The chicken yolk sac IgY receptor, a mammalian mannose receptor family member, transcytoses IgY across polarized epithelial cells

In mammals the transfer of passive immunity from mother to young is mediated by the MHC-related receptor FcRn, which transports maternal IgG across epithelial cell barriers. In birds, maternal IgY in egg yolk is transferred across the yolk sac to passively immunize chicks during gestation and early independent life. The chicken yolk sac IgY receptor (FcRY) is the ortholog of the mammalian phospholipase A2 receptor, a mannose receptor family member, rather than an FcRn or MHC homolog. FcRn and FcRY both exhibit ligand binding at the acidic pH of endosomes and ligand release at the slightly basic pH of blood. Here we show that FcRY expressed in polarized mammalian epithelial cells functioned in endocytosis, bidirectional transcytosis, and recycling of chicken FcY/IgY. Confocal immunofluorescence studies demonstrated that IgY binding and endocytosis occurred at acidic but not basic pH, mimicking pH-dependent uptake of IgG by FcRn. Colocalization studies showed FcRY-mediated internalization via clathrin-coated pits and transport involving early and recycling endosomes. Disruption of microtubules partially inhibited apical-to-basolateral and basolateral-to-apical transcytosis, but not recycling, suggesting the use of different trafficking machinery. Our results represent the first cell biological evidence of functional equivalence between FcRY and FcRn and provide an intriguing example of how evolution can give rise to systems in which similar biological requirements in different species are satisfied utilizing distinct protein folds.     

Apical to basolateral transcytosis and apical recycling of immunoglobulin G in trophoblast-derived BeWo cells: effects of low temperature, nocodazole, and cytochalasin D

The murine neonatal Fc receptor, FcRn, carries out two functions: materno-fetal IgG delivery and maintenance of serum IgG homeostasis. During human pregnancy maternal IgG is transferred across placental syncytiotrophoblasts presumably by the human homolog of FcRn, hFcRn. Trophoblast-derived BeWo cells express hFcRn endogenously and can be considered as a model system to investigate IgG transport in syncytiotrophoblasts. Using a pulse-chase protocol, we here demonstrate that polarized BeWo cells exhibit not only apical to basolateral transcytosis but also apical IgG recycling. Thus, for the first time we demonstrate that epithelial cells can be involved in both materno-fetal IgG transmission and regulation of serum IgG levels. Lowering the temperature from 37 to 16 degrees C reduced, but did not block, IgG recycling and transcytosis. Microtubule-disruption by nocodazole did not influence transcytosis or apical recycling. Disassembly of filamentous actin by cytochalasin D stimulated apical endocytosis and recycling, while transcytosis remained unaffected. In summary, in BeWo cells apically internalized IgG enters both a transcytotic and recycling pathway. While the transcytotic route is temperature-sensitive but independent from microtubules and actin filaments, the apical recycling pathway is temperature-influenced and stimulated by actin disassembly, suggestive for the involvement of distinct endosome subcompartments in transcytosis and recycling.     

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.     

The recycling and transcytotic pathways for IgG transport by FcRn are distinct and display an inherent polarity

The Fc receptor FcRn traffics immunoglobulin G (IgG) in both directions across polarized epithelial cells that line mucosal surfaces, contributing to host defense. We show that FcRn traffics IgG from either apical or basolateral membranes into the recycling endosome (RE), after which the actin motor myosin Vb and the GTPase Rab25 regulate a sorting step that specifies transcytosis without affecting recycling. Another regulatory component of the RE, Rab11a, is dispensable for transcytosis, but regulates recycling to the basolateral membrane only. None of these proteins affect FcRn trafficking away from lysosomes. Thus, FcRn transcytotic and recycling sorting steps are distinct. These results are consistent with a single structurally and functionally heterogeneous RE compartment that traffics FcRn to both cell surfaces while discriminating between recycling and transcytosis pathways polarized in their direction of transport.     

Tryptophan- and dileucine-based endocytosis signals in the neonatal Fc receptor

The neonatal Fc receptor, FcRn, transports immunoglobulin G across intestinal cells in suckling rats. FcRn enters these cells by endocytosis and is present on the apical and basolateral surfaces. We investigated the roles of aromatic amino acids and a dileucine motif in the cytoplasmic domain of rat FcRn. We expressed mutant FcRn in which alanine replaced Trp-311, Leu-322, and Leu-323, or Phe-340 in the inner medullary collecting duct cell line IMCD. Individual replacement of the aromatic amino acids or the dileucine motif only partially blocked endocytosis of (125)I-Fc, whereas uptake by FcRn containing alanine residues in place of both Trp-311 and the dileucine motif was reduced to the level obtained with the tailless receptor. Leu-314 was required for the function of the tryptophan-based endocytosis signal, and Asp-317 and Asp-318 were required for the dileucine-based signal. Nonvectorial delivery of newly synthesized FcRn to the two cell surfaces was unaffected by loss of the endocytosis signals. However, the steady-state distribution of endocytosis mutants was predominantly apical, unlike wild-type FcRn, which was predominantly basolateral. This shift appeared to arise because the loss of endocytosis signals inhibited apical to basolateral transcytosis of FcRn more than basolateral to apical transcytosis.     

Nonvectorial surface transport, endocytosis via a Di-leucine-based motif, and bidirectional transcytosis of chimera encoding the cytosolic tail of rat FcRn expressed in Madin-Darby canine kidney cells

Transfer of passive immunity from the mother to the fetus or newborn involves the transport of IgG across several epithelia. Depending on the species, IgG is transported prenatally across the placenta and yolk sac or is absorbed from colostrum and milk by the small intestine of the suckling newborn. In both cases apical to basolateral transepithelial transport of IgG is thought to be mediated by FcRn, an IgG Fc receptor with homology to major histocompatibility class I antigens. Here, we analyzed the intracellular routing of chimera encoding the rat FcRn tail fused to the ecto- and transmembrane domain of the macrophage FcgammaRIIb. Newly synthesized chimera were delivered in a nonvectorial manner to the apical and basolateral cell surface, from where the chimera were able to internalize and transcytose. Apical to basolateral and basolateral to apical transcytosis were differently regulated. This intracellular routing of the chimera is similar to that of the native FcRn, indicating that the cytosolic tail of the receptor is necessary and sufficient to endow an unrelated FcR with the intracellular transport behavior of FcRn. Furthermore, the di-leucine motif in the cytosolic domain of FcRn was required for rapid and efficient endocytosis but not for basolateral sorting of the chimera.     

Regulation of vesicle trafficking in madin-darby canine kidney cells by Rab11a and Rab25

Polarized epithelial cells maintain the polarized distribution of basolateral and apical membrane proteins through a process of receptor-mediated endocytosis, sorting, and then recycling to the appropriate membrane domain. We have previously shown that the small GTP-binding proteins, Rab11a and Rab25, are associated with the apical recycling system of Madin-Darby canine kidney cells. Here we have utilized inducible expression of wild-type, dominant negative, and constitutively active mutants to directly compare the functions of Rab25 and Rab11a in postendocytic vesicular transport. We found that a Rab11a mutant deficient in GTP binding, Rab11aS25N, potently inhibited both transcytosis and apical recycling yet failed to inhibit transferrin recycling. Similarly, expression of either wild type Rab25 or the active mutant Rab25S21V inhibited both apical recycling and transcytosis of IgA by greater than 50% but had no effect on basolateral recycling of transferrin. Interestingly, the GTPase-deficient mutant Rab11aS20V inhibited basolateral to apical transcytosis of IgA, but had no effect on either apical or basolateral recycling. These results indicate that neither Rab11a nor Rab25 function in the basolateral recycling of transferrin in polarized Madin-Darby canine kidney cells cells, consistent with recent morphological observations by others. Thus, transferrin receptors must be recycled to the plasma membrane prior to sorting of apically directed cargoes into Rab11a/Rab25-positive apical recycling endosomes.     

Transcytosis of NgCAM in epithelial cells reflects differential signal recognition on the endocytic and secretory pathways

NgCAM is a cell adhesion molecule that is largely axonal in neurons and apical in epithelia. In Madin-Darby canine kidney cells, NgCAM is targeted to the apical surface by transcytosis, being first inserted into the basolateral domain from which it is internalized and transported to the apical domain. Initial basolateral transport is mediated by a sequence motif (Y(33)RSL) decoded by the AP-1B clathrin adaptor complex. This motif is a substrate in vitro for tyrosine phosphorylation by p60src, a modification that disrupts NgCAM's ability to interact with clathrin adaptors. Based on the behavior of various NgCAM mutants, it appears that after arrival at the basolateral surface, the AP-1B interaction site is silenced by phosphorylation of Tyr(33). This slows endocytosis and inhibits basolateral recycling from endosomes, resulting in NgCAM transcytosis due to a cryptic apical targeting signal in its extracellular domain. Thus, transcytosis of NgCAM and perhaps other membrane proteins may reflect the spatial regulation of recognition by adaptors such as AP-1B.     

Localization of the human neonatal Fc receptor (FcRn) in human nasal epithelium

The airway epithelium is a central player in the defense against pathogens including efficient mucociliary clearance and secretion of immunoglobulins, mainly polymeric IgA, but also IgG. Pulmonary administration of therapeutic antibodies on one hand, and intranasal immunization on the other, are powerful tools to treat airway infections. In either case, the airway epithelium is the primary site of antibody transfer. In various epithelia, bi-polar transcytosis of IgG and IgG immune complexes is mediated by the human neonatal Fc receptor, FcRn, but FcRn expression in the nasal epithelium had not been demonstrated, so far. We prepared affinity-purified antibodies against FcRn α-chain and confirmed their specificity by Western blotting and immunofluorescence microscopy. These antibodies were used to study the localization of FcRn α-chain in fixed nasal tissue. We here demonstrate for the first time that ciliated epithelial cells, basal cells, gland cells, and endothelial cells in the underlying connective tissue express the receptor. A predominant basolateral steady state distribution of the receptor was observed in ciliated epithelial as well as in gland cells. Co-localization of FcRn α-chain with IgG or with early sorting endosomes (EEA1-positive) but not with late endosomes/lysosomes (LAMP-2-positive) in ciliated cells was observed. This is indicative for the presence of the receptor in the recycling/transcytotic pathway but not in compartments involved in lysosomal degradation supporting the role of FcRn in IgG transcytosis in the nasal epithelium.     

Antibodies in the small intestine: mucosal synthesis and deposition of anti-glycosyl IgA, IgM, and IgG in the enterocyte brush border

Synthesis and deposition of immunoglobulins in the brush border was studied in organ-cultured pig small intestinal mucosal explants. Surprisingly, comparable amounts of IgM and IgA were synthesized during a 6-h pulse, and also newly made IgG was detected in media and explants, including the microvillar fraction. For IgA and IgM, this subcellular distribution is consistent with basolateral-to-apical transcytosis, mediated by the polymeric immunoglobulin receptor. IgG is a ligand for the Fc receptor FcRn, and beta2-microglobulin, the light chain of FcRn, coclustered in immunogold double labeling with IgG in subapical endosomes and in the basolateral membrane of enterocytes. In addition, beta2-microglobulin was copurified with IgG on protein G-Sepharose. Apical endocytosis of IgG, as judged by internalization of fluorescent protein G, was not detectable except in a few isolated cells. This suggests that IgG in the adult small intestine is transported across the enterocyte mainly in the basolateral to apical direction. Significant fractions of all immunoglobulins bound to lactoseagarose, indicating that "anti-glycosyl" antibodies, raised against commensal gut bacteria, are synthesized locally in the small intestine. By partial deposition in the brush border, these antibodies therefore may have a protective function by preventing lectin-like pathogens from gaining access to the brush border surface.     

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.     

The Na+/H+ Exchanger NHE6 in the Endosomal Recycling System Is Involved in the Development of Apical Bile Canalicular Surface Domains in HepG2 Cells

Polarized epithelial cells develop and maintain distinct apical and basolateral surface domains despite a continuous flux of membranes between these domains. The Na⁺/H⁺exchanger NHE6 localizes to endosomes but its function is unknown. Here, we demonstrate that polarized hepatoma HepG2 cells express an NHE6.1 variant that localizes to recycling endosomes and colocalizes with transcytosing bulk membrane lipids. NHE6.1 knockdown or overexpression decreases or increases recycling endosome pH, respectively, and inhibits the maintenance of apical, bile canalicular plasma membranes and, concomitantly, apical lumens. NHE6.1 knockdown or overexpression has little effect on the de novo biogenesis of apical surface domains. NHE6.1 knockdown does not inhibit basolateral-to-apical transcytosis of bulk membrane lipids, but it does promote their progressive loss from the apical surface, leaving cells unable to efficiently retain bulk membrane and bile canalicular proteins at the apical surface. The data suggest that a limited range of endosome pH mediated by NHE6.1 is important for securing the polarized distribution of membrane lipids at the apical surface and maintenance of apical bile canaliculi in HepG2 cells and hence cell polarity. This study underscores the emerging role of the endosomal recycling system in apical surface development and identifies NHE6 as a novel regulatory protein in this process.     

Neonatal Fc receptor mediates internalization of Fc in transfected human endothelial cells

The neonatal Fc receptor, FcRn mediates an endocytic salvage pathway that prevents degradation of IgG, thus contributing to the homeostasis of circulating IgG. Based on the low affinity of IgG for FcRn at neutral pH, internalization of IgG by endothelial cells is generally believed to occur via fluid-phase endocytosis. To investigate the role of FcRn in IgG internalization, we used quantitative confocal microscopy to characterize internalization of fluorescent Fc molecules by HULEC-5A lung microvascular endothelia transfected with GFP fusion proteins of human or mouse FcRn. In these studies, cells transfected with FcRn accumulated significantly more intracellular Fc than untransfected cells. Internalization of FcRn-binding forms of Fc was proportional to FcRn expression level, was enriched relative to dextran internalization in proportion to FcRn expression level, and was blocked by incubation with excess unlabeled Fc. Because we were unable to detect either surface expression of FcRn or surface binding of Fc, these results suggest that FcRn-dependent internalization of Fc may occur through sequestration of Fc by FcRn in early endosomes. These studies indicate that FcRn-dependent internalization of IgG may be important not only in cells taking up IgG from an extracellular acidic space, but also in endothelial cells participating in homeostatic regulation of circulating IgG levels.     

The receptor recycling pathway contains two distinct populations of early endosomes with different sorting functions

Receptor recycling involves two endosome populations, peripheral early endosomes and perinuclear recycling endosomes. In polarized epithelial cells, either or both populations must be able to sort apical from basolateral proteins, returning each to its appropriate plasma membrane domain. However, neither the roles of early versus recycling endosomes in polarity nor their relationship to each other has been quantitatively evaluated. Using a combined morphological, biochemical, and kinetic approach, we found these two endosome populations to represent physically and functionally distinct compartments. Early and recycling endosomes were resolved on Optiprep gradients and shown to be differentially associated with rab4, rab11, and transferrin receptor; rab4 was enriched on early endosomes and at least partially depleted from recycling endosomes, with the opposite being true for rab11 and transferrin receptor. The two populations were also pharmacologically distinct, with AlF4 selectively blocking export of transferrin receptor from recycling endosomes to the basolateral plasma membrane. We applied these observations to a detailed kinetic analysis of transferrin and dimeric IgA recycling and transcytosis. The data from these experiments permitted the construction of a testable, mathematical model which enabled a dissection of the roles of early and recycling endosomes in polarized receptor transport. Contrary to expectations, the majority (>65%) of recycling to the basolateral surface is likely to occur from early endosomes, but with relatively little sorting of apical from basolateral proteins. Instead, more complete segregation of basolateral receptors from receptors intended for transcytosis occurred upon delivery to recycling endosomes.     

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.     

Effect of monensin on ricin and fluid phase transport in polarized MDCK cells.

The effect of monensin on endocytosis, transcytosis, recycling and transport to the Golgi apparatus in filter-grown Madin-Darby canine kidney (MDCK) cells was investigated using 125I-labeled ricin as a marker for membrane transport, and horseradish peroxidase (HRP) as a marker for fluid phase transport. Monensin (10 microM) stimulated transcytosis of both markers about 3-fold in the basolateral to apical direction. Transcytosis of HRP in the opposite direction, apical to basolateral, was reduced to approximately 50% of the control by monensin, whereas that of ricin was slightly increased. Recycling of markers endocytosed from the apical surface was reduced in the presence of monensin and there was an increased accumulation of both ricin and HRP in the cells. Transport of ricin to the Golgi apparatus increased to the same extent as the increase in intracellular accumulation. No change in recycling or accumulation was observed with monensin when the markers were added basolaterally, but transport of ricin to the Golgi apparatus increased almost 3-fold. Our results indicate that basolateral to apical transcytosis is increased in the absence of low endosomal pH, and they suggest that apical to basolateral transcytosis of a membrane-bound marker (ricin) is affected by monensin differently from that of a fluid phase marker (HRP).     

Endocytic traffic in polarized epithelial cells: role of the actin and microtubule cytoskeleton

The cytoskeleton is required for multiple cellular events including endocytosis and the transfer of cargo within the endocytic system. Polarized epithelial cells are capable of endocytosis at either of their distinct apical or basolateral plasma membrane domains. Actin plays a role in internalization at both cell surfaces. Microtubules and actin are required for efficient transcytosis and delivery of proteins to late endosomes and lysosomes. Microtubules are also important in apical recycling pathways and, in some polarized cell types, basolateral recycling requires actin. The microtubule motor proteins dynein and kinesin and the class I unconventional myosin motors play a role in many of these trafficking steps. This review examines the endocytic pathways of polarized epithelial cells and focuses on the emerging roles of the actin cytoskeleton in these processes.     

Basolateral to apical transcytosis in polarized cells is indirect and involves BFA and trimeric G protein sensitive passage through the apical endosome

We have used temperature and nocodazole blocks in an in vivo basolateral to apical transcytosis assay to dissociate the early transcytotic steps occurring during the formation of transcytotic vesicles and their microtubule-dependent translocation into the apical region, from the late steps when transcytotic cargo is delivered into the apical media. We found that polarized MDCK cells transfected with rabbit polymeric IgA receptor (pIgA-R) internalize basolaterally added pIgA-R ligand ([Fab]2 fragment of IgG against the receptor's ectodomain) at 17 degrees C but do not deliver it to the apical PM. Instead, the ligand accumulates in an apically localized transcytotic compartment, distal to the basolateral endosome and the microtubule- requiring translocation step. We have characterized this compartment and show that it is distinct from basolateral transferrin recycling endosomes, basolateral early endosomes or late endosomes or lysosomes. The apical transcytotic compartment colocalizes with the compartment containing apically recycling membrane markers (ricin and apically internalized pIgA-R ligand) but is distinct from the compartment receiving apically internalized fluid phase marker (BSA). This compartment is an intermediate station of the overall pathway since transcytotic ligand can exit the compartment and be released into the apical medium when cells preloaded at 17 degrees C are subsequently incubated at 37 degrees C. We have used this system to examine the effect of Brefeldin A (BFA) and the involvement of trimeric GTPases in the late (post apical transcytotic compartment) steps of the transcytotic pathway. We found that addition of BFA or cholera toxin, a known activator of Gs alpha, to cells preloaded with transcytotic ligand at 17 degrees C significantly inhibits the exit of ligand from the apical transcytotic compartment. General structure and function of the apical endosome are not affected since neither BFA nor cholera toxin inhibit the recycling of apically internalized membrane markers (ricin and pIgA-R ligand) from the same compartment. The data suggest that transcytosis connects the "membrane-sorting" sub-domain of the basolateral endosome with a homologous sub-domain of the apical endosome and that exit of transcytosing cargo from the apical endosome is controlled by a BFA and trimeric G protein sensitive mechanism, distinct from that used for recycling of apically internalized proteins (ricin or pIgA-R).     

Effect of Calmodulin Antagonists on Endocytosis and Intracellular Transport of Ricin in Polarized MDCK Cells

The effect of calmodulin antagonists on endocytosis, transcytosis, recycling, and transport to the Golgi apparatus from both the apical and the basolateral plasma membrane of polarized Madin–Darby canine kidney cells has been investigated by using the plant toxin ricin as a membrane marker. The calmodulin antagonists trifluoperazine andN-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) stimulated apical endocytosis of ricin, whereas basolateral endocytosis was unaffected. A stimulation of the apical uptake of the fluid-phase marker horseradish peroxidase by calmodulin antagonists was also found both by biochemical and by ultrastructural studies. Furthermore, W-7 reduced the recycling of ricin to the apical plasma membrane, whereas the recycling to the basolateral plasma membrane was not changed. Transport of ricin to the Golgi apparatus was also selectively affected by the calmodulin antagonist W-7. After basolateral endocytosis of ricin, transport to the Golgi apparatus was reduced, whereas after apical endocytosis the fraction of endocytosed ricin transport to the Golgi apparatus was increased. Transcytosis of ricin from the basolateral to the apical pole was increased in the presence of calmodulin antagonists, whereas these compounds did not have any significant effect on the apical to basolateral transcytosis. Thus, the results obtained indicate that calmodulin is involved in regulation of apical endocytosis and recycling as well as in transcytosis of ricin from the basolateral plasma membrane. Furthermore, the data suggest that calmodulin plays a role in regulation of ricin transport to the Golgi apparatus.     

Receptor-mediated transcytosis of IgA in MDCK cells is via apical recycling endosomes

Classically, the polymeric immunoglobulin receptor and its ligand, IgA, are thought to be sorted from basolateral early endosomes into transcytotic vesicles that directly fuse with the apical plasma membrane. In contrast, we have found that in MDCK cells IgA is delivered from basolateral endosomes to apical endosomes and only then to the apical cell surface. When internalized from the basolateral surface of MDCK cells IgA is found to accumulate under the apical plasma membrane in a compartment that is accessible to two apically added membrane markers: anti-secretory component Fab fragments, and avidin internalized from the biotinylated apical pole of the cell. This accumulation occurs in the presence of apical trypsin, which prevents internalization of the ligand from the apical cell surface. Using a modification of the diaminobenzidine density-shift assay, we estimate that approximately 80% of basolaterally internalized IgA resides in the apical endosomal compartment. In addition, approximately 50% of basolaterally internalized transferrin, a basolateral recycling protein, has access to this apical endosomal compartment and is efficiently recycled back to the basolateral surface. Microtubules are required for the organization of the apical endosomal compartment and it is dispersed in nocodazole-treated cells. Moreover, this compartment is largely inaccessible to fluid-phase markers added to either pole of the cell, and therefore seems analogous to the recycling endosome described in nonpolarized cells. We propose a model in which transcytosis is not a specialized pathway that uses unique transcytotic vesicles, but rather combines portions of pathways used by non- transcytosing molecules.