PubMed Health⌕ Search

Biomedical subjects

E Rodriguez-Boulan

Publications and source records attributed to E Rodriguez-Boulan.

At least 73 records · Page 4Linked to original sources
PubMed1992

Polarized budding of vesicular stomatitis and influenza virus from cultured human and bovine retinal pigment epithelium.

The retinal pigment epithelium (RPE) is able to perform a variety of functions because of its high degree of plasma membrane polarity. Some aspects of this polarity such as the localization of the majority of Na-K ATPase to the apical membrane distinguish the RPE from kidney cells and most other transporting epithelia. The polarized budding of enveloped viruses such as vesicular stomatitis and influenza from the basolateral and apical membrane, respectively, has been used to study mechanisms underlying the domain-specific sorting of membrane proteins in cultured epithelial cell lines. These processes also serve as a useful index of the degree of polarization in epithelial cell cultures. Viral budding from apical and basolateral RPE membranes was used in this study to determine whether the sorting of viral envelope membrane proteins by the RPE is reversed in polarity from that of kidney cells and, if so, whether this might predict a fundamental difference in membrane protein sorting for RPE. The results clearly indicate that the polarity of viral membrane sorting and subsequent viral budding is the same in RPE as in other polarized epithelial cell lines examined to date.

Animals↗
PubMed1991

The role of phosphorylation in development of tight junctions in cultured renal epithelial (MDCK) cells.

We have explored the effect of the protein kinase inhibitor H7 on tight junction formation in a MDCK cell model for the development of cell-cell contact, tight junctions and epithelial polarity: the "Ca++ switch" model. In this developmental model, which is thought to mimic processes during the early morphogenesis of epithelial tissues, the protein kinase inhibitor H7 markedly inhibits the development of transepithelial resistance of confluent MDCK cells during the "switch" from low (1-5 microM) to normal (1.8 mM) Ca++ media compared with control MDCK cells. Moreover, indirect immunofluorescence using specific antisera against two tight junctional proteins, ZO1 and cingulin, revealed that H7 inhibits the sorting of these proteins from an intracellular site to the lateral surfaces of MDCK cells when the Ca++ in the medium is raised. These data suggest protein kinase mediation in sorting events that lead to the assembly of tight junctions.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Targeting of transmembrane and GPI-anchored forms of N-CAM to opposite domains of a polarized epithelial cell.

The calcium-independent neural cell adhesion molecule N-CAM is expressed transiently during development in many tissues, including epithelia. The three naturally occurring principal isoforms of N-CAM differ in the way in which they associate with the membrane and in their cytoplasmic domains. These isoforms are generated by developmentally regulated alternative splicing of a single gene: the large cytoplasmic domain (ld) form (relative molecular mass 180,000 (Mr 180K] is specific for post-mitotic neurons; the 120K small cytoplasmic domain (ssd) and 140K small surface domain (sd) forms also occur on other cell types. One function of the different isoforms could be to specify cellular localization; for example, glycosyl phosphatidyl inositol (GPI)-membrane anchoring acts as a targeting signal for expression on the apical surface of polarized epithelial cells. Neurons and epithelial cells may use similar mechanisms for polarizing their plasma membrane proteins. We have therefore investigated the targeting of GPI-anchored (ssd N-CAM, 120K) and transmembrane forms of N-CAM (sd N-CAM, 140K; ld N-CAM, 180K) by comparing the expression of each after transfection of the appropriate complementary DNAs into polarized epithelial cells. We find that isoforms with alternative modes of membrane association are targeted to different surfaces of polarized epithelial cells: ssd N-CAM is expressed on the apical surface, whereas sd and ld N-CAM are expressed on the basolateral surface. These results suggest that the different isoforms of N-CAM determine their own diverse cellular destinations. They also support the hypothesis that the GPI anchor acts as an apical targeting signal in epithelia.

Animals↗
PubMed1991

Evidence for nuclear targeting of prothymosin and parathymosin synthesized in situ.

To test the hypothesis that prothymosin and parathymosin contain amino acid sequences that cause them to be targeted to the cell nucleus, expression vectors were constructed containing a simian virus 40 promoter and cDNAs that would code for chimeric proteins composed of truncated human growth hormone (hGH) linked to the NH2 terminus of prothymosin or parathymosin. The truncated hGH lacked the signal peptide sequence required for its secretion. After transfection of these constructs into HeLa S3 cells, which do not normally synthesize hGH, the use of indirect immunofluorescence staining to follow the localization of the hGH chimeras demonstrated that both prothymosin and parathymosin caused targeting to the cell nucleus. Controls with a construct coding for native hGH only, and one coding for the truncated hGH lacking the signal peptide, revealed secretion into culture medium and staining in the endoplasmic reticulum and Golgi apparatus in the first case, and diffuse staining throughout the cytoplasm in the second. The results provide direct evidence, with proteins synthesized in situ, for the presence of nuclear localization signals in both prothymosin and parathymosin.

Base Sequence↗
PubMed1991

Mannosamine, a novel inhibitor of glycosylphosphatidylinositol incorporation into proteins.

Mannosamine (2-amino-2-deoxy D-mannose) is shown here to block the incorporation of glycosylphosphatidylinositol (GPI) into GPI-anchored proteins. The amino sugar drastically reduced the surface expression of a recombinant GPI-anchored protein in polarized MDCK cells, converted this apical membrane-bound protein to an unpolarized secretory product and blocked the expression of endogenous GPI-anchored proteins. Furthermore, it specifically inhibited the incorporation of [3H]ethanolamine (a GPI component) into mammalian and trypanosomal GPI-anchored proteins and into a well characterized GPI-lipid of Trypanosoma brucei. These results suggest that mannosamine converted an apical GPI-anchored protein to a non-polarized secretory product by depleting transfer competent GPI-precursor lipids. Our inhibitor studies provide new independent evidence for the apical targeting role of GPI in polarized epithelia and open the way towards a greater understanding of the functional role of GPI in membrane trafficking and cell regulation.

Animals↗
PubMed1991

Apical polarity of Na,K-ATPase in retinal pigment epithelium is linked to a reversal of the ankyrin-fodrin submembrane cytoskeleton.

In striking contrast to most other transporting epithelia (e.g., urinary or digestive systems), where Na,K-ATPase is expressed basolaterally, the retinal pigment epithelium (RPE) cells display Na,K-ATPase pumps on the apical membrane. We report here studies aimed to identify the mechanisms underlying this polarity "reversal" of the RPE Na,K-ATPase. By immunofluorescence on thin frozen sections, both alpha and beta subunits were localized on the apical surface of both freshly isolated rat RPE monolayers and RPE monolayers grown in culture. The polarity of the RPE cell is not completely reversed, however, since aminopeptidase, an apically located protein in kidney epithelia, was also found on the apical surface of RPE cells. We used subunit- and isoform-specific cDNA probes to determine that RPE Na,K-ATPase has the same isoform (alpha 1) as the one found in kidney. Ankyrin and fodrin, proteins of the basolateral membrane cytoskeleton of kidney epithelial cells known to be associated with the Na,K-ATPase (Nelson, W. J., and R. W. Hammerton. 1989. J. Cell Biol. 110:349-357) also displayed a reversed apical localization in RPE and were intimately associated to Na,K-ATPase, as revealed by cross-linking experiments. These results indicate that an entire membrane-cytoskeleton complex is assembled with opposite polarity in RPE cells. We discuss our observations in the context of current knowledge on protein sorting mechanisms in epithelial cells.

Animals↗
PubMed1991

Microtubular organization and its involvement in the biogenetic pathways of plasma membrane proteins in Caco-2 intestinal epithelial cells.

We characterized the three-dimensional organization of microtubules in the human intestinal epithelial cell line Caco-2 by laser scanning confocal microscopy. Microtubules formed a dense network approximately 4-microns thick parallel to the cell surface in the apical pole and a loose network 1-micron thick in the basal pole. Between the apical and the basal bundles, microtubules run parallel to the major cell axis, concentrated in the vicinity of the lateral membrane. Colchicine treatment for 4 h depolymerized 99.4% of microtubular tubulin. Metabolic pulse chase, in combination with domain-selective biotinylation, immune and streptavidin precipitation was used to study the role of microtubules in the sorting and targeting of four apical and one basolateral markers. Apical proteins have been recently shown to use both direct and transcytotic (via the basolateral membrane) routes to the apical surface of Caco-2 cells. Colchicine treatment slowed down the transport to the cell surface of apical and basolateral proteins, but the effect on the apical proteins was much more drastic and affected both direct and indirect pathways. The final effect of microtubular disruption on the distribution of apical proteins depended on the degree of steady-state polarization of the individual markers in control cells. Aminopeptidase N (APN) and sucrase-isomaltase (SI), which normally reach a highly polarized distribution (110 and 75 times higher on the apical than on the basolateral side) were still relatively polarized (9 times) after colchicine treatment. The decrease in the polarity of APN and SI was mostly due to an increase in the residual basolateral expression (10% of control total surface expression) since 80% of the newly synthesized APN was still transported, although at a slower rate, to the apical surface in the absence of microtubules. Alkaline phosphatase and dipeptidylpeptidase IV, which normally reach only low levels of apical polarity (four times and six times after 20 h chase, nine times and eight times at steady state) did not polarize at all in the presence of colchicine due to slower delivery to the apical surface and increased residence time in the basolateral surface. Colchicine-treated cells displayed an ectopic localization of microvilli or other apical markers in the basolateral surface and large intracellular vacuoles. Polarized secretion into apical and basolateral media was also affected by microtubular disruption. Thus, an intact microtubular network facilitates apical protein transport to the cell surface of Caco-2 cells via direct and indirect routes; this role appears to be crucial for the final polarity of some apical plasma membrane proteins but only an enhancement factor for others.

Alkaline Phosphatase↗
PubMed1991

An internal deletion in the cytoplasmic tail reverses the apical localization of human NGF receptor in transfected MDCK cells.

A cDNA encoding the full-length 75-kD human nerve growth factor receptor was transfected into MDCK cells and its product was found to be expressed predominantly (80%) on the apical membrane, as a result of vectorial targeting from an intracellular site. Apical hNGFR bound NGF with low affinity and internalized it inefficiently (6% of surface bound NGF per hour). Several mutant hNGFRs were analyzed, after transfection in MDCK cells, for polarized surface expression, ligand binding, and endocytosis. Deletionof juxta-membrane attachment sites for a cluster of O-linked sugars did not alter apical localization. A mutant receptor lacking the entire cytoplasmic tail (except for the five proximal amino acids) was also expressed on the apical membrane, suggesting that information for apical sorting was contained in the ectoplasmic or transmembrane domains. However, a 58 amino acid deletion in the hNGFR tail that moved a cytoplasmic tyrosine (Tyr 308) closer to the membrane into a more charged environment resulted in a basolateral distribution of the mutant receptor and reversed vectorial (basolateral) targeting. The basolateral mutant receptor also internalized 125I-NGF rapidly (90% of surface bound NGF per hour), exhibited a larger intracellular fraction and displayed a considerably shortened half-life (approximately 3 h). We suggest that hNGFR with the internal cytoplasmic deletion expresses a basolateral targeting signal, related to endocytic signals, that is dominant over apical targeting information in the ecto/transmembrane domains. These results apparently contradict a current model that postulates that basolateral targeting is a default mechanism.

Amino Acid Sequence↗
PubMed1991

An endogenous MDCK lysosomal membrane glycoprotein is targeted basolaterally before delivery to lysosomes.

Using surface immunoprecipitation at 37 degrees C to "catch" the transient apical or basolateral appearance of an endogenous MDCK lysosomal membrane glycoprotein, the AC17 antigen, we demonstrate that the bulk of newly synthesized AC17 antigen is polarly targeted from the Golgi apparatus to the basolateral plasma membrane or early endosomes and is then transported to lysosomes via the endocytic pathway. The AC17 antigen exhibits very similar properties to members of the family of lysosomal-associated membrane glycoproteins (LAMPs). Parallel studies of an avian LAMP, LEP100, transfected into MDCK cells revealed colocalization of the two proteins to lysosomes, identical biosynthetic and degradation rates, and similar low levels of steady-state expression on both the apical (0.8%) and basolateral (2.1%) membranes. After treatment of the cells with chloroquine, newly synthesized AC17 antigen, while still initially targeted basolaterally, appears stably in both the apical and basolateral domains, consistent with the depletion of the AC17 antigen from lysosomes and its recycling in a nonpolar fashion to the cell surface.

Animals↗
PubMed1991

Induction of vacuolar apical compartments in the Caco-2 intestinal epithelial cell line.

Complete disruption of the microtubular network by colchicine or nocodazole in Caco-2 intestinal epithelial cells results in the appearance of basolateral microvilli and brush border-containing intracellular vacuoles (vacuolar apical compartment: VAC). These vacuoles are surrounded by a terminal web, express apical markers and exclude basolateral markers. The vacuoles do not originate from internalized apical or basolateral plasma membrane and their development is blocked by protein synthesis inhibitors, suggesting that they are newly synthesized. After removal of the microtubule inhibitors, VACs are usually degraded and/or released into the lateral intercellular space. Rarely was fusion with the apical membrane observed. These experiments support a role for microtubules in the biogenesis of the apical surface and indicate that, under some conditions, apical plasma membrane assembly may occur in the cytoplasm, as observed in some human pathological states.

Cell Line↗
PubMed1991

Fusion proteins containing a minimal GPI-attachment signal are apically expressed in transfected MDCK cells.

We have shown that addition of the C-terminal 37 amino acids of decay-accelerating factor (DAF) to secretory proteins leads to glycosyl-phosphatidyl-inositol (GPI) anchoring and apical surface expression in MDCK cells. Theoretically, transferred apical sorting information may reside in the glycolipid-anchor moiety or the DAF sequence (9 amino acids) that remains after signal cleavage and GPI attachment. We show here that removal of eight of these nine remaining amino acids, thereby creating a minimal GPI-attachment signal, results in apical expression of GPI-anchored human growth hormone. These data argue that the apical sorting information conveyed by the C terminus of DAF is related to its ability to direct GPI attachment, rather than to a specific sequence that remains in the fusion protein.

Amino Acid Sequence↗
PubMed1991

New techniques lead to advances in epithelial cell polarity.

We have utilized cell surface biotinylation assays to study protein targeting signals and pathways in polarized epithelial cells. These studies have revealed that in MDCK cells, most proteins are sorted intracellularly and are targeted directly to the surface; in other cell types, protein targeting may be mediated by a selective retrieval event. Studies on both intact and permeabilized cells demonstrate that microtubules facilitate apical but not basolateral delivery. Recent transfection studies in MDCK cells have identified glycosyl phosphatidyl inositol (GPI) as an apical targeting signal; interaction of the GPI moiety with glycolipids preferentially expressed on the apical surface may mediate this process. Several proteinaceous basolateral targeting signals have also been recently described.

Animals↗
PubMed1990

Polarized expression of functional rat liver asialoglycoprotein receptor in transfected Madin-Darby canine kidney cells.

The rat liver asialoglycoprotein receptor or rat hepatic lectin (RHL) consists of two polypeptide species, a major one designated RHL-1 and a minor one designated RHL-2/3, which exists in two differentially glycosylated forms. We have studied the biosynthesis, targeting, and function of the different forms after transfection of their cDNAs into the polarized Madin-Darby canine kidney cell line. In cells expressing only RHL-1, newly synthesized protein undergoes rapid intracellular degradation and is not detected at the cell surface. In contrast, RHL-2/3 when transfected alone is much more stable and is expressed at the basolateral surface of fiber-grown cells. When both forms are expressed together, newly synthesized RHL-1 escapes rapid degradation and is detected at the basolateral surface. In double transfectants a functional receptor is formed that specifically endocytoses and degrades ligand at the basolateral side.

Animals↗
PubMed1990

Preferred apical distribution of glycosyl-phosphatidylinositol (GPI) anchored proteins: a highly conserved feature of the polarized epithelial cell phenotype.

We use a sensitive biotin polarity assay to survey the surface distribution of glycosyl-phosphatidylinositol (GPI) anchored proteins in five model epithelial cell lines derived from different species (dog, pig, man) and tissues, i.e., kidney (MDCK I, MDCK II, LLC-PK1) and intestine (Caco-2 and SK-CO15). After biotinylation of apical or basolateral surfaces of confluent monolayers grown on polycarbonate filters, GPI-anchored proteins are identified by their shift from a Triton X-114 detergent-rich phase to a detergent-poor phase in the presence of phosphatidylinositol-specific phospholipase C. All GPI-anchored proteins detected (3-9 per cell type, at least 13 different proteins) are found to be apically polarized; no GPI-anchored protein is observed preferentially localized to the basal surface. One of the GPI-anchored proteins is identified as carcinoembryonic antigen (CEA). Survey of MDCK II-RCAr, a mutant cell line with a pleiotropic defect in galactosylation of glycoproteins and glycolipids (that presumably affects GPI anchors) also reveals an apical polarization of all GPI-anchored proteins. In contrast, analysis of MDCK II-ConAr (a mutant cell line with an unknown defect in glycosylation) revealed five GPI-anchored proteins, two of which appeared relatively unpolarized. Our results indicate that the polarized apical distribution of GPI-anchored proteins is highly conserved across species and tissue-type and may depend on glycosylation.

Animals↗
PubMed1990

Polarized release of enveloped viruses in the embryonic chick heart: demonstration of epithelial polarity in the presumptive myocardium.

The presumptive myocardium of the embryonic vertebrate heart is composed of cells which exhibit the morphology of a cuboidal epithelium. To examine the functional polarity of these developing myocytes, embryonic chick hearts (Hamburger-Hamilton stages 10-13) were infected with either influenza virus (FLU) or vesicular stomatitis virus (VSV). These viruses have been shown to sort vectorially to either apical (FLU) or basolateral (VSV) membrane surfaces in monolayers of polarized kidney (MDCK) cells. Our results demonstrate that these viruses bud with comparable polarity from differentiating myocytes. However, there appear to be stage-dependent differences in the polarized budding of the two viruses: restricted basolateral release of VSV is present before or shortly after the formation of the heart tube, whereas polarized budding of FLU is established later in development. These results are discussed in terms of plasma membrane organization during the early stages of cardiac development.

Animals↗
PubMed1990

Glycophospholipid membrane anchoring provides clues to the mechanism of protein sorting in polarized epithelial cells.

The sorting signals that guide proteins to apical and basolateral surfaces of epithelial cells have remained elusive. Current evidence suggests a hierarchy of sorting information with multiple sorting signals (apical and basolateral) present in different domains of a given plasma membrane protein. The observation that covalently attached glycosyl-phosphatidylinositol (GPI) acts as a 'dominant' apical targeting signal is compatible with the involvement of glycolipids in epithelial protein sorting.

Animals↗