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E Rodriguez-Boulan

Publications and source records attributed to E Rodriguez-Boulan.

At least 91 records · Page 5Linked to original sources
PubMed1990

Vectorial apical delivery and slow endocytosis of a glycolipid-anchored fusion protein in transfected MDCK cells.

To characterize the mechanisms that determine the apical polarity of proteins anchored by glycosylphosphatidylinositol (GPI), we studied the targeting of a GPI-anchored form of a herpes simplex glycoprotein, gD-1, in transfected MDCK cells. Using a biotin-based targeting assay, we found that GPI-anchored gD-1 was sorted intracellularly and delivered directly to the apical surface. Endocytosis of GPI-anchored gD-1 occurred slowly and preferentially from the apical domain, while transcytosis of the basolateral fraction did not occur at a significant rate (incompatible with being a precursor to the apical pool). Prevention of tight junction formation by incubation in medium with micromolar Ca2+ resulted in expression of GPI-anchored gD-1 on the free surface, but not on the attached surface of the cell. Our results indicate that the apical polarity of a GPI-anchored protein is generated by vectorial delivery to the apical membrane, where its distribution is maintained by slow endocytosis and by a retention system not necessarily involving the tight junction.

Animals↗
PubMed1990

Vectorial targeting of an endogenous apical membrane sialoglycoprotein and uvomorulin in MDCK cells.

We studied the cell-surface delivery pathways of newly synthesized membrane glycoproteins in MDCK cells and for this purpose we characterized an endogenous apical integral membrane glycoprotein. By combining a pulse-chase protocol with domain-selective cell-surface biotinylation, immune precipitation, and streptavidin-agarose precipitation (Le Bivic et al. 1989. Proc. Natl. Acad. Sci USA. 86:9313-9317), we followed the appearance at the cell surface of a major apical sialoglycoprotein, gp114, a basolateral protein, uvomorulin, and a transcytosing protein, the polyimmunoglobulin receptor (pIg-R). We determined that both gp114 and uvomorulin appeared to be delivered directly to their respective surface, with mistargeting levels of 8 and 2%, respectively. Using the same technique, the pIg-R was first detected on the basolateral domain and then on the apical domain, to be finally released into the apical medium, as described (Mostov, K. E., and D. L. Deitcher. 1986. Cell. 46:613-621). To directly determine whether the gp114 pool present on the basolateral surface was a precursor of the apical gp114, we compared it with the equivalent pIg-R pool, by labeling with sulfo-NHS-SS-biotin, a cleavable, tight junction-impermeable probe, and by following the fraction of this probe that became resistant to basal glutathione and accessible to apical glutathione during incubation at 37 degrees C. We found that, contrary to pIg-R, basolateral gp114 was poorly endocytosed and was not transcytosed to the apical side. These results demonstrate that an endogenous apical integral membrane glycoprotein of Madin-Darby canine kidney cells is sorted intracellularly and is vectorially targeted to the apical surface.

Animals↗
PubMed1990

Biogenetic pathways of plasma membrane proteins in Caco-2, a human intestinal epithelial cell line.

We studied the sorting and surface delivery of three apical and three basolateral proteins in the polarized epithelial cell line Caco-2, using pulse-chase radiolabeling and surface domain-selective biotinylation (Le Bivic, A., F. X. Real, and E. Rodriguez-Boulan. 1989. Proc. Natl. Acad. Sci. USA. 86:9313-9317). While the basolateral proteins (antigen 525, HLA-I, and transferrin receptor) were targeted directly and efficiently to the basolateral membrane, the apical markers (sucrase-isomaltase [SI], aminopeptidase N [APN], and alkaline phosphatase [ALP]) reached the apical membrane by different routes. The large majority (80%) of newly synthesized ALP was directly targeted to the apical surface and the missorted basolateral pool was very inefficiently transcytosed. SI was more efficiently targeted to the apical membrane (greater than 90%) but, in contrast to ALP, the missorted basolateral pool was rapidly transcytosed. Surprisingly, a distinct peak of APN was detected on the basolateral domain before its accumulation in the apical membrane; this transient basolateral pool (at least 60-70% of the enzyme reaching the apical surface, as measured by continuous basal addition of antibodies) was efficiently transcytosed. In contrast with their transient basolateral expression, apical proteins were more stably localized on the apical surface, apparently because of their low endocytic capability in this membrane. Thus, compared with two other well-characterized epithelial models, MDCK cells and the hepatocyte, Caco-2 cells have an intermediate sorting phenotype, with apical proteins using both direct and indirect pathways, and basolateral proteins using only direct pathways, during biogenesis.

Alkaline Phosphatase↗
PubMed1989

The distribution of glycosyl-phosphatidylinositol anchored proteins is differentially regulated by serum and insulin.

Glycosyl-phosphatidylinositol (GPI) anchored proteins are surveyed in two insulin sensitive cell types by surface labeling and phospholipase C-induced release into the medium. Serum starvation selectively increases both the number and intensity of a subset of GPI-anchored proteins. After serum starvation, loss of cell-surface GPI-anchored proteins is induced acutely by either serum re-exposure or insulin, suggesting that hormonal treatment may promote the release of these proteins from the cell surface.

Animals↗
PubMed1989

Morphogenesis of the polarized epithelial cell phenotype.

Polarized epithelial cells play fundamental roles in the ontogeny and function of a variety of tissues and organs in mammals. The morphogenesis of a sheet of polarized epithelial cells (the trophectoderm) is the first overt sign of cellular differentiation in early embryonic development. In the adult, polarized epithelial cells line all body cavities and occur in tissues that carry out specialized vectorial transport functions of absorption and secretion. The generation of this phenotype is a multistage process requiring extracellular cues and the reorganization of proteins in the cytoplasm and on the plasma membrane; once established, the phenotype is maintained by the segregation and retention of specific proteins and lipids in distinct apical and basal-lateral plasma membrane domains.

Animals↗
PubMed1989

Integral and peripheral protein composition of the apical and basolateral membrane domains in MDCK cells.

Selective biotinylation of the apical or basolateral domains of confluent MDCK monolayers grown on polycarbonate filters with a water soluble biotin analog, sulfo-NHS-biotin, was employed to reveal strikingly distinct patterns of endogenous "peripheral" and "integral" membrane proteins. "Peripheral" proteins were found to be approximately fivefold more abundant with this procedure than "integral" membrane proteins, both on the apical and on the basolateral surface. The distinct apical and basal patterns were shown to depend upon the integrity of the monolayer; when the tight junctions were disrupted by preincubation in calcium-depleted medium, the patterns appeared practically indistinguishable. Two-dimensional gel electrophoresis demonstrated that only a very small percentage of the biotinylated proteins were found in similar amounts on both apical and basolateral domains. These results indicate that the sorting mechanisms that segregate apical and basolateral epithelial proteins are very strict. The simple procedure described here has clear advantages over other methods available to label apical and basal epithelial surface domains, namely, higher accessibility of the biotin probe to the basolateral membrane, possibility of purifying biotinylated proteins via immobilized streptavidin and minimal exposure of the researcher to isotopes. It should be very useful in characterizing the apical and basolateral protein compositions of other epithelial cells and in studies on the development of epithelial cell polarity.

Animals↗
PubMed1989

Vectorial targeting of apical and basolateral plasma membrane proteins in a human adenocarcinoma epithelial cell line.

We studied the surface delivery pathways followed by newly synthesized plasma membrane proteins in intestinal cells. To this end, we developed an assay and characterized an epithelial cell line (SK-CO-15) derived from human colon adenocarcinoma. Polarized confluent monolayers (2000 omega.cm2), grown on polycarbonate filter chambers, were pulsed with radioactive methionine/cysteine and, at different times of chase, the protein fraction reaching the apical or basolateral surface was recovered by domain-selective biotinylation, immunoprecipitation, and immobilized streptavidin precipitation. Both an apical and a basolateral marker were found to be delivered vectorially to the respective surface, with a sorting efficiency of 50:1 for the basolateral marker and 14:1 for the apical marker.

Adenocarcinoma↗
PubMed1989

Steady-state distribution and biogenesis of endogenous Madin-Darby canine kidney glycoproteins: evidence for intracellular sorting and polarized cell surface delivery.

We used domain-selective biotinylation/125I-streptavidin blotting (Sargiacomo, M., M. P. Lisanti, L. Graeve, A. Le Bivic, and E. Rodriguez-Boulan. 1989 J. Membr. Biol. 107:277-286), in combination with lectin precipitation, to analyze the apical and basolateral glycoprotein composition of Madin-Darby canine kidney (MDCK) cells and to explore the role of glycosylation in the targeting of membrane glycoproteins. All six lectins used recognized both apical and basolateral glycoproteins, indicating that none of the sugar moieties detected were characteristic of the particular epithelial cell surface. Pulse-chase experiments coupled with domain-selective glycoprotein recovery were designed to detect the initial appearance of newly synthesized glycoproteins at the apical or basolateral cell surface. After a short pulse with a radioactive precursor, glycoproteins reaching each surface were biotinylated, extracted, and recovered via precipitation with immobilized streptavidin. Several basolateral glycoproteins (including two sulfated proteins) and at least two apical glycoproteins (one of them the major sulfated protein of MDCK cells) appeared at the corresponding surface after 20-40 min of chase, but were not detected in the opposite surface, suggesting that they were sorted intracellularly and vectorially delivered to their target membrane. Several "peripheral" apical proteins were detected at maximal levels on the apical surface immediately after the 15-min pulse, suggesting a very fast intracellular transit. Finally, domain-selective labeling of surface carbohydrates with biotin hydrazide (after periodate oxidation) revealed strikingly different integral and peripheral glycoprotein patterns, resembling the Con A pattern, after labeling with sulfo-N-hydroxy-succinimido-biotin. The approaches described here should be useful in characterizing the steady-state distribution and biogenesis of endogenous cell surface components in a variety of epithelial cell lines.

Animals↗
PubMed1989

A glycophospholipid membrane anchor acts as an apical targeting signal in polarized epithelial cells.

Glycosyl-phosphatidylinositol- (GPI) anchored proteins contain a large extracellular protein domain that is linked to the membrane via a glycosylated form of phosphatidylinositol. We recently reported the polarized apical distribution of all endogenous GPI-anchored proteins in the MDCK cell line (Lisanti, M. P., M. Sargiacomo, L. Graeve, A. R. Saltiel, and E. Rodriguez-Boulan. 1988. Proc. Natl. Acad. Sci. USA. 85:9557-9561). To study the role of this mechanism of membrane anchoring in targeting to the apical cell surface, we use here decay-accelerating factor (DAF) as a model GPI-anchored protein. Endogenous DAF was localized on the apical surface of two human intestinal cell lines (Caco-2 and SK-CO15). Recombinant DAF, expressed in MDCK cells, also assumed a polarized apical distribution. Transfer of the 37-amino acid DAF signal for GPI attachment to the ectodomain of herpes simplex glycoprotein D (a basolateral antigen) and to human growth hormone (a regulated secretory protein) by recombinant DNA methods resulted in delivery of the fusion proteins to the apical surface of transfected MDCK cells. These results are consistent with the notion that the GPI anchoring mechanism may convey apical targeting information.

Animals↗
PubMed1989

Polarized endocytosis by Madin-Darby canine kidney cells transfected with functional chicken liver glycoprotein receptor.

We have studied the expression of the chicken hepatic glycoprotein receptor (chicken hepatic lectin [CHL]) in Madin-Darby canine kidney (MDCK) cells, by transfection of its cDNA under the control of a retroviral promotor. Transfected cell lines stably express 87,000 surface receptors/cell with a kd = 13 nM. In confluent monolayers, approximately 40% of CHL is localized at the plasma membrane. 98% of the surface CHL is expressed at the basolateral surface where it performs polarized endocytosis and degradation of glycoproteins carrying terminal N-acetylglucosamine at a rate of 50,000 ligand molecules/h. Studies of the half-life of metabolically labeled receptor and of the stability of biotinylated cell surface receptor after internalization indicate that transfected CHL performs several rounds of uptake and recycling before it gets degraded. The successful expression of a functional basolateral receptor in MDCK cells opens the way for the characterization of the mechanisms that control targeting and recycling of proteins to the basolateral membrane of epithelial cells.

Animals↗
PubMed1989

Repolarization of Na+-K+ pumps during establishment of epithelial monolayers.

Madin-Darby canine kidney (MDCK) cells plated at confluence and incubated for 20 h in low (5 microM) Ca2+ have no tight junctions (TJs), and their Na+-K+-ATPase is randomly distributed over the surface. On transfer to normal Ca2+ levels (1.8 mM) ("Ca2+ switch"), TJs and transepithelial resistance develop quickly, trapping a considerable fraction (35%) of the surface Na+-K+-ATPase on the apical (incorrect) side. This misplaced enzyme is subsequently removed from this region or inactivated, demonstrating that polarization proceeds despite TJs. Simultaneously, the amount of Na+-K+-ATPase on the basolateral side increases in a higher proportion (125%), than could be accounted for by relocation of the misplaced apical enzyme. This incorporation is prevented by cycloheximide, ammonium chloride, primaquine, or chloroquine, suggesting that Na+-K+-ATPase originates in an intracellular pool and that its surface insertion requires synthesis of new enzyme or of a protein factor, since it is carried to the surface membrane through a mechanism of exocytosis. In summary, asymmetric distribution of ion pumps depends 1) on polarized insertion of Na+-K+-ATPase as well as 2) on removal or inactivation of misplaced enzyme.

Animals↗
PubMed1988

A polarized epithelial cell mutant deficient in translocation of UDP-galactose into the Golgi complex.

Two lectin-resistant mutants derived from a polarized epithelial cell line have been described (Meiss, H.K., Green, R.F., and Rodriguez-Boulan, E.J. (1982) Mol. Cell. Biol. 2, 1287-1294). One of these mutants, the Madin-Darby canine kidney strain II cell line resistant to Ricinus communis agglutinin (MDCKII-RCAr), has been further characterized, and the biochemical defect leading to its altered phenotype has been determined. MDCKII-RCAr cells are shown to be enriched in cell-surface glycoconjugates bearing terminal N-acetylglucosamine residues by in vitro exogalactosylation and by labeling with fluorescent lectins. Binding assays with a sialic acid-specific lectin reveal a 70-75% reduction in sialylation of cell-surface glycoconjugates. The defect is pleiotropic in nature, affecting glycoproteins as well as glycosphingolipids. Analysis of glycosphingolipids shows a strong reduction of galactose-containing glycosphingolipids. Almost 90% of the glycosphingolipids are identified as glucosyl-ceramide. The mutant is not deficient in galactosyl- and sialytransferase activities. However, Golgi vesicles isolated from MDCKII-RCAr cells translocate UDP-galactose at only 2% of the rate observed for vesicles from wild-type MDCKII cells. The deficiency is specific, because translocation rates of UDP-N-acetylglucosamine and CMP-sialic acid are comparable for vesicles isolated from MDCKII-RCAr cells and wild-type cells. Despite the inability to translocate UDP-galactose into the lumen of the Golgi apparatus, MDCKII-RCAr cells are able to form monolayers with normal apical and basolateral polarity as shown by plasma membrane domain-restricted exogalactosylation.

Animals↗
PubMed1988

Insulin-stimulated release of lipoprotein lipase by metabolism of its phosphatidylinositol anchor.

Lipoprotein lipase (LPL) plays a critical role in the metabolism of plasma lipoproteins. In 3T3-L1 adipocytes, insulin elicits the rapid release of LPL through mechanisms that are independent of energy metabolism and protein synthesis. Some of the metabolic actions of insulin may be mediated by the activation of a specific phospholipase that hydrolyzes a glycosyl phosphatidylinositol (PI) molecule. The insulin-sensitive glycosyl-PI is structurally similar to the glycolipid membrane anchor of a number of proteins. LPL appears to be anchored to the 3T3-L1 cell surface by glycosyl-PI, and its rapid release by insulin may be due to activation of a glycosyl-PI-specific phospholipase C.

Adipose Tissue↗
PubMed1988

Polarized apical distribution of glycosyl-phosphatidylinositol-anchored proteins in a renal epithelial cell line.

Polarized epithelial cell monolayers contain two distinct plasma membrane domains as delineated by the presence of tight junctions--i.e., an apical surface that faces the external environment and a basolateral surface that functions both in cell-cell contact and cell-substrate attachment. Central to the understanding of epithelial cell polarity is the question of how such cell-surface specializations are generated. A different class of membrane glycoproteins has recently emerged that may yield new insight into the mechanism underlying the biogenesis of this polarity. Members of this class contain a large extracellular protein domain linked to the membrane via glycosyl-phosphatidylinositol. Using a polarized renal epithelial cell line (Madin-Darby canine kidney), we identified endogenous glycosyl-phosphatidylinositol-anchored proteins through release by a phosphatidylinositol-specific phospholipase C. Six glycosyl-phosphatidylinositol-anchored proteins of 110, 85, 70, 55, 38, and 35 kDa were identified and appeared to be restricted to the apical surface. Our data are consistent with the notion that the glycosyl-phosphatidylinositol membrane anchor may contain the necessary information for "targeting" to the apical surface.

Animals↗
PubMed1988

Isolation and characterization of the apical surface of polarized Madin-Darby canine kidney epithelial cells.

We have developed a fast and efficient method to isolate the apical surface of Madin-Darby canine kidney epithelial cells. After confluent cell monolayers were coated with alternate layers of cationized colloidal silica and a polyanion, 60% of the apical surface was recovered as large membrane sheets through the application of a polylysine-coated glass surface. Scanning electron microscopy of the cytoplasmic aspect of the apical surface revealed a honeycomb pattern given by the cell borders fractured at or above the level of the tight junctions. By transmission electron microscopy, the apical preparation appeared to be composed of plasma membrane and a thin layer of cytoplasm. Enzyme assays and immunoblots demonstrated a 6- to 7-fold enrichment of an apical marker and a low level of contamination by cytoplasmic and basolateral markers. After removal of cytosolic contaminants and peripheral membrane proteins by alkaline extraction, apical integral membrane proteins were characterized by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (NaDodSO4/PAGE). Lectin blotting and [3H]glucosamine labeling identified two major sialoglycoproteins of apparent Mr 200,000 and 100,000. The apical membrane sheets here described provide a useful model for systematic characterization of the molecular components of the membrane, for reconstitution of lipid and protein transport in cell-free systems, and for study of the interactions of submembranous cytoskeletal proteins with the apical plasma membrane domain.

Animals↗