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E Rodriguez-Boulan

Publications and source records attributed to E Rodriguez-Boulan.

At least 55 records · Page 3Linked to original sources
PubMed1995

Cyclic AMP modulates the rate of 'constitutive' exocytosis of apical membrane proteins in Madin-Darby canine kidney cells.

Madin-Darby canine kidney and other epithelial cell lines (e.g. Caco-2, MCF-10A and MCF-7) develop intracellular vacuoles composed of apical membrane displaying microvilli (VACs) when impaired from forming normal cell-to-cell contacts. In a previous publication, we showed that VACs are rapidly exocytosed upon treatment with 8-Br-3',5'-cyclic adenosine monophosphate (8-Br-cAMP), a membrane-permeable analog of cAMP, and that this exocytosis correlates with variations in the cellular cAMP concentration in response to the cell-cell contacts. In the present work, we tested the hypothesis that cAMP may be a positive modulator of the 'constitutive' exocytic pathway. To mimic conditions in cells with incomplete intercellular contacts, the intracellular levels of cAMP were decreased by means of two independent approaches: (i) pores were induced in the plasma membrane with the polypeptidic antibiotic subtilin, thus allowing small molecules (including cAMP) to permeate and move out of the cytoplasm; and (ii) adenylate cyclase and protein kinase A were blocked with specific inhibitors. In all cases, the intracellular levels of cAMP were measured and, in porated cells, equilibrated to simulate the corresponding physiological intracellular concentrations. The decrease in cAMP within the physiological range resulted in a decreased rate of transport of an apical marker of the constitutive pathway (influenza virus hemagglutinin) from the trans-Golgi network to the apical plasma membrane. Likewise, the delivery of a number of cellular apical proteins to the plasma membrane was retarded at low cAMP concentrations. The inhibitors of adenylate cyclase failed to block basolateral delivery of vesicular stomatitis virus G protein. This differential modulatory effect may represent a differentiation-dependent control of the insertion of apical membrane in epithelial cells.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1995

Epithelial cell polarity: new perspectives.

All epithelial cells possess two distinct plasma membrane domains. The apical and basolateral domains differ in protein and lipid composition, and this allows the cell to perform a variety of vectorial functions. Structures involved in generating and maintaining these distinct membrane domains include the tight junction, which serves to restrict lateral diffusion within the membrane, and the cortical cytoskeleton, which can selectively bind and retain transmembrane proteins at a particular surface. A major means to generating membrane asymmetry lies in the ability of the cell to sort apical and basolateral proteins and target them to appropriate destinations. This sorting occurs predominantly at two intracellular sites: the trans-Golgi network, and the basolateral endosome. Constitutive protein traffic in epithelial cells has recently been shown to be regulated via classical signal transduction pathways involving heterotrimeric G proteins and protein kinases. The diversion of apical and basolateral proteins into specific pathways can be mediated by signals contained within these proteins. Apical sorting information is thought to be localized in the luminal domain of transmembrane proteins, and in the case of proteins anchored to the membrane via a GPI anchor, apical sorting information is provided by the lipid moiety. In contrast, basolateral signals have been identified in the cytoplasmic domain of transmembrane proteins. Shared similarities between basolateral signals and those required for endocytosis have suggested that these two sorting processes are mechanistically related.

Animals↗
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

TGN38 recycles basolaterally in polarized Madin-Darby canine kidney cells.

Sorting of newly synthesized plasma membrane proteins to the apical or basolateral surface domains of polarized cells is currently thought to take place within the trans-Golgi network (TGN). To explore the relationship between protein localization to the TGN and sorting to the plasma membrane in polarized epithelial cells, we have expressed constructs encoding the TGN marker, TGN38, in Madin-Darby canine kidney (MDCK) cells. We report that TGN38 is predominantly localized to the TGN of these cells and recycles via the basolateral membrane. Analyses of the distribution of Tac-TGN38 chimeric proteins in MDCK cells suggest that the cytoplasmic domain of TGN38 has information leading to both TGN localization and cycling through the basolateral surface. Mutations of the cytoplasmic domain that disrupt TGN localization also lead to nonpolarized delivery of the chimeric proteins to both surface domains. These results demonstrate an apparent equivalence of basolateral and TGN localization determinants and support an evolutionary relationship between TGN and plasma membrane sorting processes.

Amino Acid Sequence↗
PubMed1994

Permeabilization of MDCK cells with cholesterol binding agents: dependence on substratum and confluency.

We studied systematically the susceptibility of Madin-Darby canine kidney (MDCK) cells to permeabilization by two cholesterol binding agents, digitonin and streptolysin-O (SLO), under different culture conditions. Monolayers grown on polycarbonate filter chambers (Transwells) required twice the concentration of digitonin effective on monolayers grown on glass or plastic (80 vs. 40 micrograms/ml) to allow antibody penetration or the release of 90% of the cytosolic protein lactate dehydrogenase (LDH). Neither the apical nor the basolateral surface showed preferential susceptibility to digitonin. Confluent MDCK cells, cultured either on filters or on impermeable substrates, showed poor antibody permeability after addition of commercial SLOs, even when used at concentrations 100 times higher (20 U/ml) than those effective on nonepithelial Chinese hamster ovary cells. Surprisingly, culture conditions that prevent tight junction formation and the acquisition of a polarized phenotype (< 10 microM Ca2+) increased dramatically the susceptibility to permeabilization by SLO. On restoration of normal Ca2+ levels, susceptibility to SLO quickly decreased. Thus conditions that lead to the full establishment of polarity result in decreased sensitivity to disruption by digitonin and SLO.

Animals↗
PubMed1994

Brefeldin A causes structural and functional alterations of the trans-Golgi network of MDCK cells.

The trans-Golgi network (TGN) of MDCK cells is exquisitely sensitive to the fungal metabolite brefeldin A (BFA), in contrast to the refractory Golgi stack of these cells. At a concentration of 1 microgram/ml, BFA promoted extensive tubulation of the TGN while the medical Golgi marker alpha-mannosidase II was not affected. Tubules emerging minutes after addition of the drug contained both the apical marker influenza hemagglutinin (HA), previously accumulated at 20 degrees C, and the fusion protein interleukin receptor/TGN38 (TGG), a TGN marker that recycles basolaterally, indicating that, in contrast to TGN vesicles, TGN-derived tubules cannot sort apical and basolateral proteins. After 60 minutes treatment with BFA, HA and TGG tubules formed extensive networks widely spread throughout the cell, different from the focused centrosomal localization previously described in non-polarized cells. The TGG network partially codistributed with an early endosomal tubular network loaded with transferrin, suggesting that the TGG and endosomal networks had fused or that TGG had entered the endosomal network via surface recycling and endocytosis. The extensive structural alterations of the TGN were accompanied by functional disruptions, such as the extensive mis-sorting of influenza HA, and by the release of the TGN marker gamma-adaptin. Our results suggest the involvement of BFA-sensitive adaptor proteins in TGN-->surface transport.

Adaptor Protein Complex gamma Subunits↗
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

Ricin-resistant Madin-Darby canine kidney cells missort a major endogenous apical sialoglycoprotein.

gp114 is a major sialoglycoprotein expressed on the apical membrane of Madin-Darby canine kidney (MDCK) II cells. We investigated its distribution in two lectin-resistant mutant cell lines derived from MDCKII cells, MDCKII-RCAr and MDCKII-ConAr cells. gp114 was present on the apical membrane of MDCKII-ConAr cells but was predominantly basolateral in MDCKII-RCAr cells. No change of polarity was observed for several apical and basolateral markers in this cell line. Reversal of polarity of gp114 mainly resulted from a modification of its intracellular sorting. gp114 showed altered endocytosis in MDCKII-RCAr cells. In MDCKII cells gp114 was slowly endocytosed, whereas in MDCKII-RCAr cells endocytosis of gp114 was highly increased. Using mannosidase I and II inhibitors we found that N-glycosylation only slightly affects gp114 sorting and endocytosis. Our results suggest that gp114 or an associated component in MDCK-RCAr fails to express apical information or that a mutation creates a basolateral sorting signal which could be related to endocytic signals.

Animals↗
PubMed1993

Correctly sorted molecules of a GPI-anchored protein are clustered and immobile when they arrive at the apical surface of MDCK cells.

Glycosyl-phosphatidylinositol (GPI)-anchored proteins are sorted to the apical surface of many epithelial cell types. To better understand the mechanism for apical segregation of these proteins, we analyzed the lateral mobility and molecular associations of a model GPI-anchored protein, herpes simplex virus gD1 fused to human decay accelerating factor (gD1-DAF) (Lisanti, M. P., I. W. Caras, M. A. Davitz, and E. Rodriguez-Boulan. 1989. J. Cell Biol. 109:2145-2156) shortly after arrival and after long-term residence at the surface of confluent, polarized MDCK cells. FRAP measurements of lateral diffusion showed that the mobile fraction of newly arrived gD1-DAF molecules was much less than the mobile fraction of long-term resident molecules (40 vs. 80-90%). Fluorescence resonance energy transfer measurements showed that the newly arrived molecules were clustered, while resident molecules were not. Newly delivered gD1-DAF molecules were clustered but not immobilized in mutant, Concanavalin A-resistant MDCK cells that failed to sort gD1-DAF. Our results indicate that GPI-anchored proteins in MDCK cells are clustered before delivery to the surface. However, clustering alone does not target molecules for apical delivery. The immobilization observed when gD1-DAF is correctly sorted suggests that the clusters must associate some component of the cell's cytoplasm.

Animals↗
PubMed1993

Apical polarization of N-CAM in retinal pigment epithelium is dependent on contact with the neural retina.

The retinal pigment epithelium (RPE) is unique among epithelia in that its apical surface does not face a lumen, but, instead, is specialized for interaction with the neural retina. The molecules involved in the interaction of the RPE with the neural retina are not known. We show here that the neural cell adhesion molecule (N-CAM) is found both on the apical surface of RPE in situ and on the outer segments of photoreceptors, fulfilling an important requisite for an adhesion role between both structures. Strikingly, culture of RPE results in rapid redistribution of N-CAM to the basolateral surface. This is not due to an isoform shift, since the N-CAM expressed by cultured cells (140 kD) is the same as that expressed by RPE in vivo. Rather, the reversed polarity of N-CAM appears to result from the disruption of the contact between the RPE and the photoreceptors of the neural retina. We suggest that N-CAM in RPE and photoreceptors participate in these interactions.

Animals↗
PubMed1993

Glycosylphosphatidylinositol-anchored proteins are preferentially targeted to the basolateral surface in Fischer rat thyroid epithelial cells.

Glycosylphosphatidylinositol (GPI) acts as an apical targeting signal in MDCK cells and other kidney and intestinal cell lines. In striking contrast with these model polarized cell lines, we show here that Fischer rat thyroid (FRT) epithelial cells do not display a preferential apical distribution of GPI-anchored proteins. Six out of nine detectable endogenous GPI-anchored proteins were localized on the basolateral surface, whereas two others were apical and one was not polarized. Transfection of several model GPI proteins, previously shown to be apically targeted in MDCK cells, also led to unexpected results. While the ectodomain of decay accelerating factor (DAF) was apically secreted, 50% of the native, GPI-anchored form, of this protein was basolateral. Addition of a GPI anchor to the ectodomain of Herpes simplex gD-1, secreted without polarity, led to basolateral localization of the fusion protein, gD1-DAF. Targeting experiments demonstrated that gD1-DAF was delivered vectorially from the Golgi apparatus to the basolateral surface. These results indicate that FRT cells have fundamental differences with MDCK cells with regard to the mechanisms for sorting GPI-anchored proteins: GPI is not an apical signal but, rather, it behaves as a basolateral signal. The "mutant" behavior of FRT cells may provide clues to the nature of the mechanisms that sort GPI-anchored proteins in epithelial cells.

Animals↗
PubMed1993

Increased LAMP-2 polylactosamine glycosylation is associated with its slower Golgi transit during establishment of a polarized MDCK epithelial monolayer.

An endogenous Madin-Darby canine kidney (MDCK) lysosomal membrane glycoprotein that exhibits a basolateral targeting pathway to the lysosome is shown here to exhibit significant N-terminal amino acid sequence identity to lysosomal associated membrane proteins (LAMP-2) of other species. During establishment of the MDCK monolayer after only 1 d of culture, this canine LAMP-2 has a larger molecular size (110 kDa) than following formation of a confluent monolayer after 3 d of culture (100 kDa) due to the increased presence of N-linked polylactosamine oligosaccharide chains. Neither polylactosamine glycosylation of LAMP-2 in MDCK cells nor truncation of N-linked oligosaccharide chains of LAMP-2 in a ricin-resistant MDCK-RCAR cell line influenced the basolateral polarity of its targeting. However, the rate of basolateral delivery of LAMP-2 in MDCK cells plated for 3 d was significantly faster (t1/2 = 28 min) than in 1-d cells (t1/2 = 40 min); in MDCK-RCAR cells the rate of basolateral delivery at both 1 and 3 d of plating was similar (t1/2 = 40 min). The rate differential in MDCK cells occurred after arrival of LAMP-2 to the Golgi apparatus because the rate of acquisition of endoglycosidase H resistance was the same (t1/2 = 25 min) at both days of plating. The rate of transit of LAMP-2 through the Golgi apparatus to the basolateral domain was therefore far more rapid (approximately 4-fold) in 3 d compared with 1-d MDCK cultures. The increased polylactosamine glycosylation of MDCK LAMP-2 at early times of plating during the establishment of a confluent epithelial monolayer may thus be related to its longer residence time in the Golgi apparatus.

Amino Acid Sequence↗
PubMed1993

Immortalization of polarized rat retinal pigment epithelium.

Rat retinal pigment epithelial (RPE) cells were immortalized by infection with a temperature-sensitive tsA SV40 virus and following cloning and selection for epithelial properties the polarized RPE-J cell line was obtained. At the permissive temperature of 33 degrees C, RPE-J cells behave as an immortalized cell line. When RPE-J cells are grown on nitrocellulose filters coated with a thin layer of Matrigel in the presence of 10(-8) M retinoic acid for 6 days at 33 degrees C and then switched for 33-36 hours to the non-permissive temperature of 40 degrees C, they acquire a differentiated polarized RPE phenotype. Under these growth conditions, RPE-J cells exhibit circumferential staining for the tight-junction protein ZO-1 and acquire a transepithelial resistance of 350 ohms cm2. Morphologically, RPE-J cells exhibit a characteristic RPE morphology with extensive apical microvilli as well as numerous dense bodies including premelanosomes and varied multilamellar structures. Ruthenium red labeling revealed the frequent basal localization of the tight junction. The cells were identified to be of rat RPE origin by their expression of the rat RPE marker RET-PE2 and their ability to phagocytose latex beads. While RPE-J cells are capable of sorting influenza and vesicular stomatitis virus to the apical and basal surfaces, respectively, the Na,K-ATPase is not polarized and the neural cell adhesion molecule, N-CAM, is localized exclusively to the lateral surface. In vivo the apical surface of RPE interacts with the adjacent neural retina and the Na,K-ATPase and N-CAM are both apical; the altered polarity of these two proteins in RPE-J cells may be a consequence of the absence of apical interaction with the neural retina in culture. Previous studies of RPE have been restricted to the use of primary cultures and the RPE-J cell line should prove an excellent model system for the study of the mechanisms determining the characteristic polarity and functions of the retinal pigment epithelium.

Animals↗
PubMed1993

Structural reorganization of the rough endoplasmic reticulum without size expansion accounts for dexamethasone-induced secretory activity in AR42J cells.

A striking reorganization of the rough endoplasmic reticulum (RER) from a tubulo-vesicular (TV-RER) to a stacked cisternal (SC-RER) configuration was observed when the secretory activity of AR42J cells, a cell line derived from a rat pancreatic acinar carcinoma, was induced by dexamethasone. Treatment with 10 nM dexamethasone resulted in a 6.6-fold increase in the intracellular and a 4.6-fold increase in the secreted amylase activity, respectively. On the basis of the morphometric analysis of thin-section electron micrographs it has been previously reported that this increase in secretory activity is accompanied by a 2.4-fold or 30-fold increase in the size of the RER. We have developed a new biochemical method to determine the size of the RER by quantifying the membrane-bound ribosomes. Using this procedure we did not detect any change in the size of the RER after induction of an active secretory state in AR42J cells. Electron microscopic observation showed the predominance of SC-RER in dexamethasone-treated cells compared to the abundance of TV-RER in control cells. Laser scanning confocal microscopy showed a patchy distribution of ER staining in dexamethasone-treated cells compared to more basal localization in control cells. On the basis of our observations we conclude that in AR42J cells the increase in secretory activity induced by dexamethasone is accompanied by a reorganization of the RER rather than by an increase in ER surface area, as reported by others. Our results suggest that SC-RER is a biosynthetically more efficient form of the RER, which is found predominantly in actively secreting cells.

Amylases↗
PubMed1993

Polarity signals in epithelial cells.

In simple epithelia, specialized vectorial functions such as transport and secretion are made possible by the segregation of proteins and lipids into opposite surface domains. This polarized distribution results from selective delivery to and retention at the appropriate domain. In the case of direct delivery, the sorting site for apical and basolateral proteins is the trans-Golgi network (TGN) where they are incorporated into distinct apical and basolateral vesicles that are targeted to the respective surfaces. The machinery that controls this simple process is in fact rather complicated. It involves many different steps from the recognition event (between 'sorting signal(s)' and 'sorting receptor(s)' to the formation of the vesicles, their budding, and the docking to the specialized plasma membrane domain. Here we summarize the latest developments in the sorting of apical and basolateral proteins, focusing in particular on the signals that are involved in this process and the current hypotheses about the mechanisms responsible for it, in both epithelia and in non-polarized cells.

Amino Acid Sequence↗
PubMed1992

Changes in intracellular calcium during the development of epithelial polarity and junctions.

The "Ca2+ switch" model with cultured Madin-Darby canine kidney (MDCK) cells is useful in studying the biogenesis of epithelial polarity and junction formation and provides insight into early steps in the morphogenesis of polarized epithelial tissues. When extracellular Ca2+ in the medium is changed from less than 5 microM to 1.8 mM, MDCK cells rapidly change from a nonpolarized state exhibiting little cell-cell contact (with the apical membrane and junctional proteins largely within the cell) to a polarized state with well-formed tight junctions and desmosomes. To examine the role of intracellular Ca2+ in the development of polarity and junctions, we made continuous spectrofluorimetric measurements of intracellular Ca2+ during the "switch," using the fluorescent indicator fura-2. Intracellular Ca2+ increased greater than 10-fold during the switch and gave a complex pattern of increase, decrease, and stabilization. In contrast, intracellular pH [monitored with 2',7'-bis(2-carboxyethyl)-5(and 6)-carboxyfluorescein (BCECF)] did not change during the period studied. When intracellular Ca2+ curves in several cells were compared, considerable heterogeneity in the rate of increase of intracellular Ca2+ levels and in peak levels was evident, perhaps reflecting the heterogeneity among cells in establishing junctions and polarity. The heterogeneity of the process was confirmed by digital imaging of intracellular Ca2+ and was present even in a "clonal" line of MDCK cells, indicating the heterogeneity was intrinsic to the process and not simply a function of slight genetic variation within the population of MDCK cells. In pairs of cells that had barely established cell-cell contact, often one cell exhibited a much greater increase in intracellular Ca2+ than the other cell in the pair. At the site of cell-cell contact, an apparent localized change (an increase over the basal level) in intracellular Ca2+ was frequently present and occasionally appeared to extend beyond the point of cell-cell contact. Since the region of cell-cell contact is also the site where junctions form and where vesicles containing apical membranes fuse during the development of polarity, we postulate a role for global and local changes in intracellular Ca2+ in these events.

Animals↗
PubMed1992

Modulation of transcytotic and direct targeting pathways in a polarized thyroid cell line.

Two biosynthetic pathways exist for delivery of membrane proteins to the apical surface of epithelial cells, direct transport from the trans-Golgi network (TGN) and transcytosis from the basolateral membrane. Different epithelial cells vary in the expression of these mechanisms. Two extremes are MDCK cells, that use predominantly the direct route and hepatocytes, which deliver all apical proteins via the basolateral membrane. To determine how epithelial cells establish a particular targeting phenotype, we studied the apical delivery of endogenous dipeptidyl peptidase IV (DPPIV) at early and late stages in the development of monolayers of a highly polarized epithelial cell line derived from Fischer rat thyroid (FRT). In 1 day old monolayers, surface delivery of DPPIV from the TGN was unpolarized (50%/50%) but a large basal to apical transcytotic component resulted in a polarized apical distribution. In contrast, after 7 days of culture, delivery of DPPIV was mainly direct (85%) with no transcytosis of the missorted component. A basolateral marker, Ag 35/40 kD, on the other hand, was directly targeted (90-98%) at all times. These results indicate that the sorting machinery for apical proteins develops independently from the sorting machinery for basolateral proteins and that the sorting site relocates progressively from the basal membrane to the TGN during development of the epithelium. The transient expression of the transcytotic pathway may serve as a salvage pathway for missorted apical proteins when the polarized phenotype is being established.

Animals↗