Targeting of an apical endosomal protein to endosomes in Madin-Darby canine kidney cells requires two sorting motifs

The efficient sorting and targeting of endocytosed macromolecules is critical for epithelial function. However, the distribution of endosomal compartments in these cells remains controversial. In this study, we show that polarized Madin–Darby canine kidney (MDCK) cells target the apical endosomal protein endotubin into an apical early endosomal compartment that is distinct from the apical recycling endosomes. Furthermore, through a panel of site-directed mutations we show that signals required for apical endosomal targeting of endotubin are composed of two distinct motifs on the cytoplasmic domain, a hydrophobic motif and a consensus casein kinase II site. Endotubin-positive endosomes in MDCK cells do not label with basolaterally internalized transferrin or ricin, do not contain the small guanosine triphosphate-binding protein rab11, and do not tubulate in response to low concentrations of brefeldin-A (BFA). Nevertheless, high concentrations of BFA reversibly inhibits the sorting of endotubin from transferrin and cause colocalization in tubular endosomes. These results indicate that, in polarized cells, endotubin targets into a distinct subset of apical endosomes, and the targeting information required both for polarity and endosomal targeting is provided by the cytoplasmic portion of the molecule.     

Zonula occludens-1 function in the assembly of tight junctions in Madin-Darby canine kidney epithelial cells

Zonula occludens (ZO)-1 was the first tight junction protein to be cloned and has been implicated as an important scaffold protein. It contains multiple domains that bind a diverse set of junction proteins. However, the molecular functions of ZO-1 and related proteins such as ZO-2 and ZO-3 have remained unclear. We now show that gene silencing of ZO-1 causes a delay of approximately 3 h in tight junction formation in Madin-Darby canine kidney (MDCK) epithelial cells, but mature junctions seem functionally normal even in the continuing absence of ZO-1. Depletion of ZO-2, cingulin, or occludin, proteins that can interact with ZO-1, had no discernible effects on tight junctions. Rescue of junction assembly using murine ZO-1 mutants demonstrated that the ZO-1 C terminus is neither necessary nor sufficient for normal assembly. Moreover, mutation of the PDZ1 domain did not block rescue. However, point mutations in the Src homology 3 (SH3) domain almost completely prevented rescue. Surprisingly, the isolated SH3 domain of ZO-1 could also rescue junction assembly. These data reveal an unexpected function for the SH3 domain of ZO-1 in regulating tight junction assembly in epithelial cells and show that cingulin, occludin, or ZO-2 are not limiting for junction assembly in MDCK monolayers.     

Localization of the tight junction protein, ZO-1, is modulated by extracellular calcium and cell-cell contact in Madin-Darby canine kidney epithelial cells

Using the monoclonal antibody R26.4, we have previously identified a approximately 225-kD peripheral membrane protein, named ZO-1, that is uniquely associated with the tight junction (zonula occludens) in a variety of epithelia including the Madin-Darby canine kidney (MDCK) epithelial cell line (Stevenson, B. R., J. D. Siliciano, M. S. Mooseker, and D. A. Goodenough. 1986. J. Cell Biol. 103:755-766). In this study we have analyzed the effects of cell-cell contact and extracellular calcium on the localization and the solubility of ZO-1. In confluent monolayers under normal calcium conditions, ZO-1 immunoreactivity is found exclusively at the plasma membrane in the region of the junctional complex. If MDCK cells are maintained in spinner culture under low calcium conditions, ZO-1 is diffusely organized within the cytoplasm. After the plating of suspension cells at high cell density in medium with normal calcium concentrations, ZO-1 becomes localized to the plasma membrane at sites of cell-cell contact within 5 h in a process that is independent of de novo protein synthesis. However, if suspension cells are plated at high density in low calcium medium or if suspension cells are plated at low cell density in normal calcium growth medium, ZO-1 remains diffusely organized. ZO-1 localization also becomes diffuse in monolayers that have been established in normal calcium medium and then subsequently switched into low calcium medium. These results suggest that both extracellular calcium and cell-cell contact are necessary for normal localization of ZO-1 to the plasma membrane. An analysis of the solubility properties of ZO-1 from suspension cells and monolayers revealed that high salt, nonionic detergent, and a buffer containing chelators were somewhat more effective at solubilizing ZO-1 from suspension cells than from monolayers.     

Modulation of the expression of an apical plasma membrane protein of Madin-Darby canine kidney epithelial cells: cell-cell interactions control the appearance of a novel intracellular storage compartment

Experimental conditions that abolish or reduce to a minimum intercellular contacts between Madin-Darby canine kidney epithelial cells result in the appearance of an intracellular storage compartment for apical membrane proteins. Subconfluent culture, incubation in 1-5 microM Ca++, or inclusion of dissociated cells within agarose or collagen gels all caused the intracellular accumulation of a 184-kD apical membrane protein within large (0.5-5 micron) vacuoles, rich in microvilli. Influenza virus hemagglutinin, an apically targeted viral glycoprotein, is concentrated within these structures but the basolateral glycoprotein G of vesicular stomatitis virus and a cellular basolateral 63-kD membrane protein of Madin-Darby canine kidney cells were excluded. This novel epithelial organelle (VAC), which we designate the vacuolar apical compartment, may play an as yet unrecognized role in the biogenesis of the apical plasma membrane during the differentiation of normal epithelia.     

Modulation of fodrin (membrane skeleton) stability by cell-cell contact in Madin-Darby canine kidney epithelial cells

During growth of Madin-Darby canine kidney (MDCK) epithelial cells, there is a dramatic change in the stability, biophysical properties, and distribution of the membrane skeleton (fodrin) which coincides temporally and spatially with the development of the polarized distribution of the Na+, K+-ATPase, a marker protein of the basolateral domain of the plasma membrane. These changes occur maximally upon the formation of a continuous monolayer of cells, indicating that extensive cell-cell contact may play an important role in the organization of polarized MDCK cells (Nelson, W. J., and P. J. Veshnock, 1986, J. Cell Biol., 103:1751-1766). To directly analyze the role of cell-cell contact in these events, we have used an assay in which the organization of fodrin and membrane proteins is analyzed in confluent monolayers of MDCK cells in the absence or presence of cell-cell contact by adjusting the concentration Ca++ in the growth medium. Our results on the stability and solubility properties of fodrin reported here show directly that there is a positive correlation between cell-cell contact and increased stability and insolubility of fodrin. Furthermore, we show that fodrin can be recruited from an unstable pool of protein to a stable pool during induction of cell-cell contact; significantly, the stabilization of fodrin is not affected by the addition of cyclohexamide, indicating that proteins normally synthesized during the induction of cell-cell contact are not required. Together these results indicate that cell-cell contact may play an important role in the development of polarity in MDCK cells by initiating the formation of a stable, insoluble matrix of fodrin with preexisting (membrane) proteins at the cell periphery. This matrix may function subsequently to trap proteins targeted to the membrane, resulting in the maintenance of membrane domains.     

Cdc42-dependent modulation of tight junctions and membrane protein traffic in polarized Madin-Darby canine kidney cells

Polarized epithelial cells maintain the asymmetric composition of their apical and basolateral membrane domains by at least two different processes. These include the regulated trafficking of macromolecules from the biosynthetic and endocytic pathway to the appropriate membrane domain and the ability of the tight junction to prevent free mixing of membrane domain-specific proteins and lipids. Cdc42, a Rho family GTPase, is known to govern cellular polarity and membrane traffic in several cell types. We examined whether this protein regulated tight junction function in Madin-Darby canine kidney cells and pathways that direct proteins to the apical and basolateral surface of these cells. We used Madin-Darby canine kidney cells that expressed dominant-active or dominant-negative mutants of Cdc42 under the control of a tetracycline-repressible system. Here we report that expression of dominant-active Cdc42V12 or dominant-negative Cdc42N17 altered tight junction function. Expression of Cdc42V12 slowed endocytic and biosynthetic traffic, and expression of Cdc42N17 slowed apical endocytosis and basolateral to apical transcytosis but stimulated biosynthetic traffic. These results indicate that Cdc42 may modulate multiple cellular pathways required for the maintenance of epithelial cell polarity.     

Dynamics of membrane-skeleton (fodrin) organization during development of polarity in Madin-Darby canine kidney epithelial cells

Madin-Darby canine kidney (MDCK) epithelial cells exhibit a polarized distribution of membrane proteins between the apical and basolateral domains of the plasma membrane. We have initiated studies to investigate whether the spectrin-based membrane skeleton plays a role in the establishment and maintenance of these membrane domains. MDCK cells express an isoform of spectrin composed of two subunits, Mr 240,000 (alpha-subunit) and Mr 235,000 (gamma-subunit). This isoform is immunologically and structurally related to fodrin in lens and brain cells, which is a functional and structural analog of alpha beta-spectrin, the major component of the erythrocyte membrane skeleton. Analysis of fodrin in MDCK cells by immunoblotting, immunofluorescence, and metabolic labeling revealed significant changes in the biophysical properties, subcellular distribution, steady-state levels, and turnover of the protein during development of a continuous monolayer of cells. The changes in the cellular organization of fodrin did not appear to coincide with the distributions of microfilaments, microtubules, or intermediate filaments. These changes result in the formation of a highly insoluble, relatively dense and stable layer of fodrin which appears to be localized to the cell periphery and predominantly in the region of the basolateral plasma membrane of MDCK cells in continuous monolayers. The formation of this structure coincides temporally and spatially with extensive cell-cell contact, and with the development of the polarized distribution of the Na+, K+-ATPase, a marker protein of the basolateral plasma membrane.     

Enhancement of cell-cell contact by claudin-4 in renal epithelial Madin-Darby canine kidney cells

Claudin-4 regulates ion permeability via a paracellular pathway in renal epithelial cells, but its other physiological functions have not been examined. We found that hyperosmotic stress increases claudin-4 expression in Madin-Darby canine kidney cells. Here, we examined whether claudin-4 affects cell motility, cell association, and the intracellular distribution of endogenous junctional proteins. Doxycycline-inducible expression of claudin-4 did not change endogenous levels of claudin-1, claudin-2, claudin-3, occludin, E-cadherin, and ZO-1. Claudin-4 overexpression increased cell association and decreased cell migration without affecting cell proliferation. Doxycycline did not change cell junctional protein levels, cell association or cell migration in mock-transfected cells. The insolubility of claudin-1 and -3 in Triton X-100 was increased by claudin-4 overexpression, but that of claudin-2, occludin, ZO-1, and E-cadherin was unchanged. Immunocytochemistry showed that claudin-4 overexpression increases the accumulation of claudin-1 and -3 in tight junctions (TJs). Furthermore, claudin-4 overexpression increased the association of claudin-4 with claudin-1 and -3. These results suggest that claudin-4 accumulates claudin-1 and -3 in TJs to enhance cell-cell contact in renal tubular epithelial cells.     

Sorting of sphingolipids in epithelial (Madin-Darby canine kidney) cells

To study the intracellular transport of newly synthesized sphingolipids in epithelial cells we have used a fluorescent ceramide analog, N-6[7-nitro-2,1,3-benzoxadiazol-4-yl] aminocaproyl sphingosine (C6-NBD-ceramide; Lipsky, N. G., and R. E. Pagano, 1983, Proc. Natl. Acad. Sci. USA, 80:2608-2612) as a probe. This ceramide was readily taken up by filter-grown Madin-Darby canine kidney (MDCK) cells from liposomes at 0 degrees C. After penetration into the cell, the fluorescent probe accumulated in the Golgi area at temperatures between 0 and 20 degrees C. Chemical analysis showed that C6-NBD-ceramide was being converted into C6-NBD-sphingomyelin and C6-NBD-glucosyl-ceramide. An analysis of the fluorescence pattern after 1 h at 20 degrees C by means of a confocal scanning laser fluorescence microscope revealed that the fluorescent marker most likely concentrated in the Golgi complex itself. Little fluorescence was observed at the plasma membrane. Raising the temperature to 37 degrees C for 1 h resulted in intense plasma membrane staining and a loss of fluorescence from the Golgi complex. Addition of BSA to the apical medium cleared the fluorescence from the apical but not from the basolateral plasma membrane domain. The basolateral fluorescence could be depleted only by adding BSA to the basal side of a monolayer of MDCK cells grown on polycarbonate filters. We conclude that the fluorescent sphingomyelin and glucosylceramide were delivered from the Golgi complex to the plasma membrane where they accumulated in the external leaflet of the membrane bilayer. The results also demonstrated that the fatty acyl labeled lipids were unable to pass the tight junctions in either direction. Quantitation of the amount of NBD-lipids delivered to the apical and the basolateral plasma membranes during incubation for 1 h at 37 degrees C showed that the C6-NBD-glucosylceramide was two- to fourfold enriched on the apical as compared to the basolateral side, while C6-NBD-sphingomyelin was about equally distributed. Since the surface area of the apical plasma membrane is much smaller than that of the basolateral membrane, both lipids achieved a higher concentration on the apical surface. Altogether, our results suggest that the NBD-lipids are sorted in MDCK cells in a way similar to their natural counterparts.     

Sorting of membrane and fluid at the apical pole of polarized Madin-Darby canine kidney cells

When fluid-phase markers are internalized from opposite poles of polarized Madin-Darby canine kidney cells, they accumulate in distinct apical and basolateral early endosomes before meeting in late endosomes. Recent evidence suggests that significant mixing of apically and basolaterally internalized membrane proteins occurs in specialized apical endosomal compartments, including the common recycling endosome and the apical recycling endosome (ARE). The relationship between these latter compartments and the fluid-labeled apical early endosome is unknown at present. We report that when the apical recycling marker, membrane-bound immunoglobulin A (a ligand for the polymeric immunoglobulin receptor), and fluid-phase dextran are cointernalized from the apical poles of Madin-Darby canine kidney cells, they enter a shared apical early endosome (相似文献   

Carboxypeptidase M, a glycosylphosphatidylinositol-anchored protein, is localized on both the apical and basolateral domains of polarized Madin-Darby canine kidney cells.

Carboxypeptidase M, a glycosylphosphatidylinositol-anchored membrane glycoprotein, is highly expressed in Madin-Darby canine kidney (MDCK) cells, where it was previously shown that the glycosylphosphatidylinositol anchor and N-linked carbohydrate are apical targeting signals. Here, we show that carboxypeptidase M has an unusual, non-polarized distribution, with up to 44% on the basolateral domain of polarized MDCK cells grown on semipermeable inserts. Alkaline phosphatase, as well as five other glycosylphosphatidylinositol-anchored proteins, and transmembrane gamma-glutamyl transpeptidase exhibited the expected apical localization. Basolateral carboxypeptidase M was readily released by exogenous phosphatidylinositol-specific phospholipase C, showing it is glycosylphosphatidylinositol-anchored, whereas apical carboxypeptidase M was more resistant to release. In contrast, the spontaneous release of carboxypeptidase M into the medium was much higher on the apical than the basolateral domain. In pulse-chase studies, newly synthesized carboxypeptidase M arrived in equal amounts within 30 min on both domains, indicating direct sorting. After 4-8 h of chase, the steady-state distribution was attained, possibly due to transcytosis from the basolateral to the apical domain. These data suggest the presence of a unique basolateral targeting signal in carboxypeptidase M that competes with its apical targeting signals, resulting in a non-polarized distribution in MDCK cells.     

The polarized distribution of an apical cell surface glycoprotein is maintained by interactions with the cytoskeleton of Madin-Darby canine kidney cells

A monoclonal antibody made against a 135-kD glycoprotein (gp135) on the plasma membrane of Madin-Darby canine kidney (MDCK) cells was used to study the development and maintenance of epithelial cell surface polarity. Immunofluorescence microscopy and immunogold electron microscopy of confluent monolayers demonstrated that gp135 had a polarized cell surface distribution and was only localized on the apical surface. The role of membrane contacts in establishing gp135 polarity was determined by plating cells in low Ca++-medium to prevent the formation of intercellular junctions. Quantitative immunogold electron microscopy demonstrated that gp135 had a polarized distribution on cells lacking membrane contacts and was observed on the apical surface at a density 24 times that of the basal membrane contacting the substratum. The possibility that gp135 was associated with components of the apical cytoskeleton was investigated using cytoskeleton-disrupting drugs. Incubation in cytochalasin D produced a clustering of both actin and gp135, and double-label fluorescence microscopy demonstrated that these proteins were colocalized. Experiments using nocodazole had no effect, suggesting that gp135 could be interacting with actin microfilaments, but not microtubules. Treatment with Triton X-100 extracted approximately 50% of the gp135 and immunofluorescence microscopy indicated that the gp135 which remained associated with the detergent-insoluble cytoskeleton had a distribution identical to that of control cells. Experiments demonstrating that gp23, a nonpolarized glycoprotein, was preferentially extracted from the apical membrane suggested that the improperly sorted apical gp23 did not interact with the cytoskeleton. These results provided evidence that the polarized cell surface distribution of gp135 was maintained through its interaction with actin in the apical cytoskeleton.     

Involvement of nectin-activated Cdc42 small G protein in organization of adherens and tight junctions in Madin-Darby canine kidney cells

Nectins, Ca2+-independent immunoglobulin-like cell-cell adhesion molecules, trans-interact and form cell-cell adhesion, which increases the velocities of the formation of the E-cadherin-based adherens junctions (AJs) and the claudin-based tight junctions (TJs) in Madin-Darby canine kidney (MDCK) cells. The trans-interactions of nectins furthermore induce activation of Cdc42 and Rac small G proteins, but the roles of these small G proteins activated in this way remain unknown. We examined here the role and the mode of action of Cdc42 in the organization of AJs and TJs in MDCK cells. We first made the NWASP-Cdc42 and Rac interactive binding (CRIB) domain, an inhibitor of activated Cdc42, fused to the Ki-Ras CAAX motif (NWASP-CRIB-CAAX; where A is aliphatic amino acid), which was targeted to the cell-cell adhesion sites. We then found that overexpression of NWASP-CRIB-CAAX reduced the velocities of the formation of AJs and TJs. Conversely, overexpression of a constitutively active mutant of Cdc42 (V12Cdc42) increased their velocities, and the inhibitory effect of NWASP-CRIB-CAAX was suppressed by co-expression with V12Cdc42. The inhibitory effect of NWASP-CRIB-CAAX on the formation of AJs and TJs was suppressed by co-expression of nectin-1 of which trans-interaction activated endogenous Cdc42. Moreover, the formation of the claudin-based TJs required a greater amount of activated Cdc42 than that of the E-cadherin-based AJs. These results indicate that the Cdc42 activated by the trans-interactions of nectins is involved in the organization of AJs and TJs in different mechanisms in MDCK cells.     

MAL mediates apical transport of secretory proteins in polarized epithelial Madin-Darby canine kidney cells.

The MAL proteolipid is an integral membrane protein identified as a component of the raft machinery for apical sorting of membrane proteins in Madin-Darby canine kidney (MDCK) cells. Previous studies have implicated lipid rafts in the transport of exogenous thyroglobulin (Tg), the predominant secretory protein of thyroid epithelial cells, to the apical surface in MDCK cells. We have examined the secretion of recombinant Tg and gp80/clusterin, a major endogenous secretory protein not detected in Triton X-100 insoluble rafts, for the investigation of the involvement of MAL in the constitutive apical secretory pathway of MDCK cells. We show that MAL depletion impairs apical secretion of Tg and causes its accumulation in the Golgi. Cholesterol sequestration, which blocks apical secretion of Tg, did not alter the levels of MAL in rafts but created a block proximal to Tg entrance into rafts. Apical secretion of gp80/clusterin was also inhibited by elimination of endogenous MAL. Our results suggest a role for MAL in the transport of both endogenously and exogenously expressed apical secretory proteins in MDCK cells.     

A hepatocyte-specific basolateral membrane protein is targeted to the same domain when expressed in Madin-Darby canine kidney cells

Different mechanisms for polarized sorting of apical and basolateral plasma membrane proteins appear to be operative in different cell types. In hepatocytes, all proteins are first transported to the basolateral surface, where sorting (probably signal-mediated) of apical proteins then takes place. In contrast, in Madin-Darby canine kidney (MDCK) cells, proteins are directly transported from the trans-Golgi network to their appropriate plasma membrane domain. In order to study the differences in the sorting requirements of the two cell types, we have expressed a hepatocyte-specific basolateral membrane protein, the asialoglycoprotein receptor H1, in MDCK cells. H1 was found to be specifically transported to the basolateral domain also in this heterologous system, suggesting that either the same basolateral targeting signal is operative in both cell types or, more likely, that basolateral transport occurs "by default," i.e. without the requirement for a sorting signal.     

A proteoglycan undergoes different modifications en route to the apical and basolateral surfaces of Madin-Darby canine kidney cells

We have grown polarized epithelial Madin-Darby canine kidney II (MDCK II) cells on filters in the presence of [(35)S]sulfate, [(3)H]glucosamine, or [(35)S]cysteine/[(35)S]methionine to study proteoglycan (PG) synthesis, sorting, and secretion to the apical and basolateral media. Whereas most of the [(35)S]sulfate label was recovered in basolateral PGs, the [(3)H]glucosamine label was predominantly incorporated into the glycosaminoglycan chains of apical PGs, indicating that basolateral PGs are more intensely sulfated than their apical counterparts. Expression of the PG serglycin with a green fluorescent protein tag (SG-GFP) in MDCK II cells produced a protein core secreted 85% apically, which was largely modified by chondroitin sulfate chains. Surprisingly, the 15% of secreted SG-GFP molecules recovered basolaterally were more heavily sulfated and displayed a different sulfation pattern than the apical counterpart. More detailed studies of the differential modification of apically and basolaterally secreted SG-GFP indicate that the protein cores have been designated to apical and basolateral transport platforms before pathway-specific, post-translational modifications have been completed.     

Development of cell surface polarity in the epithelial Madin-Darby canine kidney (MDCK) cell line.

The development of surface polarity has been studied in the epithelial Madin-Darby canine kidney (MDCK) cell line by examining two basolateral markers: a monoclonal antibody against a 58-kd protein and [35S]methionine uptake. The surface distribution of these markers was followed after plating the cells on coverslips or nitrocellulose filters. In subconfluent monolayers the apical surface of many cells was stained with the anti-58-kd antibody. Clearing of the apical surface occurred first after confluency had been reached in cells grown on coverslips. Similarly, in cells grown on filters the basolateral 58-kd protein disappeared from the apical surface concomitantly with the development of a measurable electrical resistance over the cell monolayer. The uptake of [35S]methionine was measured from both sides of filter-grown cells and began to polarize early after seeding, reaching a value of greater than 98% basolateral in the fully polarized monolayer. These results emphasize that the development of surface polarity in MDCK cells is a gradual process, and that extensive cell-cell contacts seem to be required for complete surface polarization.     

Absence of the tight junctional protein AF-6 disrupts epithelial cell-cell junctions and cell polarity during mouse development.

BACKGROUND: The establishment, maintenance and rearrangement of junctions between epithelial cells are extremely important in many developmental, physiological and pathological processes. AF-6 is a putative Ras effector; it is also a component of tight and adherens junctions, and has been shown to bind both Ras and the tight-junction protein ZO-1. In the mouse, AF-6 is encoded by the Af6 gene. As cell-cell junctions are important in morphogenesis, we generated a null mutation in the murine Af6 locus to test the hypothesis that lack of AF-6 function would cause epithelial abnormalities. RESULTS: Although cell-cell junctions are thought to be important in early embryogenesis, homozygous mutant embryos were morphologically indistinguishable from wild-type embryos through 6.5 days post coitum (dpc) and were able to establish all three germ layers. The earliest morphological abnormalities were observed in the embryonic ectoderm of mutant embryos at 7.5 dpc. The length of the most apical cell-cell junctions was reduced, and basolateral surfaces of those cells were separated by multiple gaps. Cells of the embryonic ectoderm were less polarized as assessed by histological criteria and lateral localization of an apical marker. Mutant embryos died by 10 dpc, probably as a result of placental failure. CONCLUSIONS: AF-6 is a critical regulator of cell-cell junctions during mouse development. The loss of neuroepithelial polarity in mutants is consistent with a loss of efficacy of the cell-cell junctions that have a critical role in establishing apical/basolateral asymmetry.     

Glucocorticoid down-regulation of fascin protein expression is required for the steroid-induced formation of tight junctions and cell-cell interactions in rat mammary epithelial tumor cells

Glucocorticoid hormones, which are physiological regulators of mammary epithelium development, induce the formation of tight junctions in rat Con8 mammary epithelial tumor cells. We have discovered that, as part of this process, the synthetic glucocorticoid dexamethasone strongly and reversibly down-regulated the expression of fascin, an actin-bundling protein that also interacts with the adherens junction component beta-catenin. Ectopic constitutive expression of full-length mouse fascin containing a Myc epitope tag (Myc-fascin) in Con8 cells inhibited the dexamethasone stimulation of transepithelial electrical resistance, disrupted the induced localization of the tight junction protein occludin and the adherens junction protein beta-catenin to the cell periphery, and prevented the rearrangement of the actin cytoskeleton. Ectopic expression of either the carboxyl-terminal 213 amino acids of fascin, which includes the actin and beta-catenin-binding sites, or the amino-terminal 313 amino acids of fascin failed to disrupt the glucocorticoid induction of tight junction formation. Mammary tumor cells expressing the full-length Myc-fascin remained generally glucocorticoid responsive and displayed no changes in the levels or protein-protein interactions of junctional proteins or the amount of cytoskeletal associated actin filaments. However, a cell aggregation assay demonstrated that the expression of Myc-fascin abrogated the dexamethasone induction of cell-cell adhesion. Our results implicate the down-regulation of fascin as a key intermediate step that directly links glucocorticoid receptor signaling to the coordinate control of junctional complex formation and cell-cell interactions in mammary tumor epithelial cells.     

Novel kelch-like protein, KLEIP, is involved in actin assembly at cell-cell contact sites of Madin-Darby canine kidney cells

Dynamic rearrangements of cell-cell adhesion underlie a diverse range of physiological processes, but their precise molecular mechanisms are still obscure. Thus, identification of novel players that are involved in cell-cell adhesion would be important. We isolated a human kelch-related protein, Kelch-like ECT2 interacting protein (KLEIP), which contains the broad-complex, tramtrack, bric-a-brac (BTB)/poxvirus, zinc finger (POZ) motif and six-tandem kelch repeats. KLEIP interacted with F-actin and was concentrated at cell-cell contact sites of Madin-Darby canine kidney cells, where it colocalized with F-actin. Interestingly, this localization took place transiently during the induction of cell-cell contact and was not seen at mature junctions. KLEIP recruitment and actin assembly were induced around E-cadherin-coated beads placed on cell surfaces. The actin depolymerizing agent cytochalasin B inhibited this KLEIP recruitment around E-cadherin-coated beads. Moreover, constitutively active Rac1 enhanced the recruitment of KLEIP as well as F-actin to the adhesion sites. These observations strongly suggest that KLEIP is localized on actin filaments at the contact sites. We also found that N-terminal half of KLEIP, which lacks the actin-binding site and contains the sufficient sequence for the localization at the cell-cell contact sites, inhibited constitutively active Rac1-induced actin assembly at the contact sites. We propose that KLEIP is involved in Rac1-induced actin organization during cell-cell contact in Madin-Darby canine kidney cells.