Biomedical subjects

M S Balda

Publications and source records attributed to M S Balda.

At least 19 recordsLinked to original sources

PubMed2000

Occludin modulates transepithelial migration of neutrophils.

Neutrophils cross epithelial sheets to reach inflamed mucosal surfaces by migrating along the paracellular route. To avoid breakdown of the epithelial barrier, this process requires coordinated opening and closing of tight junctions, the most apical intercellular junctions in epithelia. To determine the function of epithelial tight junction proteins in this process, we analyzed neutrophil migration across monolayers formed by stably transfected epithelial cells expressing wild-type and mutant occludin, a membrane protein of tight junctions with four transmembrane domains and both termini in the cytosol. We found that expression of mutants with a modified N-terminal cytoplasmic domain up-regulated migration, whereas deletion of the C-terminal cytoplasmic domain did not have an effect. The N-terminal cytosolic domain was also found to be important for the linear arrangement of occludin within tight junctions but not for the permeability barrier. Moreover, expression of mutant occludin bearing a mutation in one of the two extracellular domains inhibited neutrophil migration. The effects of transfected occludin mutants on neutrophil migration did not correlate with their effects on selective paracellular permeability and transepithelial electrical resistance. Hence, specific domains and functional properties of occludin modulate transepithelial migration of neutrophils.

Actins↗

PubMed1999

Occludin and the functions of tight junctions.

The tight junction or zonula occludens is the most apical structure of the epithelial junctional complex. Tight junctions from semipermeable intercellular diffusion barriers that control paracellular diffusion in a regulated manner. This intercellular junction also acts as an intramembrane fence that prevents the intermixing of apical and basolateral lipids in the exocytoplasmic leaflet of the plasma membrane. Moreover, evidence suggests that tight junction components participate in the regulation of cell growth and differentiation. Occludin was the first identified transmembrane protein of this intercellular junction and received much attention since its molecular characterization. This review discusses experiments that were done with occludin and how they influenced our current thinking of the molecular functioning of tight junctions.

Animals↗

PubMed1998

The cytoplasmic domains of a beta1 integrin mediate polarization in Madin-Darby canine kidney cells by selective basolateral stabilization.

In Madin-Darby canine kidney cells, newly synthesized apical and basolateral membrane proteins are generally transported directly to their respective cell surface domain due to targeting determinants that mediate sorting in the Golgi complex. In several basolateral membrane proteins, these targeting determinants reside in the cytoplasmic domains. We show here that basolateral expression of the human alpha5beta1 integrin in stably transfected Madin-Darby canine kidney cells is also mediated by the cytoplasmic domains. Distinct regions in both cytoplasmic domains were found to be sufficient to mediate basolateral expression independently from one another. Unexpectedly, newly synthesized wild-type alpha5beta1 and basolaterally expressed chimeras containing the cytoplasmic domain of either alpha5 or beta1 were integrated into both cell surface domains, preferentially apically, during biosynthesis. The apical pools of wild-type integrin and chimeric subunits were found to become quickly degraded, whereas the basolateral pools were stabilized. Thus, the cytoplasmic domains of the alpha5beta1 integrin are independently sufficient to mediate sorting by selective basolateral stabilization.

Animals↗

PubMed1998

Carbohydrate-mediated Golgi to cell surface transport and apical targeting of membrane proteins.

Polarized expression of most epithelial plasma membrane proteins is achieved by selective transport from the Golgi apparatus or from endosomes to a specific cell surface domain. In Madin-Darby canine kidney (MDCK) cells, basolateral sorting generally depends on distinct cytoplasmic targeting determinants. Inactivation of these signals often resulted in apical expression, suggesting that apical transport of transmembrane proteins occurs either by default or is mediated by widely distributed characteristics of membrane glycoproteins. We tested the hypothesis of N-linked carbohydrates acting as apical targeting signals using three different membrane proteins. The first two are normally not glycosylated and the third one is a glycoprotein. In all three cases, N-linked carbohydrates were clearly able to mediate apical targeting and transport. Cell surface transport of proteins containing cytoplasmic basolateral targeting determinants was not significantly affected by N-linked sugars. In the absence of glycosylation and a basolateral sorting signal, the reporter proteins accumulated in the Golgi complex of MDCK as well as CHO cells, indicating that efficient transport from the Golgi apparatus to the cell surface is signal-mediated in polarized and non-polarized cells.

Animals↗

PubMed1998

Biogenesis of tight junctions: the C-terminal domain of occludin mediates basolateral targeting.

Tight junctions form a morphological and physical border between the apical and the basolateral cell surface domains of epithelial cells; hence assembly of tight junctions could occur from both of the two plasma membrane domains. We show here that the C-terminal cytoplasmic domain of occludin, the only known transmembrane protein of tight junctions, was sufficient to mediate basolateral expression of a chimeric protein. Since this chimera was transported directly to the basolateral membrane during biosynthesis, the C-terminal domain of occludin contains a basolateral targeting signal. Additionally, the C-terminal domain of occludin was also able to mediate endocytosis. Thus, the C-terminal cytoplasmic domain appears to govern intracellular transport of occludin. To test whether the basolateral membrane is an obligatory intermediate in transport of occludin to tight junctions, we analyzed the expression of occludin molecules rendered unable to efficiently integrate into tight junctions by the introduction of N-linked glycosylation sites into the two extracellular loops. Indeed, glycosylated occludin accumulated in the basolateral membrane, supporting a model in which the biogenesis of tight junctions occurs from this cell-surface domain.

Animals↗

PubMed1998

Tight junctions.

Tight junctions are the most apical intercellular junctions of epithelial and endothelial cells and create a regulatable semipermeable diffusion barrier between individual cells. On a cellular level, they form an intramembrane diffusion fence that restricts the intermixing of apical and basolateral membrane components. In addition to these well defined functions, more recent evidence suggests that tight junctions are also involved in basic cellular processes like the regulation of cell growth and differentiation.

Animals↗

PubMed1997

Cytoskeletal rearrangement mediates human microvascular endothelial tight junction modulation by cytokines.

The tight junction (TJ) is a specialized intercellular structure responsible for the regulation of ionic and macromolecular flux across cell monolayers. Because plasma leakage is believed to occur mainly across the microvasculature, we hypothesized that microvascular endothelial cells (MVEC) may form more intact, regulatable TJ than other endothelial cell (EC) types, allowing further insight into the control of EC permeability. Primary cultures of MVEC monolayers produced transmonolayer electrical resistances (TER) of 120-155 omega.cm2, approximately 10 times that of large-vessel EC. Treatment with tumor necrosis factor and interferon-gamma caused a 50% decrease in the TER and a striking fragmentation of the basal, continuous interendothelial cell zonula occludens-1 protein (ZO-1) distribution determined by immunofluorescence. Fragmentation was inhibited by cytochalasin D, and confocal microscopy demonstrated a colocalization between F actin and ZO-1. These findings suggest that the F actin cytoskeleton plays a central role in endothelial TJ barrier regulation and that dynamic cytoskeletal alterations may primarily control vascular permeability.

1-Methyl-3-isobutylxanthine↗

PubMed1996

The SH3 domain of the tight junction protein ZO-1 binds to a serine protein kinase that phosphorylates a region C-terminal to this domain.

ZO-1 is a tight junction phosphoprotein partially homologous to a tumor suppressor in Drosophila. The homologous region contains an SH3 domain with an unidentified function. Using fusion proteins containing the SH3 domain and various N- and C-terminal sequences, we tested for association of a kinase with this protein domain in extracts of MDCK cells. We show that the SH3 domain of ZO-1 binds a serine protein kinase that phosphorylates a region immediately C-terminal to the SH3 domain. This kinase associates specifically with the SH3 domain of ZO-1 and appears to be also associated with junctional complexes extracted from MDCK cells.

Animals↗

PubMed1996

Functional dissociation of paracellular permeability and transepithelial electrical resistance and disruption of the apical-basolateral intramembrane diffusion barrier by expression of a mutant tight junction membrane protein.

Tight junctions, the most apical of the intercellular junctions that connect individual cells in a epithelial sheet, are thought to form a seal that restricts paracellular and intramembrane diffusion. To analyze the functioning of tight junctions, we generated stable MDCK strain 2 cell lines expressing either full-length or COOH-terminally truncated chicken occludin, the only known transmembrane component of tight junctions. Confocal immunofluorescence and immunoelectron microscopy demonstrated that mutant occludin was incorporated into tight junctions but, in contrast to full-length chicken occludin, exhibited a discontinuous junctional staining pattern and also disrupted the continuous junctional ring formed by endogenous occludin. This rearrangement of occludin was not paralleled by apparent changes in the junctional morphology as seen by thin section electron microscopy nor apparent discontinuities of the junctional strands observed by freeze-fracture. Nevertheless, expression of both wild-type and mutant occludin induced increased transepithelial electrical resistance (TER). In contrast to TER, particularly the expression of COOH-terminally truncated occludin led to a severalfold increase in paracellular flux of small molecular weight tracers. Since the selectivity for size or different types of cations was unchanged, expression of wild-type and mutant occludin appears to have activated an existing mechanism that allows selective paracellular flux in the presence of electrically sealed tight junctions. Occludin is also involved in the formation of the apical/basolateral intramembrane diffusion barrier, since expression of the COOH-terminally truncated occludin was found to render MDCK cells incapable of maintaining a fluorescent lipid in a specifically labeled cell surface domain.

Amino Acid Sequence↗

PubMed1995

Altered hepatic localization and expression of occludin after common bile duct ligation.

Epithelial tight junctions form a regulated barrier that seals the paracellular space and prevents mixing of luminal contents with the interstitium. This barrier is composed of a group of proteins including the putative "sealing" protein occludin that appears to bind directly to a cytoplasmic junction protein, ZO-1. To study the interaction and regulation of these two components when paracellular integrity is altered, we assessed protein expression and immunofluorescent (IF) localization of ZO-1 and occludin in a rat model of hepatocyte tight junction damage induced by common bile duct ligation (CBDL). Protein levels were detected in liver by immunoblotting and IF localization by 3-dimensional reconstruction of serial 0.5-micron confocal microscopic optical sections. As previously described, ZO-1 protein levels progressively increased to threefold control levels 9 days after CBDL. In contrast, occludin protein levels decreased by 50% within 2 days after CBDL and returned to control values by 9 days. In control IF sections, ZO-1 and occludin colocalized, forming thin continuous staining outlining canaliculi. After CBDL, ZO-1 staining appeared discontinuous, and a punctate pericanalicular accumulation of signal developed around junctional areas. Occludin staining was also discontinuous after CBDL, but, in contrast to ZO-1, was not punctate and remained localized either in a linear fashion along canalicular margins or in a homogeneous fashion in immediately surrounding areas. CBDL results in changes in the expression and localization of the putative tight junction sealing protein occludin in hepatocytes that are distinct from those observed for the peripheral membrane tight junction protein ZO-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Epidermal growth factor induces tyrosine phosphorylation and reorganization of the tight junction protein ZO-1 in A431 cells.

Addition of epidermal growth factor (EGF) to A431 human epidermal carcinoma cells results in actin reorganization and phosphorylation of several cytoskeletal proteins. In the present study, we found that EGF treatment of this cell line also results in the redistribution and tyrosine phosphorylation of ZO-1. In normal polarized epithelial cells, ZO-1 is restricted to the cytoplasmic surface of the most apical of the intercellular junctions, the tight junction. In contrast, ZO-1 in the majority of unstimulated A431 cells in small subconfluent islands colocalizes with actin along the lateral cell membranes and in rare microspikes and membrane ruffles. Exposure to EGF results in a transient redistribution of actin into an apically positioned ring. ZO-1 becomes highly focused at apical sites of cell contact and co-localizes with the newly formed band of perijunctional actin. Coincidently, ZO-1 and another tight junction protein, ZO-2, become transiently phosphorylated on tyrosine residues, as determined by anti-phosphotyrosine immunoblotting. Pre-treatment of A431 cells with cytochalasin D, which disrupts normal microfilament organization, does not affect EGF-dependent phosphorylation of the EGF receptor. However, cytochalasin D pretreatment blocks both the EGF-induced ZO-1 rearrangement and tyrosine phosphorylation, suggesting that these responses are dependent on an intact actin microfilament system. We speculate that the transient tyrosine phosphorylation of ZO-1 in response to EGF treatment may be involved in remodeling of intercellular junctions in A431 cells.

Actins↗

PubMed1993

The tight junction protein ZO-1 is homologous to the Drosophila discs-large tumor suppressor protein of septate junctions.

Tight junctions form an intercellular barrier between epithelial cells, serve to separate tissue compartments, and maintain cellular polarity. Paracellular sealing properties vary among cell types and are regulated by undefined mechanisms. Sequence of the full-length cDNA for human ZO-1, the first identified tight junction component, predicts a protein of 1736 aa. The N-terminal 793 aa are homologous to the product of the lethal(1)discs-large-1 (dlg) tumor suppressor gene of Drosophila, located in septate junctions, and to a 95-kDa protein located in the postsynaptic densities of rat brain, PSD-95. All three proteins contain both a src homology region 3 (SH3 domain), previously identified in membrane proteins involved in signal transduction, and a region homologous to guanylate kinase. ZO-1 contains an additional 943-aa C-terminal domain that is proline-rich (14.1%) and contains an alternatively spliced domain, whose expression was previously shown to correlate with variable properties of tight junctions. dlg mutations result in loss of apical-basolateral epithelial cell polarity and in neoplastic growth. These results suggest a protein family specialized for signal transduction on the cytoplasmic surface of intercellular junctions. These results also provide biochemical evidence for similarity between invertebrate septate and vertebrate tight junctions. The C-terminal domain of ZO-1, and its alternatively spliced region, appears to confer variable properties unique to tight junctions.

Alternative Splicing↗

PubMed1993

The structure and regulation of tight junctions.

Tight junctions create a paracellular barrier between both epithelial and endothelial cells. Recent advances have helped define their molecular composition and regulation. Studies in cultured cell lines provide new insights into how assembly and barrier properties may be controlled by signal transduction cascades.

Animals↗

PubMed1993

Assembly of the tight junction: the role of diacylglycerol.

Extracellular Ca2+ triggers assembly and sealing of tight junctions (TJs) in MDCK cells. These events are modulated by G-proteins, phospholipase C, protein kinase C (PKC), and calmodulin. In the present work we observed that 1,2-dioctanoylglycerol (diC8) promotes the assembly of TJ in low extracellular Ca2+, as evidenced by translocation of the TJ-associated protein ZO-1 to the plasma membrane, formation of junctional fibrils observed in freeze-fracture replicas, decreased permeability of the intercellular space to [3H]mannitol, and reorganization of actin filaments to the cell periphery, visualized by fluorescence microscopy using rhodamine-phalloidin. In contrast, diC8 in low Ca2+ did not induce redistribution of the Ca-dependent adhesion protein E-cadherin (uvomorulin). Extracellular antibodies to E-cadherin block junction formation normally induced by adding Ca2+. diC8 counteracted this inhibition, suggesting that PKC may be in the signaling pathway activated by E-cadherin-mediated cell-cell adhesion. In addition, we found a novel phosphoprotein of 130 kD which coimmunoprecipitated with the ZO-1/ZO-2 complex. Although the assembly and sealing of TJs may involve the activation of PKC, the level of phosphorylation of ZO-1, ZO-2, and the 130-kD protein did not change after adding Ca2+ or a PKC agonist. The complex of these three proteins was present even in low extracellular Ca2+, suggesting that the addition of Ca2+ or diC8 triggers the translocation and assembly of preformed TJ subcomplexes.

Actins↗

PubMed1993

Two classes of tight junctions are revealed by ZO-1 isoforms.

The tight junction forms the intercellular barrier separating tissue compartments. The characteristics of this barrier are remarkably diverse among different epithelia and endothelia and are not explained by our limited knowledge of its molecular composition. Two isoforms of the 220-kDa tight junction protein ZO-1 result from alternative RNA splicing and differ by an internal 80-amino acid domain, termed alpha (E. Willott, M. S. Balda, M. Heintzman, B. Jameson, and J. M. Anderson. Am. J. Physiol. 262 (Cell Physiol. 31): C1119-C1124, 1992). Using antibodies specific for each isoform and double-labeled immunofluorescence microscopy, we observed that the ZO-1 alpha- isoform is restricted to junctions of endothelial cells and highly specialized epithelial cells of both seminiferous tubules (Sertoli cells) and renal glomeruli (podocytes); in contrast, the ZO-1 alpha+ isoform is expressed in cells of all other epithelia examined. Both immunoblotting and ribonuclease protection analysis confirmed this pattern of expression. This distribution does not correlate with differences in junctional resistance or ultrastructural complexity. Instead, we observe a correlation with junctional plasticity; ZO-1 alpha- is expressed in structurally dynamic junctions, whereas ZO-1 alpha+ is expressed in those which are less dynamic. This is the first molecular distinction among tight junctions and reveals a fundamental dichotomy with implications for how the paracellular barriers of endothelia and epithelia are regulated.

Amino Acid Sequence↗

PubMed1992

Localization and differential expression of two isoforms of the tight junction protein ZO-1.

ZO-1 is a peripheral membrane protein of approximately 225 kDa located on the cytoplasmic side of all tight junctions. ZO-1 cDNA sequencing disclosed the presence of a 240-bp sequence in only some of the ZO-1 cDNAs studied. This 240-bp region encoded an inframe insertion of 80 amino acids, named motif-alpha. Expression of the predicted transcripts in normal rat and human tissues and in human epithelial cell lines (Caco-2, T84, Hep G2) was shown by reverse transcription of RNA and then DNA amplification. Immunoblot analysis showed both protein isoforms were present; however, in different cell lines, their amounts differed markedly relative to each other. Immunolocalization at light and ultrastructural levels, using antibodies generated against motif-alpha or shared sequences flanking it, indicated both forms localized indistinguishably to tight junctions. These observations demonstrate the existence and variable expression of ZO-1 isoforms and raise the question whether these isoforms contribute to tight junction diversity in different epithelia.

Amino Acid Sequence↗

PubMed1991

Assembly and sealing of tight junctions: possible participation of G-proteins, phospholipase C, protein kinase C and calmodulin.

The making and sealing of a tight junction (TJ) requires cell-cell contacts and Ca2+, and can be gauged through the development of transepithelial electrical resistance (TER) and the accumulation of ZO-1 peptide at the cell borders. We observe that pertussis toxin increases TER, while AIF3 and carbamil choline (carbachol) inhibit it, and 5-guanylylimidodiphosphate (GTPTs) blocks the development of a cell border pattern of ZO-1, suggesting that G-proteins are involved. Phospholipase C (PLC) and protein kinase C (PKC) probably participate in these processes since (i) activation of PLC by thyrotropin-1 releasing hormone increases TER, and its inhibition by neomycin blocks the development of this resistance; (ii) 1,2-dioctanoylglycerol, an activator of PKC, stimulates TER development, while polymyxin B and 1-(5-isoquinoline sulfonyl)-2-methyl-piperazine dihydrochloride (H7), which inhibit this enzyme, abolish TER. Addition of 3-isobutyl-1-methyl-xanthine, dB-cAMP or forskolin do not enhance the value of TER, but have just the opposite effect. Trifluoperazine and calmidazoline inhibit TER development, suggesting that calmodulin (CaM) also plays a role in junction formation. These results indicate that junction formation may be controlled by a network of reactions where G-proteins, phospholipase C, adenylate cyclase, protein kinase C and CaM are involved.

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

PubMed1989

Evidence for cholinergic innervation in dog renal tissue.

Denervation procedures that affect the sympathetic system of the kidney, as demonstrated by norepinephrine depletion of renal tissue, increased urine volume, fractional sodium excretion, and free water clearance in anesthetized water-loaded dogs. These increases were reduced by atropine, which also blocked the increase above those basal functional levels produced by acetylcholine in both innervated and denervated kidneys. An in vitro tubular cell preparation of innervated kidneys corresponding to the outer cortex showed [3H]quinuclidinyl benzilate (QNB) binding parameters characteristic of muscarinic receptors. Denervation did not change either [3H]QNB binding parameters or the ability of inner and outer cortex cells to perform the hemicholinium-3-inhibitable, sodium-dependent choline uptake and conversion of [3H]choline to [3H]acetylcholine. This cell membrane behavior is similar to that observed in tissues where cholinergic neuronal structures are present, suggesting the existence of a cholinergic innervation of the kidney, independent of the integrity of vessel-traveling nerves. Similarly, the finding of choline acetyltransferase in renal tissue, unaffected by sympathetic denervation, seems to confirm the presence of cholinergic nerve terminals. The cholinergic system may thus contribute to the regulation of tubular reabsorption of sodium and water in some conditions.

Acetylcholine↗