Search PubMed⌕ Search

Biomedical subjects

G van Meer

Publications and source records attributed to G van Meer.

At least 55 records · Page 3Linked to original sources
PubMed1994

Molecular characterization of phagosomes.

The transformation of newly formed phagosomes into mature phagolysosomes is a process that involves a complex series of interactions between phagosomes and other vacuolar organelles. The machinery required by phagosomes to mediate these interactions is poorly understood. In this study, we allowed human and various rodent cells to take up latex beads whose density facilitates a simple purification of phagosomes using discontinuous sucrose gradients. With this system, we initiated a systematic study of phagosome proteins using two-dimensional gel electrophoresis and the currently available two-dimensional gel protein data bases. By this approach, we were able to recognize a group of polypeptides associated with mouse J774 phagosomes-phagolysosomes including annexin II, annexin VI, the beta-1 and beta-2 subunits of trimeric G proteins, and a group of actin-binding proteins. While the amount of annexin II associated to phagosomes was similar at all times of latex internalization, the levels of annexin VI were higher on late phagosomes. Phospholipid analysis of J774 phagosomes isolated at early and late time points during phagolysosome formation also revealed significant differences in their lipid composition. In the human phagosomes, we resolved over 200 polypeptides on the two-dimensional gels. These included the proteins described in the mouse, as well as 32 polypeptides that were found to be highly enriched in phagosomes, 15 of which are not present in the current data bases. The results demonstrate that the use of latex bead phagosomes is a powerful system to identify key molecules involved in phagolysosome biogenesis.

Animals↗
PubMed1994

The phospholipid composition of extracellular herpes simplex virions differs from that of host cell nuclei.

Enveloped viruses of eukaryotes obtain their membrane by budding through a cellular membrane. Therefore, most frequently the lipid composition of the virion envelope reflects that of the membrane where budding took place. In the case of herpes simplex viruses, nucleocapsids assemble in the nucleus and bud through the inner nuclear membrane. The pathway from the perinuclear space to the extracellular medium is as yet poorly understood. Here we demonstrate that the phospholipid composition of extracellular herpes simplex virions differs from that of nuclei isolated from the infected cells. The viral membrane contains threefold higher concentrations of sphingomyelin and phosphatidylserine. These lipids are typically enriched in the Golgi apparatus and plasma membrane. The data are in agreement with a model in which herpes simplex virus, after budding through the inner nuclear membrane, loses its envelope by fusing with the outer nuclear membrane and obtains a new membrane by budding into a compartment late in the exocytotic pathway, very likely the Golgi apparatus or membranes derived from it. Alternatively, because the perinuclear space is continuous with the ER lumen, the virus after its first budding may be transported through the exocytotic pathway without ever leaving the lumen of the subsequent compartments. In that case, either the virions, while budding through the nuclear membrane select for sphingomyelin and phosphatidylserine, or the original lipids of the viral envelope are exchanged for lipids of an exocytotic membrane, most likely by a transient membrane continuity between the virion and the vesicle by which it is surrounded. Light particles, virus-like particles that lack capsid and DNA but contain tegument and envelope proteins, displayed the same lipid composition as complete herpes simplex virions, suggesting that they also acquired their envelope from a Golgi membrane.

Animals↗
PubMed1994

Localization of O-glycan initiation, sphingomyelin synthesis, and glucosylceramide synthesis in Vero cells with respect to the endoplasmic reticulum-Golgi intermediate compartment.

The identification of an endoplasmic reticulum-Golgi intermediate compartment (ERGIC), defined by the 53-kDa transmembrane marker protein ERGIC-53, has added to the complexity of the exocytic pathway of higher eukaryotic cells. Recently, a subcellular fractionation procedure was established for the isolation of the ERGIC from Vero cells (Schweizer, A., Matter, K., Ketcham, C. M., and Hauri, H.-P. (1991) J. Cell Biol. 113, 45-54) which provides a means to study more precisely the compartmentalization of the various enzymic functions along the early secretory pathway. Here, we have investigated if O-glycan initiation and sphingomyelin synthesis are associated with the ERGIC by analyzing both the responsible enzyme activities and their corresponding products. Moreover, the synthesis of glucosylceramide, the precursor of most glycosphingolipids, was also analyzed. In the purified ERGIC fraction UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (GalNAc transferase) was only minimally enriched, sphingomyelin synthase was not enriched, and UDP-glucose:ceramide-glucosyl transferase specific activity was lower than in the homogenate. On Percoll gradients all three enzymes cofractionated with Golgi markers rather than ERGIC-53. Accordingly, sphingomyelin concentrations were extremely low in the ERGIC fraction. Double immunofluorescence localization of core N-acetylgalactosamine, the product of GalNAc transferase, by monoclonal antibodies against GalNAc-Ser/Thr (Tn antigen) revealed only little apparent overlap with ERGIC-53. This was particularly evident in brefeldin A-treated cells which showed entirely different patterns of Tn antigens and ERGIC-53. The results suggest that in the secretory pathway of Vero cells O-glycan initiation and sphingomyelin as well as glucosylceramide synthesis mainly occur beyond the ERGIC in the Golgi apparatus.

Amino Acid Sequence↗
PubMed1994

Conversion of diacylglycerol to phosphatidylcholine on the basolateral surface of epithelial (Madin-Darby canine kidney) cells. Evidence for the reverse action of a sphingomyelin synthase.

When N-6[7-nitro-2,1,3-benzoxadiazol-4-yl]aminohexanoyl-phosphatidic acid (C6-NBD-PA) is inserted into the plasma membrane of fibroblasts, it is metabolized by the cells to C6-NBD-diacylglycerol (DG), -triacylglycerol, -phosphatidylcholine (PC), and -phosphatidylethanolamine (PE) (Pagano, R. E., Longmuir, K. J., and Martin, O. C. (1983) J. Biol. Chem. 258, 2034-2040). In Madin-Darby canine kidney (MDCK) cells incubated at 10 degrees C with C6-NBD-PA, up to 70% of the newly synthesized C6-NBD-PC but no C6-NBD-PE could be depleted from the basolateral cell surface by the addition of bovine serum albumin to the medium. Preincubation of the cells with [3H]choline for 2 h at 37 degrees C prior to C6-NBD-PA addition at 10 degrees C labeled non-depletable C6-NBD-PC with a specific activity of > 10 times that of the depletable C6-NBD-PC on the basolateral cell surface, indicating that the latter had not been synthesized by the CDP-choline pathway. C6-NBD-DG could substitute for C6-NBD-PA as substrate for both intracellular and surface C6-NBD-PC synthesis. In addition, C6-NBD-PC synthesis on the cell surface was independent of the location of the C6-NBD-chain on the 1- or 2-position, indicating that the reaction occurred by transfer of phosphorylcholine. Using C6-NBD-ceramide, C6-NBD-sphingomyelin (SM) synthesis also was discovered on the basolateral but not on the apical cell surface. The conversion of PC plus ceramide to DG and SM on the basolateral MDCK cell surface suggests that the synthesis of C6-NBD-PC on this surface occurred via the reverse reaction of a SM synthase. Indeed, the surface C6-NBD-PC synthesis was reduced to 40-50% by addition of C6-NBD-ceramide or hydrolysis of cell surface SM by exogenous neutral sphingomyelinase. Since DG activates protein kinase C and ceramide indirectly inhibits this kinase but activates other kinase(s) and phosphatase(s), the phosphocholine transferase at the cell surface may have a regulatory role in signal transduction.

4-Chloro-7-nitrobenzofurazan↗
PubMed1994

VIP21/caveolin, glycosphingolipid clusters and the sorting of glycosylphosphatidylinositol-anchored proteins in epithelial cells.

We studied the role of the association between glycosylphosphatidylinositol (GPI)-anchored proteins and glycosphingolipid (GSL) clusters in apical targeting using gD1-DAF, a GPI-anchored protein that is differentially sorted by three epithelial cell lines. Differently from MDCK cells, where both gD1-DAF and glucosylceramide (GlcCer) are sorted to the apical membrane, in MDCK Concanavalin A-resistant cells (MDCK-ConAr) gD1-DAF was mis-sorted to both surfaces, but GlcCer was still targeted to the apical surface. In both MDCK and MDCK-ConAr cells, gD1-DAF became associated with TX-100-insoluble GSL clusters during transport to the cell surface. In dramatic contrast with MDCK cells, the Fischer rat thyroid (FRT) cell line targeted both gD1-DAF and GlcCer basolaterally. The targeting differences for GSLs in FRT and MDCK cells cannot be accounted for by a differential ability to form clusters because, in spite of major differences in the GSL composition, both cell lines assembled GSLs into TX-100-insoluble complexes with identical isopycnic densities. Surprisingly, in FRT cells, gD1-DAF did not form clusters with GSLs and, therefore, remained completely soluble. This clustering defect in FRT cells correlated with the lack of expression of VIP21/caveolin, a protein localized to both the plasma membrane caveolae and the trans Golgi network. This suggests that VIP21/caveolin may have an important role in recruiting GPI-anchored proteins into GSL complexes necessary for their apical sorting. However, since MDCK-ConAr cells expressed caveolin and clustered GPI-anchored proteins normally, yet mis-sorted them, our results also indicate that clustering and caveolin are not sufficient for apical targeting, and that additional factors are required for the accurate apical sorting of GPI-anchored proteins.

Animals↗
PubMed1994

Glycosphingolipid clusters and the sorting of GPI-anchored proteins in epithelial cells.

We studied the role of the association between glycosylphosphatidylinositol (GPI)-anchored proteins and glycosphingolipid (GSL) clusters in apical targeting using gD1-DAF, a GPI-anchored protein that is sorted differentially by three epithelial cell lines. Differently from MDCK cells, where both gD1-DAF and glucosylceramide (GlcCer) are sorted to the apical membrane, in MDCK Concanavalin A-resistant cells (MDCK-ConAr) gD1-DAF was mis-sorted to both surfaces but GlcCer was still targeted to the apical surface. In both MDCK and MDCK-ConAr cells, gD1-DAF became associated with TX-100 insoluble GSL clusters during transport to the cell surface. In contrast to MDCK cells, the Fischer rat thyroid (FRT) cell line targeted both gD1-DAF and GlcCer basolaterally. Both MDCK and FRT cells had the ability to assemble GSLs into TX-100-insoluble complexes, but, surprisingly, in FRT cells, gD1-DAF did not associate with GSLs and, therefore, remained completely soluble in TX100. This clustering defect in FRT cells correlated with the absence of VIP21/caveolin, a protein localized to both the plasma membrane caveolae and the TNG. This suggests that VIP21/caveolin may have an important role in recruiting GPI-anchored proteins into GSL complexes, necessary for their apical sorting. However, since MDCK-ConAr cells expressed caveolin and clustered GPI-anchored proteins normally, yet mis-sorted them, our results also indicate that clustering and caveolin are not sufficient for apical targeting and that additional factors are required for the accurate apical sorting of GPI-anchored proteins.

4-Chloro-7-nitrobenzofurazan↗
PubMed1993

Differential effects of brefeldin A on transport of secretory and lysosomal proteins.

Brefeldin A (BFA) rapidly blocks anterograde exocytotic transport through the Golgi complex. Sustained retrograde traffic induced by brefeldin A causes redistribution of constituents of the Golgi, but not the trans-Golgi network (TGN), to the endoplasmic reticulum (ER). In the present study on HepG2 cells, we have observed a differential effect of BFA on transport from the TGN of two soluble proteins: alpha 1-antitrypsin as a representative of secretory proteins and cathepsin D as a prototype of lysosomal enzymes. The Golgi complex of HepG2 cells is sensitive to BFA, as within minutes after its addition nearly all activity of three resident Golgi enzymes was recovered in the ER as monitored by cell fractionation on sucrose density gradients. In accordance with this, "high mannose"-glycosylated alpha 1-antitrypsin was retained in or transported back to the ER. "Complex"-glycosylated alpha 1-antitrypsin was neither secreted into the medium nor transported back to the ER. Most of it was retained in vesicles with the buoyant density of Golgi. These vesicles contained the fluid phase endocytotic marker horseradish peroxidase when this was added to the culture medium prior to the BFA, suggesting that the vesicles derived from the TGN. After BFA addition, the compartment became inaccessible to endocytosed horseradish peroxidase. In contrast to blocking transport of complex alpha 1-antitrypsin, BFA did not affect processing of newly synthesized complex-glycosylated procathepsin D (53 kDa) to the mature 31-kDa form. Neither did it interfere with processing of endocytosed procathepsin D. That the mature cathepsin D had indeed reached the lysosomes was verified by Percoll density gradient fractionation. In conclusion, in HepG2 cells, BFA induces two blocks in the secretory pathway: one at the level of the ER-Golgi juncture and the other in the TGN. In contrast, transport from the Golgi complex to the lysosomes and from the plasma membrane to the lysosomes continued.

Biological Transport↗
PubMed1993

Transport and sorting of membrane lipids.

The lipid composition of cellular membranes may seem unnecessarily complex. However, the lipid composition of each membrane is carefully regulated by local metabolism and specificity in transport, marking the functional significance for the cell. Recent research has revealed unexpected discoveries concerning the topology of lipid synthesis, specificity in lipid transport, and the function of lipid and protein microdomains in sorting.

Animals↗
PubMed1993

Assembly of vaccinia virus: role of the intermediate compartment between the endoplasmic reticulum and the Golgi stacks.

Vaccinia virus, the prototype of the Poxviridae, is a large DNA virus which replicates in the cytoplasm of the host cell. The assembly pathway of vaccinia virus displays several unique features, such as the production of two structurally distinct, infectious forms. One of these, termed intracellular naked virus (INV), remains cells associated while the other, termed extracellular enveloped virus (EEV), is released from the cell. In addition, it has long been believed that INVs acquire their lipid envelopes by a unique example of de novo membrane biogenesis. To examine the structure and assembly of vaccinia virus we have used immunoelectron microscopy using antibodies to proteins of different subcellular compartments as well as a phospholipid analysis of purified INV and EEV. Our data are not consistent with the de novo model of viral membrane synthesis but rather argue that the vaccinia virus DNA becomes enwrapped by a membrane cisterna derived from the intermediate compartment between the ER and the Golgi stacks, thus acquiring two membranes in one step. Phospholipid analysis of purified INV supports its derivation from an early biosynthetic compartment. This unique assembly process is repeated once more when the INV becomes enwrapped by an additional membrane cisterna, in agreement with earlier reports. The available data suggest that after fusion between the outer envelope and the plasma membrane, mature EEV is released from the cell.

Endoplasmic Reticulum↗
PubMed1993

Epithelial sphingolipid sorting is insensitive to reorganization of the Golgi by nocodazole, but is abolished by monensin in MDCK cells and by brefeldin A in Caco-2 cells.

In epithelial MDCK and Caco-2 cells, short-chain analogs of glucosylceramide and sphingomyelin are delivered from the Golgi to the cell surface with different apical/basolateral polarities, which results in an apical enrichment of the glycolipid glucosylceramide over the phospholipid sphingomyelin. Here, we have interfered with the integrity of the Golgi complex in various ways and tested the effects on lipid transport and sorting. Nocodazole, which depolymerizes microtubules, dispersed the Golgi over the cytoplasm of MDCK cells and reduced transport of newly synthesized C6-NBD-(N-6[7-nitro-2,1,3-benzoxadiazol-4-yl]aminocaproyl)-glucosy lceramide and C6-NBD-sphingomyelin to the apical surface by 40%. The lipids were not mistargeted to the basolateral surface and upon removal of nocodazole, apical transport recovered. Nocodazole did not affect the apical enrichment of glucosylceramide over sphingomyelin. The ionophore monensin led to swelling of the Golgi of MDCK cells and inhibited lipid transport to the cell surface by 30-50%. Whereas sphingomyelin transport to both surface domains was equally affected, monensin mainly inhibited apical transport of glucosylceramide. At 10-20 microM of monensin, the two lipids displayed the same polarity of delivery: sorting between the two lipids was abolished. Brefeldin A at 1 microgram/ml, which resulted in disruption of the Golgi in HepG2 cells and completely inhibited protein secretion, had no inhibitory effect on transport of the C6-NBD-lipids to the surface. The same was observed in Caco-2 cells. However, brefeldin A selectively shifted transport of sphingomyelin towards the apical direction which abolished the apical enrichment of glucosylceramide over sphingomyelin. Caco-2 cells were used because in MDCK cells brefeldin A did not change Golgi structure nor lipid transport and sorting. In summary, modification of the Golgi by monensin and brefeldin A, but not nocodazole, interfered with the sorting event by which glucosylceramide is enriched over sphingomyelin in the transport pathway from the Golgi to the apical surface.

Animals↗
PubMed1992

Epithelial sphingolipid sorting allows for extensive variation of the fatty acyl chain and the sphingosine backbone.

In kidney MDCK and intestinal Caco-2 epithelial cells, glucosylceramide (GlcCer) and sphingomyelin (SPH) synthesized from the short-chain sphingolipid analogue N-6-[7-nitro-2,1,3-benzoxadiazol-4-yl]aminodecanoyl (C6-NBD)-ceramide are delivered to the cell surface with apical/basolateral polarities of 2-4 and 0.6-0.9 respectively. We have tested how variations in the lipid backbone affect these polarities. First, the C6-NBD moiety was replaced by a bare [14C]octanoyl chain or by the even more bulky fluorophores 8-bimanoylthio-octanoyl (C8-bimane) and 8-diethylaminocoumarin-octanoyl (C8-DECA). In addition, the sphingosine in C6-NBD-ceramide was changed in stereoconfiguration (L-threo) or saturation (dihydro). In all cases, GlcCer and SPH were produced and appeared on the cell surface at 37 degrees C, as assayed by back-exchange. The apical/basolateral polarity of the delivery of GlcCer was variable, but always exceeded 1. GlcCer was apically enriched over SPH (2-6 times for MDCK and 3-9 times for Caco-2). Even GlcCer synthesized from a highly water-soluble truncated ceramide (octanoyl-D-erythro-sphingosine analogue with C8 backbone) was enriched apically by a factor of greater than or equal to 2 both in absolute polarity and compared with SPH. Sphingolipid sorting was quantitatively but not qualitatively affected by dramatic changes in the lipid backbone.

Animals↗
PubMed1992

Glucosylceramide is synthesized at the cytosolic surface of various Golgi subfractions.

In our attempt to assess the topology of glucosylceramide biosynthesis, we have employed a truncated ceramide analogue that permeates cell membranes and is converted into water soluble sphingolipid analogues both in living and in fractionated cells. Truncated sphingomyelin is synthesized in the lumen of the Golgi, whereas glucosylceramide is synthesized at the cytosolic surface of the Golgi as shown by (a) the insensitivity of truncated sphingomyelin synthesis and the sensitivity of truncated glucosylceramide synthesis in intact Golgi membranes from rabbit liver to treatment with protease or the chemical reagent DIDS; and (b) sensitivity of truncated sphingomyelin export and insensitivity of truncated glucosylceramide export to decreased temperature and the presence of GTP-gamma-S in semiintact CHO cells. Moreover, subfractionation of rat liver Golgi demonstrated that the sphingomyelin synthase activity was restricted to fractions containing marker enzymes for the proximal Golgi, whereas the capacity to synthesize truncated glucosylceramide was also found in fractions containing distal Golgi markers. A similar distribution of glucosylceramide synthesizing activity was observed in the Golgi of the human liver derived HepG2 cells. The cytosolic orientation of the reaction in HepG2 cells was confirmed by complete extractability of newly formed NBD-glucosylceramide from isolated Golgi membranes or semiintact cells by serum albumin, whereas NBD-sphingomyelin remained protected against such extraction.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1992

Sphingolipid trafficking--sorted out?

Studies of intracellular membrane traffic have traditionally focused on the protein components of membranes, but what about lipids? Recent findings have drawn attention to the transport of one type of lipid, the sphingolipids. Their unique physical properties may allow them to aggregate into microdomains in membranes that concentrate sphingolipids into specific transport pathways. Gerrit van Meer and Koert Burger consider here the routes of sphingolipid biosynthesis and transport, and the role of proteins in their targeting. The following article by Deborah Brown turns the tables to review the evidence suggesting that sphingolipid domains are important in specific targeting of GPI-anchored proteins to the plasma membrane.

Journal Article↗
PubMed1991

Subcellular localization of Forssman glycolipid in epithelial MDCK cells by immuno-electronmicroscopy after freeze-substitution.

Forssman antigen, a neutral glycosphingolipid carrying five monosaccharides, was localized in epithelial MDCK cells by the immunogold technique. Labeling with a well defined mAb and protein A-gold after freeze-substitution and low temperature embedding in Lowicryl HM20 of aldehyde-fixed and cryoprotected cells, resulted in high levels of specific labeling and excellent retention of cellular ultrastructure compared to ultra-thin cryosections. No Forssman glycolipid was lost from the cells during freeze-substitution as measured by radio-immunostaining of lipid extracts. Redistribution of the glycolipid between membranes did not occur. Forssman glycolipid, abundantly expressed on the surface of MDCK II cells, did not move to neighboring cell surfaces in cocultures with Forssman negative MDCK I cells, even though they were connected by tight junctions. The labeling density on the apical plasma membrane was 1.4-1.6 times higher than basolateral. Roughly two-thirds of the gold particles were found intracellularly. The Golgi complex was labeled for Forssman as were endosomes, identified by endocytosed albumin-gold, and lysosomes, defined by double labeling for cathepsin D. In most cases, the nuclear envelope was Forssman positive, but the labeling density was 10-fold less than on the plasma membrane. Mitochondria and peroxisomes, the latter identified by catalase, remained free of label, consistent with the notion that they do not receive transport vesicles carrying glycosphingolipids. The present method of lipid immunolabeling holds great potential for the localization of other antigenic lipids.

Animals↗
PubMed1991

Preservation and immunogold localization of lipids by freeze-substitution and low temperature embedding.

The success of post-embedding immunocytochemistry depends largely on the preparation methods. The requirements for structural preservation and immunocytochemistry are in some cases contradictory. This is especially the case in the study of lipid-rich structures and the localization of lipid components. Earlier work on freeze-substitution has shown that this method is very promising for the preservation of lipids and the immunocytochemical localization of lipids at the electron microscopical level. In this study we show that freeze-substitution in combination with low temperature embedding in Lowicryl HM20 has fulfilled this promise. Lamellar bodies in alveolar type II cells contain about 90% lipids and are very difficult to preserve in ultrathin cryosections. Lowicryl sections of freeze-substituted lung tissue shows excellent preservation of lamellar bodies in combination with immunogold localization of a hydrophobic surfactant protein. With an antibody against the Forssman glycolipid we demonstrate a highly reproducible intracellular localization of this glycolipid with high specificity and resolution. This method results in the retention of lipids and glycolipids and allows postembedding immunogold labeling.

Acrylic Resins↗
PubMed1990

Generation of lipid polarity in intestinal epithelial (Caco-2) cells: sphingolipid synthesis in the Golgi complex and sorting before vesicular traffic to the plasma membrane.

Generation of intestinal epithelial lipid polarity was studied in Caco-2 cells. Confluent monolayers on filters incorporated the exchangeable lipid N-6-NBD-aminocaproyl-sphingosine (C6-NBD-ceramide) from liposomes. The fluorescent ceramide was converted equally to C6-NBD-glucosylceramide and C6-NBD-sphingomyelin, analogues of lipids enriched on the apical and basolateral surface, respectively, of intestinal cells in vivo. Below 16 degrees C, where vesicular traffic is essentially blocked, each fluorescent product accumulated in the Golgi area. At 37 degrees C, 50% had been transported to the cell surface within 0.5 h, as measured by selective extraction of the fluorescent lipids onto BSA in the medium ("back-exchange") at 10 degrees C. Transport to the two surfaces could be assayed separately, as a diffusion barrier existed for both NBD-lipids and BSA. C6-NBD-glucosylceramide was enriched twofold apically, whereas C6-NBD-sphingomyelin was equally distributed over both domains. Polarities did not decrease when 37 degrees C incubations were carried out in the presence of increasing BSA concentrations to trap the fluorescent lipids immediately after their arrival at the cell surface. Within 10 min from the start of synthesis, both products displayed their typical surface polarity. Lipid transcytosis displayed a half time of hours. In conclusion, newly synthesized sphingolipids in Caco-2 cells are sorted before reaching the cell surface. Transcytosis is not required for generating the in vivo lipid polarity.

Animals↗