Isoforms of the Lutheran/basal cell adhesion molecule glycoprotein are differentially delivered in polarized epithelial cells. Mapping of the basolateral sorting signal to a cytoplasmic di-leucine motif.

Lu and Lu(v13) are two glycoprotein (gp) isoforms that belong to the immunoglobulin superfamily and carry both the Lutheran (Lu) blood group antigens and the basal cell adhesion molecule epithelial cancer antigen. Lu (85 kDa) and Lu(v13) (78 kDa) gps, which differ only in the length of their cytoplasmic domain, are adhesion molecules that bind laminin. In nonerythroid tissues, the Lu/basal cell adhesion molecule antigens are predominantly expressed in the endothelium of blood vessel walls and in the basement membrane region of normal epithelial cells, whereas they exhibit a nonpolarized expression in some epithelial cancers. Here, we analyzed the polarization of Lu and Lu(v13) gps in epithelial cells by confocal microscopy and domain-selective biotinylation assays. Differentiated human colon carcinoma Caco-2 cells exhibited a polarized expression of endogenous Lu antigens associated with a predominant expression of the Lu isoform at the basolateral domain of the plasma membrane and a very low expression of the Lu(v13) isoform at both the apical and basolateral domains. Analysis of transfected Madin-Darby canine kidney cells revealed a basolateral expression of Lu gp and a nonpolarized expression of Lu(v13) gp. Delivery of Lu(v13) to both apical and basolateral surfaces showed similar kinetics, indicating that this isoform is directly transported to each surface domain. A dileucine motif at position 608-609, specific to the Lu isoform, was characterized as a dominant basolateral sorting signal that prevents Lu gp from taking the apical delivery pathway.     

Steady-state distribution and biogenesis of endogenous Madin-Darby canine kidney glycoproteins: evidence for intracellular sorting and polarized cell surface delivery

We used domain-selective biotinylation/125I-streptavidin blotting (Sargiacomo, M., M. P. Lisanti, L. Graeve, A. Le Bivic, and E. Rodriguez-Boulan. 1989 J. Membr. Biol. 107:277-286), in combination with lectin precipitation, to analyze the apical and basolateral glycoprotein composition of Madin-Darby canine kidney (MDCK) cells and to explore the role of glycosylation in the targeting of membrane glycoproteins. All six lectins used recognized both apical and basolateral glycoproteins, indicating that none of the sugar moieties detected were characteristic of the particular epithelial cell surface. Pulse-chase experiments coupled with domain-selective glycoprotein recovery were designed to detect the initial appearance of newly synthesized glycoproteins at the apical or basolateral cell surface. After a short pulse with a radioactive precursor, glycoproteins reaching each surface were biotinylated, extracted, and recovered via precipitation with immobilized streptavidin. Several basolateral glycoproteins (including two sulfated proteins) and at least two apical glycoproteins (one of them the major sulfated protein of MDCK cells) appeared at the corresponding surface after 20-40 min of chase, but were not detected in the opposite surface, suggesting that they were sorted intracellularly and vectorially delivered to their target membrane. Several "peripheral" apical proteins were detected at maximal levels on the apical surface immediately after the 15-min pulse, suggesting a very fast intracellular transit. Finally, domain-selective labeling of surface carbohydrates with biotin hydrazide (after periodate oxidation) revealed strikingly different integral and peripheral glycoprotein patterns, resembling the Con A pattern, after labeling with sulfo-N-hydroxy-succinimido-biotin. The approaches described here should be useful in characterizing the steady-state distribution and biogenesis of endogenous cell surface components in a variety of epithelial cell lines.     

Postendocytotic sorting of the ligand for the polymeric immunoglobulin receptor in Madin-Darby canine kidney cells

The polymeric immunoglobulin receptor (pIg-R) is responsible for the receptor-mediated transcytosis of polymeric immunoglobulins (IgA and IgM) across various epithelia. We have expressed the cDNA for the pIg-R in Madin-Darby canine kidney (MDCK) cells and found that this system mimics that found in vivo (Mostov, K. E., and D. L. Deitcher. 1986. Cell. 46:613-621). We have now investigated the postendocytotic pathway of the ligand for the pIg-R. After a 5-min internalization at the basolateral surface, approximately 45% of internalized ligand recycles to the basolateral medium and 30% is transcytosed to the apical medium. We have also examined why transcytosis of ligand is unidirectional, going only from basolateral to apical, but not from apical to basolateral. Several factors could explain this, such as proteolytic cleavage of the pIg-R at the apical surface, decreased apical endocytosis of ligand, or an intracellular sorting event. In this report, we show that the protease inhibitor, leupeptin, inhibits the cleavage of the pIg-R but does not alter the unidirectionality of transcytosis. In addition, we demonstrate that there is a significant amount of apical endocytosis of ligand (70% of that observed basolaterally). Finally, we demonstrate that apically endocytosed ligand can return only to the apical surface. Thus, once ligand reaches the apical surface, it is "trapped" and cannot return to the basolateral surface. We propose that the unidirectionality of transcytosis is the result of intracellular sorting, and that this results from a signal(s) present on the pIg-R.     

Differential microtubule requirements for transcytosis in MDCK cells.

Given the role of microtubules in directing the transport of many intracellular organelles, we investigated whether intact microtubules were also required for transcytosis across epithelia. Using polarized MDCK cells expressing receptors for the Fc domain of IgG (FcRII-B2) or polymeric immunoglobulin (pIg-R), we examined the involvement of microtubules in apical to basolateral and basolateral to apical transcytosis, respectively. While depolymerization of microtubules with nocodozole had no effect on apical to basolateral transcytosis via FcR, basolateral to apical transcytosis of dimeric IgA via pIg-R was almost completely blocked. Inhibition due to nocodozole was selective for basolateral to apical transcytosis, since neither endocytosis nor receptor recycling was significantly affected at either plasma membrane domain. As shown by confocal microscopy, the block in transcytosis was due to the inability of MDCK cells to translocate IgA-containing vesicles from the basolateral to the apical cytoplasm in the absence of an intact microtubule network. The nocodazole sensitive step could be partially by-passed, however, by allowing cells to internalize IgA at 17 degrees C prior to nocodazole treatment. Although incubation at 17 degrees C blocked release of IgA into the apical medium, it did not prevent translocation of IgA-containing vesicles to the apical cytoplasm. Thus, receptor-mediated transcytosis in opposite directions exhibits distinct requirements for microtubules, a feature which reflects the spatial organization of MDCK cells.     

The influence of pH on the vesicular traffic to the surface of the polarized epithelial cell, MDCK

The effect of pH on the secretion of the gp 80 glycoprotein complex and lysozyme from MDCK cells was examined by treatment of the cells with either NH4Cl, chloroquine or monensin. In untreated cells gp 80 is sorted with approximately 75% efficiency into the apical pathway. Lysozyme is secreted in a nonpolar fashion at both cell surfaces. Treatment of the cells with the drugs had nearly identical effects on the transport kinetics and on the ratio of the proteins released at the two plasma membrane domains. At increasing drug concentrations, the transport of both proteins to the apical and the basolateral cell surface was equally retarded. Furthermore, we observed a dose-dependent decrease in the amount of gp 80 and lysozyme released at the basolateral cell surface, which was accompanied by a nearly equivalent increase in the secretion of the two proteins at the apical plasma membrane domain. A twofold rise in the apical to basolateral ratio was already found at drug concentrations which only marginally affected the kinetics of transport. These results show that an increase in intravesicular pH not only redirects secretory proteins sorted into the basolateral pathway (Caplan et al. Nature, 329, 632 (1987] but also secretory proteins devoid of sorting information for that pathway, presumably by modulating the vesicular traffic to the basolateral cell surface.     

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.     

Sorting of an apical plasma membrane glycoprotein occurs before it reaches the cell surface in cultured epithelial cells

In Madin-Darby canine kidney (MDCK) cells (a polarized epithelial cell line) infected with influenza virus, the hemagglutinin behaves as an apical plasma membrane glycoprotein. To determine biochemically the domain on the plasma membrane, apical or basolateral, where newly synthesized hemagglutinin first appears, cells were cultured on Millipore filters to make both cell surface domains independently accessible. Hemagglutinin in virus-infected cells was pulse-labeled, chased, and detected on the plasma membrane with a sensitive trypsin assay. Under all conditions tested, newly made hemagglutinin appeared simultaneously on both domains, with the bulk found in the apical membrane. When trypsin was continuously present on the basolateral surface during the chase, little hemagglutinin was cleaved relative to the amount transported apically. In addition, specific antibodies against the hemagglutinin placed basolaterally had no effect on transport to the apical domain. These observations suggested that most newly synthesized hemagglutinin does not transiently appear on the basolateral surface but rather is delivered directly to the apical surface in amounts that account for its final polarized distribution.     

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.     

Deletions in the cytoplasmic domain of the polymeric immunoglobulin receptor differentially affect endocytotic rate and postendocytotic traffic

We have examined the function of the cytoplasmic domain of the polymeric immunoglobulin receptor (pIg-R) by producing two separate deletions in the cytoplasmic domain of the pIg-R, expressing the mutant receptors in polarized MDCK cells, and analyzing each for their effects on receptor and ligand traffic. Deletion of the C-terminal 30 amino acids (726-755) reduces the rate of internalization of receptor-bound ligand from the basolateral surface. However, this mutation has no effect on delivery of receptor from the Golgi to the basolateral surface or the post-endocytotic traffic of receptor and ligand. Mutation of a tyrosine at position 734 to serine produces a receptor with a similar phenotype. If residues 670-707 are deleted from the middle of the cytoplasmic domain, both basolateral delivery and internalization are unaffected. However, unlike wild type, after endocytosis from the basolateral surface, both receptor and ligand are largely degraded. We reported previously that deletion of the entire cytoplasmic domain prevents the basolateral delivery of newly synthesized receptor (Mostov, K.E., de Bruyn Kops, A., and Deitcher, D.L. (1986) Cell 47, 359-364). In contrast, the mutants reported here are delivered to the basolateral surface, suggesting that only residues 653-669 and/or 708-725 are necessary for basolateral delivery. Thus, different deletions in the cytoplasmic domain of the pIg-R can produce mutant receptors which alter different aspects of receptor traffic.     

Membrane domains of intestinal epithelial cells: distribution of Na+,K+- ATPase and the membrane skeleton in adult rat intestine during fetal development and after epithelial isolation

The organization of the basolateral membrane domain of highly polarized intestinal absorptive cells was studied in adult rat intestinal mucosa, during development of polarity in fetal intestine, and in isolated epithelial sheets. Semi-thin frozen sections of these tissues were stained with a monoclonal antibody (mAb 4C4) directed against Na+,K+-ATPase, and with other reagents to visualize distributions of the membrane skeleton (fodrin), an epithelial cell adhesion molecule (uvomorulin), an apical membrane enzyme (aminopeptidase), and filamentous actin. In intact adult epithelium, Na+,K+-ATPase, membrane-associated fodrin, and uvomorulin were concentrated in the lateral, but not basal, subdomain. In the stratified epithelium of fetal intestine, both fodrin and uvomorulin were localized in areas of cell-cell contact at 16 and 17 d gestation, a stage when Na+,K+-ATPase was not yet expressed. These molecules were excluded from apical domains and from cell surfaces in contact with basal lamina. When Na+,K+-ATPase appeared at 18-19 d, it was codistributed with fodrin. Detachment of epithelial sheets from adult intestinal mucosa did not disrupt intercellular junctions or lateral cell contacts, but cytoplasmic blebs appeared at basal cell surfaces, and a diffuse pool of fodrin and actin accumulated in them. At the same time, Na+,K+-ATPase moved into the basal membrane subdomain, and extensive endocytosis of basolateral membrane, including Na+,K+-ATPase, occurred. Endocytosis of uvomorulin was not detected and no fodrin was associated with endocytic vesicles. Uvomorulin, along with some membrane-associated fodrin and some Na+,K+-ATPase, remained in the lateral membrane as long as intercellular contacts were maintained. Thus, in this polarized epithelium, interaction of lateral cell-cell adhesion molecules as well as basal cell-substrate interactions are required for maintaining the stability of the lateral membrane skeleton and the position of resident membrane proteins concentrated in the lateral membrane domain.     

Sorting of endogenous plasma membrane proteins occurs from two sites in cultured human intestinal epithelial cells (Caco-2)

We studied the postsynthetic sorting of endogenous plasma membrane proteins in a polarized epithelial cell line, Caco-2. Pulse-chase radiolabeling was combined with domain-specific cell surface assays to monitor the arrival of three apical and one basolateral protein at the apical and basolateral cell surface. Apical proteins were inserted simultaneously into both membrane domains. The fraction targeted to the basolateral domain was different for the three apical proteins and was subsequently sorted to the apical domain by transcytosis at different rates. In contrast, a basolateral protein was found in the basolateral membrane only. Thus, sorting of plasma membrane proteins occurred from two sites: the Golgi apparatus and the basolateral membrane. These data explain apparently conflicting results of earlier studies.     

Expression of the human immunodeficiency virus envelope glycoprotein is restricted to basolateral surfaces of polarized epithelial cells.

Polarized epithelial cells exhibit apical (lumenal) and basolateral (serosal) membrane domains that are separated by circumferential tight junctions. In such cells, enveloped viruses that mature by budding at cell surfaces are released at particular membrane domains. We have used a vaccinia virus recombinant to investigate the site of surface expression of the human immunodeficiency virus type 1 envelope glycoprotein in Madin-Darby canine kidney cells. Cells were infected with the vaccinia virus recombinant, and surface expression of the glycoprotein was analyzed by indirect immunofluorescence, 125I-protein A binding, and immunoelectron microscopy. The glycoprotein appeared exclusively at the basolateral surface as early as 2 h postinfection and reached a maximum level at 8 h postinfection. The gp120 glycoprotein was found to be secreted efficiently into culture medium, and this secretion occurred exclusively at the basolateral surface.     

The proamphiregulin cytoplasmic domain is required for basolateral sorting, but is not essential for constitutive or stimulus-induced processing in polarized Madin-Darby canine kidney cells

In this study, the role of the amphiregulin precursor (pro-AR) cytoplasmic domain in the basolateral sorting and cell-surface processing of pro-AR in polarized epithelial cells has been investigated using Madin-Darby canine kidney cells stably expressing various human pro-AR forms. Our results demonstrate that newly synthesized wild-type pro-AR (50 kDa) is delivered directly to the basolateral membrane domain with >95% efficiency, where it is sequentially cleaved within the ectodomain to release several soluble amphiregulin (AR) forms. Analyses of a pro-AR cytoplasmic domain truncation mutant (ARTL27) and two pro-AR secretory mutants (ARsec184 and ARsec190) indicated that the pro-AR cytoplasmic domain is not required for efficient delivery to the plasma membrane, but does contain essential basolateral sorting information. We show that the pro-AR cytoplasmic domain truncation mutant (ARTL27) is not sorted in polarized Madin-Darby canine kidney cells, with approximately 65% of the newly synthesized protein delivered to the apical cell surface. Under base-line conditions, ARTL27 was preferentially cleaved from the basolateral surface with 4-fold greater efficiency compared with cleavage from the apical membrane domain. However, ARTL27 ectodomain cleavage could be stimulated equivalently from either membrane domain by a variety of different stimuli. The metalloprotease inhibitor BB-94 could inhibit both base-line and stimulus-induced ectodomain cleavage of wild-type pro-AR and ARTL27. These results indicate that the pro-AR cytoplasmic domain is required for basolateral sorting, but is not essential for ectodomain processing. Preferential constitutive cleavage of ARTL27 from the basolateral cell surface also suggests that the metalloprotease activity involved in base-line and stimulus-induced ARTL27 ectodomain cleavage may be regulated differently in the apical and basolateral membrane domains of polarized epithelial cells.     

Apical sorting by galectin-3-dependent glycoprotein clustering

Epithelial cells are characterized by their polarized organization based on an apical membrane that is separated from the basolateral membrane domain by tight junctions. Maintenance of this morphology is guaranteed by highly specific sorting machinery that separates lipids and proteins into different carrier populations for the apical or basolateral cell surface. Lipid-raft-independent apical carrier vesicles harbour the beta-galactoside-binding lectin galectin-3, which interacts directly with apical cargo in a glycan-dependent manner. These glycoproteins are mistargeted to the basolateral membrane in galectin-3-depleted cells, dedicating a central role to this lectin in raft-independent sorting as apical receptor. Here, we demonstrate that high-molecular-weight clusters are exclusively formed in the presence of galectin-3. Their stability is sensitive to increased carbohydrate concentrations, and cluster formation as well as apical sorting are perturbed in glycosylation-deficient Madin-Darby canine kidney (MDCK) II cells. Together, our data suggest that glycoprotein cross-linking by galectin-3 is required for apical sorting of non-raft-associated cargo.     

Rat liver dipeptidylpeptidase IV contains competing apical and basolateral targeting information.

Madin-Darby canine kidney (MDCK) cells deliver endogenous apical and basolateral proteins directly to the appropriate domains. We are investigating the molecular signals on a model plasma membrane hydrolase, dipeptidylpeptidase IV (DPPIV). Most newly synthesized rat liver DPPIV is delivered directly to the apical surface of transfected MDCK cells; however, about 20% is delivered first to the basolateral surface and reaches the apical surface via transcytosis (Casanova, J. E., Mishumi, Y., Ikehara, Y., Hubbard, A. L., and Mostov, K. E. (1991) J. Biol. Chem. 266, 24428-24432). A soluble form of DPPIV (solDPPIV) containing only the lumenal domain of the protein was efficiently transported and secreted by stably transfected MDCK cells. If this domain contains apical sorting information, we would expect 80% of the soluble protein to be secreted apically. Surprisingly, 95% of the secreted solDPPIV was found in the apical medium. The high efficiency of apical secretion suggested that the transmembrane domain and cytoplasmic tail of DPPIV might contain competing basolateral targeting information. To test this hypothesis, we investigated the trafficking of a chimera in which the cytoplasmic tail and transmembrane domains of DPPIV were joined to lysozyme, an exogenous protein which should not contain sorting information. This protein was delivered predominantly to the basolateral surface. Our results suggest that the lumenal domain of DPPIV carries dominant apical sorting information while the transmembrane domain and cytoplasmic tail of the molecule contains competing basolateral sorting information.     

Selective inhibition of protein targeting to the apical domain of MDCK cells by brefeldin A

Dipeptidyl peptidase IV (DPPIV) is mainly vectorially targeted to the apical surface in MDCK cells. BFA was found to abolish the apical targeting of DPPIV. This BFA effect could be achieved under conditions where the ER to Golgi transport and the total surface expression of DPPIV were essentially unaffected. BFA executed its effect during the transport from the trans-Golgi network (TGN) to the surface. The inhibition of apical targeting resulted in enhanced mis-targeting to the basolateral surface. The mistargeted DPPIV was transcytosed back to the apical domain only after BFA withdrawal. In contrast, the basolateral targeting of uvomorulin was unaffected by BFA. These results established that the apical targeting of DPPIV was selectively abolished by BFA.     

Gp135/podocalyxin and NHERF-2 participate in the formation of a preapical domain during polarization of MDCK cells

Epithelial polarization involves the segregation of apical and basolateral membrane domains, which are stabilized and maintained by tight junctions and membrane traffic. We report that unlike most apical and basolateral proteins in MDCK cells, which separate only after junctions have formed, the apical marker gp135 signifies an early level of polarized membrane organization established already in single cells. We identified gp135 as the dog orthologue of podocalyxin. With a series of domain mutants we show that the COOH-terminal PSD-95/Dlg/ZO-1 (PDZ)-binding motif is targeting podocalyxin to the free surface of single cells as well as to a subdomain of the terminally polarized apical membrane. This special localization of podocalyxin is shared by the cytoplasmic PDZ-protein Na+/H+ exchanger regulatory factor (NHERF)-2. Depleting podocalyxin by RNA interference caused defects in epithelial polarization. Together, our data suggest that podocalyxin and NHERF-2 function in epithelial polarization by contributing to an early apical scaffold based on PDZ domain-mediated interactions.     

Oncostatin M regulates membrane traffic and stimulates bile canalicular membrane biogenesis in HepG2 cells

Hepatocytes are the major epithelial cells of the liver and they display membrane polarity: the sinusoidal membrane representing the basolateral surface, while the bile canalicular membrane is typical of the apical membrane. In polarized HepG2 cells an endosomal organelle, SAC, fulfills a prominent role in the biogenesis of the canalicular membrane, reflected by its ability to sort and redistribute apical and basolateral sphingolipids. Here we show that SAC appears to be a crucial target for a cytokine-induced signal transduction pathway, which stimulates membrane transport exiting from this compartment promoting apical membrane biogenesis. Thus, oncostatin M, an IL-6-type cytokine, stimulates membrane polarity development in HepG2 cells via the gp130 receptor unit, which activates a protein kinase A-dependent and sphingomyelin-marked membrane transport pathway from SAC to the apical membrane. To exert its signal transducing function, gp130 is recruited into detergent-resistant membrane microdomains at the basolateral membrane. These data provide a clue for a molecular mechanism that couples the biogenesis of an apical plasma membrane domain to the regulation of intracellular transport in response to an extracellular, basolaterally localized stimulus.     

Nonpolarized expression of a secreted murine leukemia virus glycoprotein in polarized epithelial cells

Vaccinia virus recombinants were generated which express the intact gp70/p15E of Friend mink cell focus inducing virus (F-MCFV) or truncated forms of the glycoprotein that lack the transmembrane and cytoplasmic domains. The transport of the intact and truncated envelope glycoproteins to apical or basolateral surfaces was studied in the polarized epithelial MDCK cell line. Infection of MDCK cells with the recombinant expressing the intact F-MCFV envelope glycoprotein resulted in transport exclusively to the basolateral surfaces, whereas the recombinant expressing the truncated glycoprotein was found to be secreted from both the apical and basolateral surfaces. Thus removal of the transmembrane and cytoplasmic domains of the p15E protein results in a loss of directional transport to the basolateral membrane of polarized epithelial cells.     

A Highlights from MBoC Selection: Neuralized Promotes Basal to Apical Transcytosis of Delta in Epithelial Cells

Notch receptors mediate short-range signaling controlling many developmental decisions in metazoans. Activation of Notch requires the ubiquitin-dependent endocytosis of its ligand Delta. How ligand endocytosis in signal-sending cells regulates receptor activation in juxtaposed signal-receiving cells remains largely unknown. We show here that a pool of Delta localizes at the basolateral membrane of signal-sending sensory organ precursor cells in the dorsal thorax neuroepithelium of Drosophila and that Delta is endocytosed in a Neuralized-dependent manner from this basolateral membrane. This basolateral pool of Delta is segregated from Notch that accumulates apically. Using a compartimentalized antibody uptake assay, we show that murine Delta-like 1 is similarly internalized by mNeuralized2 from the basolateral membrane of polarized Madin-Darby canine kidney cells and that internalized ligands are transcytosed to the apical plasma membrane where mNotch1 accumulates. Thus, endocytosis of Delta by Neuralized relocalizes Delta from the basolateral to the apical membrane domain. We speculate that this Neuralized-dependent transcytosis regulates the signaling activity of Delta by relocalizing Delta from a membrane domain where it cannot interact with Notch to another membrane domain where it can bind and activate Notch.