Differential involvement of endocytic compartments in the biosynthetic traffic of apical proteins

Newly synthesized basolateral markers can traverse recycling endosomes en route to the surface of Madin-Darby canine kidney cells; however, the routes used by apical proteins are less clear. Here, we functionally inactivated subsets of endocytic compartments and examined the effect on surface delivery of the basolateral marker vesicular stomatitis virus glycoprotein (VSV-G), the raft-associated apical marker influenza hemagglutinin (HA), and the non-raft-associated protein endolyn. Inactivation of transferrin-positive endosomes after internalization of horseradish peroxidase (HRP)-containing conjugates inhibited VSV-G delivery, but did not disrupt apical delivery. In contrast, inhibition of protein export from apical recycling endosomes upon expression of dominant-negative constructs of myosin Vb or Sec15 selectively perturbed apical delivery of endolyn. Ablation of apical endocytic components accessible to HRP-conjugated wheat germ agglutinin (WGA) disrupted delivery of HA but not endolyn. However, delivery of glycosylphosphatidylinositol-anchored endolyn was inhibited by >50% under these conditions, suggesting that the biosynthetic itinerary of a protein is dependent on its targeting mechanism. Our studies demonstrate that apical and basolateral proteins traverse distinct endocytic intermediates en route to the cell surface, and that multiple routes exist for delivery of newly synthesized apical proteins.     

Biogenetic pathways of plasma membrane proteins in Caco-2, a human intestinal epithelial cell line

We studied the sorting and surface delivery of three apical and three basolateral proteins in the polarized epithelial cell line Caco-2, using pulse-chase radiolabeling and surface domain-selective biotinylation (Le Bivic, A., F. X. Real, and E. Rodriguez-Boulan. 1989. Proc. Natl. Acad. Sci. USA. 86:9313-9317). While the basolateral proteins (antigen 525, HLA-I, and transferrin receptor) were targeted directly and efficiently to the basolateral membrane, the apical markers (sucrase-isomaltase [SI], aminopeptidase N [APN], and alkaline phosphatase [ALP]) reached the apical membrane by different routes. The large majority (80%) of newly synthesized ALP was directly targeted to the apical surface and the missorted basolateral pool was very inefficiently transcytosed. SI was more efficiently targeted to the apical membrane (greater than 90%) but, in contrast to ALP, the missorted basolateral pool was rapidly transcytosed. Surprisingly, a distinct peak of APN was detected on the basolateral domain before its accumulation in the apical membrane; this transient basolateral pool (at least 60-70% of the enzyme reaching the apical surface, as measured by continuous basal addition of antibodies) was efficiently transcytosed. In contrast with their transient basolateral expression, apical proteins were more stably localized on the apical surface, apparently because of their low endocytic capability in this membrane. Thus, compared with two other well-characterized epithelial models, MDCK cells and the hepatocyte, Caco-2 cells have an intermediate sorting phenotype, with apical proteins using both direct and indirect pathways, and basolateral proteins using only direct pathways, during biogenesis.     

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.     

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

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

Interactions between the exocytic and endocytic pathways in polarized Madin-Darby canine kidney cells

The compartments involved in polarized exocytosis of membrane proteins are not well defined. In this study we hypothesized that newly synthesized polymeric immunoglobulin receptors are targeted from the trans-Golgi network to endosomes prior to their appearance on the basolateral cell surface of polarized Madin-Darby canine kidney cells. To examine this hypothesis, we have used an assay designed to measure the meeting of newly synthesized receptors with a selective population of apical or basolateral endosomes loaded with horseradish peroxidase. We found that in the course of basolateral exocytosis, the wild-type polymeric immunoglobulin receptor is targeted from the trans-Golgi network to apical and basolateral endosomes. Phosphorylation of a Ser residue in the cytoplasmic tail of the receptor is implicated in this process. The biosynthetic pathway of apically sorted polymeric immunoglobulin receptor mutants similarly traversed apical endosomes, raising the possibility that apical receptors are segregated from basolateral receptors in apical endosomes. The post-endocytic pathway of transcytosing and recycling receptors also passed through apical endosomes. Together, these observations are consistent with the possibility that the biosynthetic and endocytic routes merge into endosomes and justify a model suggesting that endosomal recycling processes govern polarized trafficking of proteins traveling in both pathways.     

Different biosynthetic transport routes to the plasma membrane in BHK and CHO cells

The question of how membrane proteins are delivered from the TGN to the cell surface in fibroblasts has received little attention. In this paper we have studied how their post-Golgi delivery routes compare with those in epithelia] cells. We have analyzed the transport of the vesicular stomatitis virus G protein, the Semliki Forest virus spike glycoprotein, both basolateral in MDCK cells, and the influenza virus hemagglutinin, apical in MDCK cells. In addition, we also have studied the transport of a hemagglutinin mutant (Cys543Tyr) which is basolateral in MDCK cells. Aluminum fluoride, a general activator of heterotrimeric G proteins, inhibited the transport of the basolateral cognate proteins, as well as of the hemagglutinin mutant, from the TGN to the cell surface in BHK and CHO cells, while having no effect on the surface delivery of the wild-type hemagglutinin. Only wild-type hemagglutinin became insoluble in the detergent CHAPS during transport through the BHK and CHO Golgi complexes, whereas the basolateral marker proteins remained CHAPS-soluble. We also have developed an in vitro assay using streptolysin O-permeabilized BHK cells, similar to the one we have previously used for analyzing polarized transport in MDCK cells (Pimplikar, S.W., E. Ikonen, and K. Simons. 1994. J. Cell Biol. 125:1025-1035). In this assay anti-NSF and rab-GDI inhibited transport of Semliki Forest virus spike glycoproteins from the TGN to the cell surface while having little effect on transport of the hemagglutinin. Altogether these data suggest that fibroblasts have apical and basolateral cognate routes from the TGN to the plasma membrane.     

Transport of vesicular stomatitis virus G protein to the cell surface is signal mediated in polarized and nonpolarized cells

Current model propose that in nonpolarized cells, transport of plasma membrane proteins to the surface occurs by default. In contrast, compelling evidence indicates that in polarized epithelial cells, plasma membrane proteins are sorted in the TGN into at least two vectorial routes to apical and basolateral surface domains. Since both apical and basolateral proteins are also normally expressed by both polarized and nonpolarized cells, we explored here whether recently described basolateral sorting signals in the cytoplasmic domain of basolateral proteins are recognized and used for post TGN transport by nonpolarized cells. To this end, we compared the inhibitory effect of basolateral signal peptides on the cytosol-stimulated release of two basolateral and one apical marker in semi-intact fibroblasts (3T3), pituitary (GH3), and epithelial (MDCK) cells. A basolateral signal peptide (VSVGp) corresponding to the 29-amino acid cytoplasmic tail of vesicular stomatitis virus G protein (VSVG) inhibited with identical potency the vesicular release of VSVG from the TGN of all three cell lines. On the other hand, the VSVG peptide did not inhibit the vesicular release of HA in MDCK cells not of two polypeptide hormones (growth hormone and prolactin) in GH3 cells, whereas in 3T3 cells (influenza) hemagglutinin was inhibited, albeit with a 3x lower potency than VSVG. The results support the existence of a basolateral-like, signal-mediated constitutive pathway from TGN to plasma membrane in all three cell types, and suggest that an apical-like pathway may be present in fibroblast. The data support cargo protein involvement, not bulk flow, in the formation of post-TGN vesicles and predict the involvement of distinct cytosolic factors in the assembly of apical and basolateral transport vesicles.     

Requirement for galectin-3 in apical protein sorting

The central aspect of epithelial cells is their polarized structure, characterized by two distinct domains of the plasma membrane, the apical and the basolateral membrane. Apical protein sorting requires various signals and different intracellular routes to the cell surface. The first apical targeting motif identified is the membrane anchoring of a polypeptide by glycosyl-phosphatidyl-inositol (GPI). A second group of apical signals involves N- and O-glycans, which are exposed to the luminal side of the sorting organelle. Sucrase-isomaltase (SI) and lactase-phlorizin hydrolase (LPH), which use separate transport platforms for trafficking, are two model proteins for the study of apical protein sorting. In contrast to LPH, SI associates with sphingolipid/cholesterol-enriched membrane microdomains or "lipid rafts". After exit form the trans-Golgi network (TGN), the two proteins travel in distinct vesicle populations, SAVs (SI-associated vesicles) and LAVs (LPH-associated vesicles) . Here, we report the identification of the lectin galectin-3 delivering non-raft-dependent glycoproteins in the lumen of LAVs in a carbohydrate-dependent manner. Depletion of galectin-3 from MDCK cells results in missorting of non-raft-dependent apical membrane proteins to the basolateral cell pole. This suggests a direct role of galectin-3 in apical sorting as a sorting receptor.     

A polarized human endometrial cell line that binds and transports polymeric IgA

Summary We have demonstrated that a human endometrial cell line, HEC-1, maintains a high transepithelial electrical resistance, directionally transports fluids across the cell monolayer, and releases enveloped viruses at distinct plasma membrane domains: influenza virus is released at the apical surfaces and vesicular stomatitis virus (VSV) at the basolateral surfaces. In addition, we have examined the expression of domain-specific endogenous proteins, including the polyimmunoglobulin receptor. Multiple endogenous polypeptides were found to be secreted into the culture medium at basolateral surfaces, whereas no secretion of specific polypeptides was observed from apical cell surfaces. Distinct patterns of endogenous proteins were also observed on apical and basolateral cell surfaces, with a much more complex polypeptide pattern on the basolateral membranes. Using surface biotinylation and immunofluorescence, the polyimmunoglobulin receptor was found to be expressed on the basolateral surfaces of HEC-1 monolayers. The specific binding of poly-immunoglobulin A (pIgA) was found to occur on the basolateral surface, and was followed by transcytosis to the apical surface and release into the apical medium. The observed characteristics indicate that the endometrium-derived HEC-1 epithelial cell line can be employed as a model for studies of protein transport in polarized epithelial cells of human endometrial tissues, as well as for studies of the interaction of microorganisms with epithelial cells in the genital tract.     

N-glycans mediate apical recycling of the sialomucin endolyn in polarized MDCK cells

Apical and basolateral proteins are maintained within distinct membrane subdomains in polarized epithelial cells by biosynthetic and postendocytic sorting processes. Sorting of basolateral proteins in these processes has been well studied; however, the sorting signals and mechanisms that direct proteins to the apical surface are less well understood. We previously demonstrated that an N-glycan-dependent sorting signal directs the sialomucin endolyn to the apical surface in polarized Madin-Darby canine kidney cells. Terminal processing of a subset of endolyn's N-glycans is key for polarized biosynthetic delivery to the apical membrane. Endolyn is subsequently internalized, and via a cytoplasmic tyrosine-based sorting motif is targeted to lysosomes from where it constitutively cycles to the cell surface. Here, we examine the polarized sorting of endolyn along the postendocytic pathway in polarized cells. Our results suggest that similar N-glycan sorting determinants are required for apical delivery of endolyn along both the biosynthetic and the postendocytic pathways.     

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.     

Iron repletion relocalizes hephaestin to a proximal basolateral compartment in polarized MDCK and Caco2 cells

While intestinal cellular iron entry in vertebrates employs multiple routes including heme and non-heme routes, iron egress from these cells is exclusively channeled through the only known transporter, ferroportin. Reduced intestinal iron export in sex-linked anemia mice implicates hephaestin, a ferroxidase, in this process. Polarized cells are exposed to two distinct environments. Enterocytes contact the gut lumen via the apical surface of the cell, and through the basolateral surface, to the body. Previous studies indicate both local and systemic control of iron uptake. We hypothesized that differences in iron availability at the apical and/or basolateral surface may modulate iron uptake via cellular localization of hephaestin. We therefore characterized the localization of hephaestin in two models of polarized epithelial cell lines, MDCK and Caco2, with varying iron availability at the apical and basolateral surfaces. Our results indicate that hephaestin is expressed in a supra-nuclear compartment in non-polarized cells regardless of the iron status of the cells and in iron deficient and polarized cells. In polarized cells, we found that both apical (as FeSO(4)) and basolateral iron (as the ratio of apo-transferrin to holo-transferrin) affect mobilization of hephaestin from the supra-nuclear compartment. We find that the presence of apical iron is essential for relocalization of hephaestin to a cellular compartment in close proximity but not overlapping with the basolateral surface. Surface biotinylation studies indicate that hephaestin in the peri-basolateral location is accessible to the extra-cellular environment. These results support the hypothesis that hephaestin is involved in iron mobilization of iron from the intestine to circulation.     

Membrane proteins follow multiple pathways to the basolateral cell surface in polarized epithelial cells

Newly synthesized apical and basolateral membrane proteins are sorted from one another in polarized epithelial cells. The trans-Golgi network participates in this sorting process, but some basolateral proteins travel from the Golgi to recycling endosomes (REs) before their surface delivery. Using a novel system for pulse–chase microscopy, we have visualized the postsynthetic route pursued by a newly synthesized cohort of Na,K-ATPase. We find that the basolateral delivery of newly synthesized Na,K-ATPase occurs via a pathway distinct from that pursued by the vesicular stomatitis virus G protein (VSV-G). Na,K-ATPase surface delivery occurs at a faster rate than that observed for VSV-G. The Na,K-ATPase does not pass through the RE compartment en route to the plasma membrane, and Na,K-ATPase trafficking is not regulated by the same small GTPases as other basolateral proteins. Finally, Na,K-ATPase and VSV-G travel in separate post-Golgi transport intermediates, demonstrating directly that multiple routes exist for transport from the Golgi to the basolateral membrane in polarized epithelial cells.     

Analysis of epithelial cell surface polarity with monoclonal antibodies

The hybridoma technique of K?hler and Milstein was utilized to isolate hybrid cell lines secreting monoclonal antibodies against cell surface proteins on the Madin-Darby canine kidney (MDCK) epithelial cell line. These antibodies were employed as high-affinity ligands to study the development and maintenance of epithelial cell polarity in MDCK cells and for the identification of nephron segment-specific proteins. Using standard procedures, we were able to immunoprecipitate glycoproteins with molecular weights of 25,000 ( 25K ), 35,000 ( 35K ), and 50,000 (50K). Immunofluorescence and immunoelectron microscopy of MDCK demonstrated that the 35K and 50K proteins could be localized on both the apical and basolateral membranes of subconfluent cells but primarily on the basolateral membranes of confluent cells. By determining the cell surface distribution of the 35K and 50K proteins on MDCK cells during growth into a confluent monolayer, and after the experimental disruption of tight junctions, evidence was obtained that the polarized distribution of these cell surface glycoproteins required the presence of tight junctions. We propose that confluent MDCK cells have a mechanism that is responsible for the establishment and maintenance of epithelial apical and basolateral membranes as distinct cell surface domains. These monoclonal antibodies were also used to localize the 25K and 35K glycoproteins in the kidney. The distribution of these proteins was mapped by immunofluorescence and immunoelectron microscopy and was determined to be on the basolateral membranes of epithelial cells in only certain tubular segments of the nephron. The possible functional implications of these distributions are discussed.     

Membrane traffic in polarized epithelial cells

Epithelial cells contain apical and basolateral surfaces with distinct compositions. Sorting of certain proteins to the basolateral surface involves the epithelial-specific mu 1b clathrin adaptor subunit. Recent results have shown that targeting to the basolateral surface utilizes the exocyst, whereas traffic to the apical surface uses syntaxin 3. Endocytosis at the apical surface is regulated by ARF6. Transcytosis of IgA is regulated by the p62Yes tyrosine kinase.     

Membrane protein trafficking through the common apical endosome compartment of polarized Caco-2 cells.

By raising monoclonal antibodies to the apical surface of Caco-2 cells we have identified a membrane protein (p100) that internalizes and recycles constitutively between the apical plasma membrane and endosomes in the apical cytoplasm. By applying tracers bound to the transferrin receptor, which internalizes and recycles back to the basolateral border, we demonstrate that the apical endosomes containing p100 include a subset of multivesticular bodies (MVB), which are also accessible to proteins arriving from the basolateral endosome. Tracers bound to EGF receptors and alpha-2-macroglobulin, which internalize from the basolateral border and are degraded, probably in lysosomes, also pass through the p100-containing MVB. These studies therefore suggest that the apical cytoplasm of Caco-2 cells contains a population of MVB capable of receiving membrane proteins trafficking in from both apical and basolateral borders and then routing them to a variety of cell surface and intracellular destinations. The differential distribution of apical and basolateral tracers within the 50-nm-diameter tubules connected to these p100-positive apical MVB suggests that the destination of proteins trafficking from the MVB back to apical and basolateral surfaces is determined by the tubules to which they gain access.     

Sorting of plasma membrane proteins in epithelial cells.

Proteins follow two routes to reach the correct surface (apical or basolateral) of a polarized epithelial cell: direct sorting from the trans-Golgi network and transcytosis from early endosomes. Several signals have been identified recently that control these sorting events, namely a glycosyl-phosphatidylinositol anchor for apical targeting, a 14-residue cytoplasmic segment of the polymeric immunoglobulin receptor for basolateral targeting, and phosphorylation of a Ser residue for transcytosis of this receptor. The machinery involved is still poorly understood.     

Aminopeptidase N is directly sorted to the apical domain in MDCK cells

In different epithelial cell types, integral membrane proteins appear to follow different sorting pathways to the apical surface. In hepatocytes, several apical proteins were shown to be transported there indirectly via the basolateral membrane, whereas in MDCK cells a direct sorting pathway from the trans-Golgi-network to the apical membrane has been demonstrated. However, different proteins had been studied in these cells. To compare the sorting of a single protein in both systems, we have expressed aminopeptidase N, which already had been shown to be sorted indirectly in hepatocytes, in transfected MDCK cells. As expected, it was predominantly localized to the apical domain of the plasma membrane. By monitoring the appearance of newly synthesized aminopeptidase N at the apical and basolateral surface, it was found to be directly sorted to the apical domain in MDCK cells, indicating that the sorting pathways are indeed cell type-specific.     

Comparison of FcRn‐ and pIgR‐Mediated Transport in MDCK Cells by Fluorescence Confocal Microscopy

Protein delivery across polarized epithelia is controlled by receptor‐mediated transcytosis. Many studies have examined basolateral‐to‐apical trafficking of polymeric IgA (pIgA) by the polymeric immunoglobulin receptor (pIgR). Less is known about apical‐to‐basolateral transcytosis, the direction the neonatal Fc receptor (FcRn) transports maternal IgGs across intestinal epithelia. To compare apical‐to‐basolateral and basolateral‐to‐apical transcytosis, we co‐expressed FcRn and pIgR in Madin‐Darby canine kidney (MDCK) cells and used pulse‐chase experiments with confocal microscopy to examine transport of apically applied IgG Fcγ and basolaterally applied pIgA. Fcγ and pIgA trafficking routes were initially separate but intermixed at later chase times. Fcγ was first localized near the apical surface, but became more equally distributed across the cell, consistent with concomitant transcytosis and recycling. By contrast, pIgA transport was strongly unidirectional: pIgA shifted from near the basolateral surface to an apical location with increasing time. Some Fcγ and pIgA fluorescence colocalized in early (EEA1‐positive), recycling (Rab11a‐positive), and transferrin (Tf)‐positive common/basolateral recycling endosomes. Fcγ became more enriched in Tf‐positive endosomes with time, whereas pIgA was sorted from these compartments. Live‐cell imaging revealed that vesicles containing Fcγ or pIgA shared similar mobility characteristics and were equivalently affected by depolymerizing microtubules, indicating that both trafficking routes depended to roughly the same extent on intact microtubules.     

Microtubule perturbation retards both the direct and the indirect apical pathway but does not affect sorting of plasma membrane proteins in intestinal epithelial cells (Caco-2).

Endogenous plasma membrane proteins are sorted from two sites in the human intestinal epithelial cell line Caco-2. Apical proteins are transported from the Golgi apparatus to the apical domain along a direct pathway and an indirect pathway via the basolateral membrane. In contrast, basolateral proteins never appear in the apical plasma membrane. Here we report on the effect of the microtubule-active drug nocodazole on the post-synthetic transport and sorting of plasma membrane proteins. Pulse-chase radiolabeling was combined with domain-specific cell surface assays to monitor the appearance of three apical and one basolateral protein in plasma membrane domains. Nocodazole was found to drastically retard both the direct transport of apical proteins from the Golgi apparatus and the indirect transport (transcytosis) from the basolateral membrane to the apical cell surface. In contrast, neither the transport rates of the basolateral membrane nor the sorting itself were significantly affected by the nocodazole treatment. We conclude that an intact microtubular network facilitates, but is not necessarily required for, the transport of apical membrane proteins along the two post-Golgi pathways to the brush border.