Exocytotic pathways exist to both the apical and the basolateral cell surface of the polarized epithelial cell MDCK

Secretion of a foreign protein--chicken oviduct lysozyme--and of endogenous proteins was studied in the polarized epithelial Madin-Darby Canine Kidney (MDCK) cell line. Cell clones that secrete enzymatically active chicken lysozyme were generated by transforming the cells with lysozyme cDNA inserted in a SV40-pBR322 recombinant vector and a dominant selectable marker gene. The kinetics and polarity of lysozyme secretion from one transformed cell clone were studied using cell monolayers grown on nitrocellulose filters. Lysozyme was secreted into the apical and the basolateral medium, demonstrating the existence of direct transport pathways to each cell surface. Control experiments excluded the effects of monolayer leakiness, reabsorption, transepithelial transport, and depolarization. In contrast, the secretion of a set of endogenous proteins of MW 30-40 kd was found to be strictly apical showing that polarized secretion also occurs in this cell line. The latter group of proteins appear to be generated from larger precursor molecules by intracellular cleavage.     

Microtubule- and motor-dependent fusion in vitro between apical and basolateral endocytic vesicles from MDCK cells

The pathways of endocytosis from the apical and the basolateral domains of epithelial MDCK cells are known to converge at the level of late endosomes in vivo. We have now reconstituted the meeting process in a cell-free assay that measures the fusion of apically and basolaterally derived endocytic vesicles with late endosomes. Our results show that this in vitro process requires the presence of polymerized microtubules, as does the convergence of the two pathways in vivo, and also depends on the presence of microtubule binding proteins, in particular the mechanochemical motors kinesin and cytoplasmic dynein.     

Distinct transport vesicles mediate the delivery of plasma membrane proteins to the apical and basolateral domains of MDCK cells

Immunoisolation techniques have led to the purification of apical and basolateral transport vesicles that mediate the delivery of proteins from the trans-Golgi network to the two plasma membrane domains of MDCK cells. We showed previously that these transport vesicles can be formed and released in the presence of ATP from mechanically perforated cells (Bennett, M. K., A. Wandinger-Ness, and K. Simons, 1988. EMBO (Euro. Mol. Biol. Organ.) J. 7:4075-4085). Using virally infected cells, we have monitored the purification of the trans-Golgi derived vesicles by following influenza hemagglutinin or vesicular stomatitis virus (VSV) G protein as apical and basolateral markers, respectively. Equilibrium density gradient centrifugation revealed that hemagglutinin containing vesicles had a slightly lower density than those containing VSV-G protein, indicating that the two fractions were distinct. Antibodies directed against the cytoplasmically exposed domains of the viral spike glycoproteins permitted the resolution of apical and basolateral vesicle fractions. The immunoisolated vesicles contained a subset of the proteins present in the starting fraction. Many of the proteins were sialylated as expected for proteins existing the trans-Golgi network. The two populations of vesicles contained a number of proteins in common, as well as components which were enriched up to 38-fold in one fraction relative to the other. Among the unique components, a number of transmembrane proteins could be identified using Triton X-114 phase partitioning. This work provides evidence that two distinct classes of vesicles are responsible for apical and basolateral protein delivery. Common protein components are suggested to be involved in vesicle budding and fusion steps, while unique components may be required for specific recognition events such as those involved in protein sorting and vesicle targeting.     

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.     

Effect of nocodazole on vesicular traffic to the apical and basolateral surfaces of polarized MDCK cells

A polarized cell, to maintain distinct basolateral and apical membrane domains, must tightly regulate vesicular traffic terminating at either membrane domain. In this study we have examined the extent to which microtubules regulate such traffic in polarized cells. Using the polymeric immunoglobulin receptor expressed in polarized MDCK cells, we have examined the effects of nocodazole, a microtubule-disrupting agent, on three pathways that deliver proteins to the apical surface and two pathways that deliver proteins to the basolateral surface. The biosynthetic and transcytotic pathways to the apical surface are dramatically altered by nocodazole in that a portion of the protein traffic on each of these two pathways is misdirected to the basolateral surface. The apical recycling pathway is slowed in the presence of nocodazole but targeting is not disrupted. In contrast, the biosynthetic and recycling pathways to the basolateral surface are less affected by nocodazole and therefore appear to be more resistant to microtubule disruption.     

Determination of the Na permeability of the tight junctions of MDCK cells by fluorescence microscopy

The kinetics of Na movement across the tight junctions of MDCK cells, grown on coverslips and perfused with HEPES or bicarbonate Ringer at 37°C, were investigated after filling the lateral intercellular spaces (LIS) of the epithelium with SBFO, an Na-sensitive fluorescent dye. Dilution and bi-ionic potential measurements showed that MDCK cell tight junctions, although cation-selective, were poorly permeable to N-methyl-D-glucamine Cl (NMDG) but freely permeable to Li. In previous experiments in which Na was replaced by NMDG, a very slow decrease in LIS Na concentration (time constant = 4.8 min) resulted. In the present study, reduction of perfusate Na from 142 to 14 or 24 mm with Na replaced by Li caused LIS Na concentration to decrease with a time constant of 0.43 min. The time constant for Na increase of the LIS was 0.28 min, significantly shorter than that for Na decrease because of the additional component of transcellular Na influx. Ouabain eliminated the transcellular component and equalized the time constants for Na influx and efflux. These results were incorporated into a mathematical model which enabled calculation of the transcellular and paracellular Na fluxes during fluid reabsorption. Regulation of the Na permeability of individual tight junctions by protein kinase A (PKA) was evaluated by treating the monolayers with the Sp-cAMPS, a cAMP substitute, or Rp-cAMPS, a specific inhibitor of PKA. Stimulation of PKA strikingly increased tight junctional permeability while PKA inhibition diminished junctional Na permeability.We thank Carter Gibson, Gennady Slobodov and Cuong Vo for valuable technical assistance.     

Exocyst requirement for endocytic traffic directed toward the apical and basolateral poles of polarized MDCK cells

The octameric exocyst complex is associated with the junctional complex and recycling endosomes and is proposed to selectively tether cargo vesicles directed toward the basolateral surface of polarized Madin-Darby canine kidney (MDCK) cells. We observed that the exocyst subunits Sec6, Sec8, and Exo70 were localized to early endosomes, transferrin-positive common recycling endosomes, and Rab11a-positive apical recycling endosomes of polarized MDCK cells. Consistent with its localization to multiple populations of endosomes, addition of function-blocking Sec8 antibodies to streptolysin-O-permeabilized cells revealed exocyst requirements for several endocytic pathways including basolateral recycling, apical recycling, and basolateral-to-apical transcytosis. The latter was selectively dependent on interactions between the small GTPase Rab11a and Sec15A and was inhibited by expression of the C-terminus of Sec15A or down-regulation of Sec15A expression using shRNA. These results indicate that the exocyst complex may be a multipurpose regulator of endocytic traffic directed toward both poles of polarized epithelial cells and that transcytotic traffic is likely to require Rab11a-dependent recruitment and modulation of exocyst function, likely through interactions with Sec15A.     

Connexin-occludin chimeras containing the ZO-binding domain of occludin localize at MDCK tight junctions and NRK cell contacts.

Occludin is a transmembrane protein of the tight junction that functions in creating both an intercellular permeability barrier and an intramembrane diffusion barrier. Creation of the barrier requires the precise localization of occludin, and a distinct family of transmembrane proteins called claudins, into continuous linear fibrils visible by freeze-fracture microscopy. Conflicting evidence exists regarding the relative importance of the transmembrane and extracellular versus the cytoplasmic domains in localizing occludin in fibrils. To specifically address whether occludin's COOH-terminal cytoplasmic domain is sufficient to target it into tight junction fibrils, we created chimeras with the transmembrane portions of connexin 32. Despite the gap junction targeting information present in their transmembrane and extracellular domains, these connexin-occludin chimeras localized within fibrils when expressed in MDCK cells, as assessed by immunofluorescence and immunogold freeze-fracture imaging. Localization of chimeras at tight junctions depends on the COOH-terminal ZO-binding domain and not on the membrane proximal domain of occludin. Furthermore, neither endogenous occludin nor claudin is required for targeting to ZO-1-containing cell-cell contacts, since in normal rat kidney fibroblasts targeting of chimeras again required only the ZO-binding domain. These results suggest an important role for cytoplasmic proteins, presumably ZO-1, ZO-2, and ZO-3, in localizing occludin in tight junction fibrils. Such a scaffolding and cytoskeletal coupling function for ZO MAGUKs is analogous to that of other members of the MAGUK family.     

Reconstitution of transcytosis in SLO-permeabilized MDCK cells: existence of an NSF-dependent fusion mechanism with the apical surface of MDCK cells.

Recently, it was demonstrated that delivery from the trans-Golgi network (TGN) to the basolateral surface of Madin-Darby canine kidney (MDCK) cells required N-ethylmaleimide-sensitive factor (NSF)-alpha soluble NSF attachment protein (SNAP)-SNAP receptor (SNARE) complexes, while delivery from the TGN to the apical surface was independent of NSF-alpha SNAP-SNARE. To determine if all traffic to the apical surface of this cell line was NSF independent, we reconstituted the transcytosis of pre-internalized IgA to the apical surface and recycling to the basolateral surface. Transcytosis and the recycling of IgA required ATP and cytosol, and both were inhibited by treatment with N-ethylmaleimide. This inhibition was reversed by the addition of recombinant NSF. Botulinum neurotoxin serotype E, which is known to cleave the 25,000 Da synaptosomal associated protein, inhibited both transcytosis and recycling, although incompletely. We conclude that membrane traffic to a target membrane is not determined by utilizing a single molecular mechanism for fusion. Rather, a target membrane, e.g. the apical plasma membrane of MDCK cells, may use multiple molecular mechanisms to fuse with incoming vesicle.     

Formation and barrier function of tight junctions in human ovarian surface epithelium

The normal ovarian surface epithelium (OSE) is a primitive epithelium made up by a single layer of mesothelial-type epithelial cells. When these cells get trapped in the ovarian stroma, expression of epithelial specific markers, such as E-cadherin, are induced. Most epithelial cells are also characterized by the ability to form tight junctions (TJ). Incomplete TJ have earlier been demonstrated in the OSE by electron microscopy studies. We have investigated expression and localization of the TJ proteins ZO-1, occludin, and claudin-1 in tissue biopsies from normal human ovaries and OSE in culture. The dynamics of TJ formation were studied in human OSE cultured on porous filters in culture inserts by measuring trans epithelial resistance (TER) including Ca(2+) switch experiments. Confluent OSE cells were also analyzed by electron microscopy. The results show that normal human OSE has expression of all three TJ proteins investigated. These proteins, ZO-1, occludin, and claudin-1, were localized to OSE cell borders both in ovarian biopsies and in cultured OSE. There was no difference in this regard between fertile and postmenopausal women. Cells in culture were polarized and presented junctional complexes seen by electron microscopy. In the Ca(2+) switch experiments, removing free Ca(2+) transiently, TER decreased significantly (P < 0.05) in the Ca(2+)-free group compared with nontreated OSE. TER was fully restored after 24 h. N-cadherin but not E-cadherin was expressed in the OSE and localized to the cell borders. We conclude that normal human OSE express and form functional TJ both in vivo and vitro. This report also describes a method to study the influence of ovarian-derived mediators on TJ in cultured OSE.     

Isolation and lipid composition of apical and basolateral membranes of colonic segments of guinea Pig

In adapting several methods of membrane isolation we established a successful way to purify apical and basolateral membranes of guinea pig colon in a parallel procedure. The conventional purification control by marker enzymes was applied. In addition, luminal membrane proteins were stained with Texas Red. Apical and basolateral enterocyte membranes were enriched 10- to 12-fold by differential precipitation and via a continuous sorbitol gradient. The membrane fractions were examined with regard to their phospholipid (PL) and fatty acid patterns and to their cholesterol content. Fluorescence polarization studies were carried out using 1,6-diphenyl-1,3, 5-hexatrien. Remarkable differences in the fatty acid pattern of the proximal and the distal colon were seen. Due to a higher content of oleic acid the saturation index of the apical membranes of the proximal colon is lower compared to that of the apical membranes of the distal colon (0.34 +/- 0.03 vs 0.42 +/- 0.05). The cholesterol content of the apical membranes of the proximal colon is markedly higher than that of the apical membranes of the distal colon (3.42 +/- 0.14 vs 1.88 +/- 0.29 mol/mol PL). There are no differences in the fluidity of these apical membranes. We assume a balancing mechanism between the cholesterol content and the amount of saturated PL-fatty acids.     

N-Glycan synthesis in the apical and basolateral secretory pathway of epithelial MDCK cells and the influence of a glycosaminoglycan domain

Different classes of glycans are implicated as mediators of apical protein sorting in the secretory pathway of epithelial cells, but recent research indicates that sorting to the apical and basolateral surfaces may occur before completion of glycan synthesis. We have previously shown that a proteoglycan (PG) core protein can obtain different glycosaminoglycan (GAG) structures in the apical and basolateral secretory routes (Tveit H, Dick G, Skibeli V, Prydz K. 2005. A proteoglycan undergoes different modifications en route to the apical and basolateral surfaces of Madin-Darby canine kidney cells. J Biol Chem. 280:29596-29603) of epithelial Madin-Darby canine kidney (MDCK) cells. We have now also determined the detailed N-glycan structures acquired by a single glycoprotein species in the same apical and basolateral secretory pathways. For this purpose, rat growth hormone (rGH) with two N-glycan sites (rGH-2N) inserted into the rGH portion (NAS and NFT) was fused to green fluorescent protein (GFP) and expressed in MDCK cells. Immunoisolated rGH variants were analyzed for site occupancy and N-glycan structure by mass spectrometry. The extent of NAS and NFT site occupancy was different, but comparable for rGH-2N secreted apically and basolaterally. Microheterogeneity existed for the glycans attached to each N-glycan site, but no major differences were observed in the apical and basolateral pathways. Transfer of the GAG modification domain from the PG serglycin to the fusion site of rGH-2N and GFP allowed polymerization of GAG chains onto the novel protein variant and influenced the microheterogeneity of the N-glycans toward more acidic glycans, but did not alter the relative site occupancy. In conclusion, no major differences were observed for N-glycan structures obtained by the expressed model proteins in the apical and basolateral secretory pathways of epithelial MDCK cells, but insertion of a GAG attachment domain shifted the N-glycans to more acidic structures.     

Viruses budding from either the apical or the basolateral plasma membrane domain of MDCK cells have unique phospholipid compositions.

Influenza virus and vesicular stomatitis virus (VSV) obtain their lipid envelope by budding through the plasma membrane of infected cells. When monolayers of Madin-Darby canine kidney (MDCK) cells, a polarized epithelial cell line, are infected with fowl plague virus (FPV), an avian influenza virus, or with VSV, new FPV buds through the apical plasma membrane whereas VSV progeny is formed by budding through the basolateral plasma membrane. FPV and VSV were isolated from MDCK host cells prelabeled with [32P]orthophosphate and their phospholipid compositions were compared. Infection was carried out at 31 degrees C to delay cytopathic effects of the virus infection, which lead to depolarization of the cell surface. 32P-labeled FPV was isolated from the culture medium, whereas 32P-labeled VSV was released from below the cell monolayer by scraping the cells from the culture dish 8 h after infection. At this time little VSV was found in the culture medium, indicating that the cells were still polarized. The phospholipid composition of the two viruses was distinctly different. FPV was enriched in phosphatidylethanolamine and phosphatidylserine and VSV in phosphatidylcholine, sphingomyelin, and phosphatidylinositol. When MDCK cells were trypsinized after infection and replated, non-infected control cells attached to reform a confluent monolayer within 4 h, whereas infected cells remained in suspension. FPV and VSV could be isolated from the cells in suspension and under these conditions the phospholipid composition of the two viruses was very similar. We conclude that the two viruses obtain their lipids from the plasma membrane in the same way and that the different phospholipid compositions of the viruses from polarized cells reflect differences in the phospholipid composition of the two plasma membrane domains.     

Vps34p differentially regulates endocytosis from the apical and basolateral domains in polarized hepatic cells

Using a microinjection approach to study apical plasma membrane protein trafficking in hepatic cells, we found that specific inhibition of Vps34p, a class III phosphoinositide 3 (PI-3) kinase, nearly perfectly recapitulated the defects we reported for wortmannin-treated cells (Tuma, P.L., C.M. Finnegan, J.-H Yi, and A.L. Hubbard. 1999. J. Cell Biol. 145:1089-1102). Both wortmannin and injection of inhibitory Vps34p antibodies led to the accumulation of resident apical proteins in enlarged prelysosomes, whereas transcytosing apical proteins and recycling basolateral receptors transiently accumulated in basolateral early endosomes. To understand how the Vps34p catalytic product, PI3P, was differentially regulating endocytosis from the two domains, we examined the PI3P binding protein early endosomal antigen 1 (EEA1). We determined that EEA1 distributed to two biochemically distinct endosomal populations: basolateral early endosomes and subapical endosomes. Both contained rab5, although the latter also contained late endosomal markers but was distinct from the transcytotic intermediate, the subapical compartment. When PI3P was depleted, EEA1 dissociated from basolateral endosomes, whereas it remained on subapical endosomes. From these results, we conclude that PI3P, via EEA1, regulates early steps in endocytosis from the basolateral surface in polarized WIF-B cells. However, PI3P must use different machinery in its regulation of the apical endocytic pathway, since later steps are affected by Vps34p inhibition.     

Apical to basolateral surface area ratio and polarity of MDCK cells grown on different supports.

We have established conditions under which Madin-Darby canine kidney cells develop a well-polarized monolayer on polycarbonate filters and on transparent filters. These filters have biochemical and mechanical advantages over the nitrocellulose filters which have been widely used. Transepithelial resistance was established 10 h after plating and stabilized after 24 h. The distribution of protein antigens was followed by surface immunofluorescence and quantitated by a surface immunoassay that we developed. Uvomorulin was localized to the lateral membrane, with low amounts detectable on the basal membrane. The 58-kDa antigen was distributed over the entire basolateral domain, including cell processes extending into the filter pores. This distribution was confirmed by immunogold labeling of frozen sections. The 114-kDa antigen was found to be present at similar surface densities on both the apical and the basolateral domain. The support used for growth had profound effects on the cell morphology. A morphometric analysis of the plasma membrane of both strains of the cell line showed an increase in the number and size of the microvilli, and a smoother basal membrane as compared to published data on nitrocellulose filters. The apical to basolateral surface area ratio was therefore modified.     

Sphingolipid transport from the trans-Golgi network to the apical surface in permeabilized MDCK cells

We have measured the transport of de novo synthesized fluorescent analogs of sphingomyelin and glucosylceramide from the trans-Golgi network (TGN) to the apical membrane in basolaterally permeabilized Madin-Darby canine kidney (MDCK) cells. Sphingolipid transport was temperature, ATP and cytosol dependent. Introduction of bovine serum albumin (BSA), which binds fluorescent sphingolipid monomer, into the permeabilized cells, did not affect lipid transport to the apical membrane. Both fluorescent sphingomyelin and glucosylceramide analogs were localized to the lumenal bilayer leaflet of isolated TGN-derived vesicles. These results strongly suggest that both sphingolipids are transported from the TGN to the apical membrane via vesicular traffic.     

Distinct apical and basolateral membrane requirements for stretch-induced membrane traffic at the apical surface of bladder umbrella cells

Epithelial cells respond to mechanical stimuli by increasing exocytosis, endocytosis, and ion transport, but how these processes are initiated and coordinated and the mechanotransduction pathways involved are not well understood. We observed that in response to a dynamic mechanical environment, increased apical membrane tension, but not pressure, stimulated apical membrane exocytosis and ion transport in bladder umbrella cells. The exocytic response was independent of temperature but required the cytoskeleton and the activity of a nonselective cation channel and the epithelial sodium channel. The subsequent increase in basolateral membrane tension had the opposite effect and triggered the compensatory endocytosis of added apical membrane, which was modulated by opening of basolateral K⁺ channels. Our results indicate that during the dynamic processes of bladder filling and voiding apical membrane dynamics depend on sequential and coordinated mechanotransduction events at both membrane domains of the umbrella cell.