocytochemically in normal and mutant human cells using monoclonal and affinity-purified polyclonal antibodies. For light microscopy, Rhodamine or Fluorescein-labelled conjugates were used, and for electron microscopy protein A-gold conjugates were employed. With the double labelling procedure, it was found that in normal fibroblasts every lysosome contained all the enzymes studied. The method described also enabled us to demonstrate the presence or absence of mutant enzyme protein in fibroblasts derived from patients with a genetic lysosomal enzyme deficiency. Immunoreactive acid hydrolases or their precursor forms were found in the rough endoplasmic reticulum, the cisternae of the Golgi complex, Golgi associated vesicles and lysosomes. This is in agreement with the present concept that the Golgi complex plays an essential role in the processing and targeting of lysosomal enzymes.

Endoplasmic Reticulum↗

PubMed1984

Intracellular receptor sorting during endocytosis: comparative immunoelectron microscopy of multiple receptors in rat liver.

Using double-label quantitative immunoelectron microscopy on ultrathin cryosections of rat liver, we have compared the endocytotic pathways of the receptors for asialoglycoprotein (ASGP-R), mannose-6-phosphate ligands (MP-R), and polymeric IgA (IgA-R). All three were found within the Golgi complex, along the entire plasma membrane, in coated pits and vesicles, and within a compartment of uncoupling of receptors and ligand ( CURL ). The receptors occurred randomly at the cell surface, in coated pits and vesicles. Within CURL tubules ASGP-R and MP-R were colocalized , but IgA-R and ASGP-R displayed dramatic microheterogeneity. Thus, in addition to its role in uncoupling and sorting recycling receptor from ligand, CURL serves as a compartment to segregate recycling receptor (e.g. ASGP-R) from receptor involved in transcytosis (e.g. IgA-R).

Animals↗

PubMed1984

Ultrastructural localization of the mannose 6-phosphate receptor in rat liver.

An affinity-purified rabbit antibody against rat liver mannose 6-phosphate receptor (MP-R) was prepared. The antibody was directed against a 215 kd-polypeptide and it recognized both ligand-occupied and free receptor. Anti-MP-R was used for immunofluorescence and immunoelectron microscopy of cryosections from rat liver. MP-R was demonstrated in all parenchymal liver cells, but not in endothelial lining cells. MP-R labeling was found at the entire plasma membrane, in coated pits and coated vesicles, in the compartment of uncoupling receptor and ligand, and in the Golgi complex. Lysosomes showed only scarce MP-R label. In double-labeling immunoelectron microscopy, MP-R co-localized with albumin in the Golgi cisternae and in secretory vesicles with lipoprotein particles. Cathepsin D was associated with MP-R in the Golgi cisternae. This finding indicates that MP-R/cathepsin D complexes traverse the Golgi complex on their way to the lysosomes. The possible involvement of CURL in lysosomal enzyme targeting is discussed.

Animals↗

PubMed1984

Immunocytochemical assays of amylase and chymotrypsinogen in rat pancreas secretory granules. Efficacy of using immunogold-labeled ultrathin cryosections to estimate relative protein concentrations.

An exploration was conducted as to whether the relative concentration of two intracellular proteins could be evaluated quantitatively from their labeling densities in ultrathin cryosections labeled with the immunogold technique. As a model rat pancreatic cells were used in which the content of amylase (Am) and chymotrypsinogen (Ch) was experimentally altered. Rats were fed either normal laboratory chow or food containing soybean trypsin inhibitor (STI), which affects the Am/Ch ratio in the tissues. The changes in Am and Ch protein levels and enzyme activities were measured biochemically in cell suspension homogenates or in zymogen granule fractions. Within 5 days a maximal change in the Am/Ch was observed as a result of adaptation to the STI diet. The Am/Ch ratio determined biochemically was compared with that from counts of gold particles bound to the respective protein in immunogold-labeled cryosections. The two data sets matched fairly well, indicating that the intensity of the immunoreaction is a reliable reflection of antigen concentration in this system.

Amylases↗

PubMed1983

Intracellular site of asialoglycoprotein receptor-ligand uncoupling: double-label immunoelectron microscopy during receptor-mediated endocytosis.

In rats infused with asialoglycoprotein for 60 min, receptor-mediated endocytosis of the ligand occurred exclusively in hepatic parenchymal cells. We have used double-label immunoelectron microscopy on ultrathin cryosections of rat liver to identify the site at which the asialoglycoprotein receptor and its ligand dissociate following their common endocytosis. Asialoglycoprotein receptor, ligand and clathrin were identified and quantitated by the use of monospecific antibodies followed by gold-protein A complexes. Both receptor and ligand were found associated with the membrane of clathrin-coated vesicles close to the cell surface. We identified other vesicles that contained ligand accumulated within the lumen. The membranes of these latter vesicles contained little receptor, but receptor was concentrated in tubular extensions that were largely free of ligand. We call this organelle CURL (compartment of uncoupling of receptor and ligand). CURL vesicles appear to transform into secondary lysosomes, wherein the ligand is degraded. The tubular vesicles are, we propose, an intermediate in recycling the receptor to the cell surface.

Animals↗

PubMed1983

Transport and topology of galactosyltransferase in endomembranes of HeLa cells.

HeLa cell membranes were studied for the distribution and orientation of the Golgi marker enzyme uridine diphosphate-galactose:beta-D-N-acetylglucosamine beta, 1-4 transferase (GT). Short pulse labeling in the presence of [35S]methionine resulted in two precursor species (Mr = 44,000 and 47,000), present in a microsomal fraction with a density of 1.18 g/ml in sucrose, presumably derived from the rough endoplasmic reticulum. Processing of the N-linked oligosaccharide(s) occurred only after the precursor molecules migrated to lighter density fractions, presumably derived from the Golgi complex. The mature GT molecules (Mr = 54,000) contain O-linked oligosaccharides as shown by beta-elimination of metabolically incorporated [3H]galactose. The O-glycosylation occurred mainly in the light density fractions. The topology of GT was studied on membrane fractions after labeling with [35S]methionine as well as immunocytochemically on ultrathin cryosections at the electron microscope level. Our results indicate that both the antigenic determinants of GT as well as polypeptide chain are present intramembraneously and at the luminal side of the membranes of the Golgi complex and rough endoplasmic reticulum.

Animals↗

PubMed1983

Vesicular stomatitis virus glycoprotein, albumin, and transferrin are transported to the cell surface via the same Golgi vesicles.

Human hepatoma cells, infected by vesicular stomatitis virus, offer a good system to study simultaneously the intracellular localization of a well defined transmembrane glycoprotein (VSV-G), a secretory glycoprotein (transferrin), and a nonglycosylated secretory protein (albumin). We used monospecific antibodies in combination with 5- and 8-nm colloidal gold particles complexed with protein A to immunolabel these proteins simultaneously in thin frozen sections of hepatoma cells. VSV-G, transferrin, and albumin are present in the same rough endoplasmic reticulum cisternae, the same Golgi compartments, and the same secretory vesicles. In the presence of the ionophore monensin intracellular transport is blocked at the trans cisternae of the Golgi complex, and VSV-G, transferrin, and albumin accumulate in dilated cisternae, which are apparently derived from the trans-Golgi elements. Glycoproteins, synthesized and secreted in the presence of monensin, are less acidic than those in control cultures. This is probably caused by a less efficient contact between the soluble secretory proteins and the membrane-bound glycosyltransferases that are present in the most monensin-affected (trans) Golgi cisternae.

Albumins↗

PubMed1983

Immunoelectron microscopic exploration of the Golgi complex.

Using immunogold labeling of ultrathin cryosections, we have studied the localization of secretory, lysosomal, and membrane proteins in the Golgi complexes of several cell types. All proteins were present in the stacks of Golgi cisternae, illustrating that the cisternae comprise a ubiquitous way station for proteins with multiple destinations. The labeling patterns support the concept that peripheral Golgi vesicles represent the main site of secretory protein concentration. Of the membrane proteins studied, the Golgi enzyme galactosyltransferase was confined to the trans-most few cisternae, whereas the receptors for asialoglycoproteins and for polymeric immunoglobulin A occurred in most cisternae, with increasing concentration approaching the trans side. The findings are discussed in relation to a cisternal cis to trans progression of Golgi cisternae and membrane specificity.

Animals↗