Apical Recycling Research Articles

Standardized Preclinical In Vitro Blood-Brain Barrier Mouse Assay Validates Endocytosis-Dependent Antibody Transcytosis Using Transferrin-Receptor-Mediated Pathways.

The presence of the blood-brain barrier (BBB) creates a nigh-on impenetrable obstacle for large macromolecular therapeutics that need to be delivered to the brain milieu to treat neurological disorders. To overcome this, one of the strategies used is to bypass the barrier with what is referred to as a "Trojan Horse" strategy, where therapeutics are designed to use endogenous receptor-mediated pathways to piggyback their way through the BBB. Even though in vivo methodologies are commonly used to test the efficacy of BBB-penetrating biologics, comparable in vitro BBB models are in high demand, as they benefit from being an isolated cellular system devoid of physiological factors that can on occasion mask the processes behind BBB transport via transcytosis. We have developed an in vitro BBB model (In-Cell BBB-Trans assay) based on the murine cEND cells that help delineate the ability of modified large bivalent IgG antibodies conjugated to the transferrin receptor binder scFv8D3 to cross an endothelial monolayer grown on porous cell culture inserts (PCIs). Following the administration of bivalent antibodies into the endothelial monolayer, a highly sensitive enzyme-linked immunosorbent assay (ELISA) is used to determine the concentration in the apical (blood) and basolateral (brain) chambers of the PCI system, allowing for the evaluation of apical recycling and basolateral transcytosis, respectively. Our results show that antibodies conjugated to scFv8D3 transcytose at considerably higher levels compared to unconjugated antibodies in the In-Cell BBB-Trans assay. Interestingly, we are able to show that these results mimic in vivo brain uptake studies using identical antibodies. In addition, we are able to transversely section PCI cultured cells, allowing for the identification of receptors and proteins that are likely involved in the transcytosis of the antibodies. Furthermore, studies using the In-Cell BBB-Trans assay revealed that transcytosis of the transferrin-receptor-targeting antibodies is dependent on endocytosis. In conclusion, we have designed a simple, reproducible In-Cell BBB-Trans assay based on murine cells that can be used to rapidly determine the BBB-penetrating capabilities of transferrin-receptor-targeting antibodies. We believe that the In-Cell BBB-Trans assay can be used as a powerful, preclinical screening platform for therapeutic neurological pathologies.

Towards new definitions of avoidable hospital admissions

Acidosis is an important complication of AKI and CKD. Renal intercalated cells (ICs) express the proton pumping vacuolar H⁺-ATPase (V-ATPase) and are extensively involved in acid-base homeostasis. H⁺ secretion in type A intercalated cells (A-ICs) is regulated by apical vesicle recycling and stimulated by cAMP. In other cell types, cAMP is increased by extracellular agonists, including adenosine, through purinergic receptors. Adenosine is a Food and Drug Administration–approved drug, but very little is known about the effect of adenosine on IC function. Therefore, we investigated the role of adenosine in the regulation of V-ATPase in ICs. Intravenous treatment of mice with adenosine or agonists of ADORA2A and ADORA2B purinergic P1 receptors induced V-ATPase apical membrane accumulation in medullary A-ICs but not in cortical A-ICs or other IC subtypes. Both receptors are located in A-IC apical membranes, and adenosine injection increased urine adenosine concentration and decreased urine pH. Cell fractionation showed that adenosine or an ADORA2A or ADORA2B agonist induced V-ATPase translocation from vesicles to the plasma membrane and increased protein kinase A (PKA)–dependent protein phosphorylation in purified medullary ICs that were isolated from mice. Either ADORA2A or ADORA2B antagonists or the PKA inhibitor mPKI blocked these effects. Finally, a fluorescence pH assay showed that adenosine activates V-ATPase in isolated medullary ICs. Our study shows that medullary A-ICs respond to luminal adenosine through ADORA2A and ADORA2B receptors in a cAMP/PKA pathway–dependent mechanism to induce V-ATPase–dependent H⁺ secretion.

An Adaptable Physiological Model of Endocytic Megalin Trafficking in Opossum Kidney Cells and Mouse Kidney Proximal Tubule.

The cells that comprise the proximal tubule (PT) are specialized for high-capacity apical endocytosis necessary to maintain a protein-free urine. Filtered proteins are reclaimed via receptor-mediated endocytosis facilitated by the multiligand receptors megalin and cubilin. Despite the importance of this pathway, we lack a detailed understanding of megalin trafficking kinetics and how they are regulated. Here, we utilized biochemical and quantitative imaging methods in a highly differentiated model of opossum kidney (OK) cells and in mouse kidney in vivo to develop mathematical models of megalin traffic. A preliminary model based on biochemically quantified kinetic parameters was refined by colocalization of megalin with individual apical endocytic compartment markers. Our model predicts that megalin is rapidly internalized, resulting in primarily intracellular distribution of the receptor at steady state. Moreover, our data show that early endosomes mature rapidly in PT cells and suggest that Rab11 is the primary mediator of apical recycling of megalin from maturing endocytic compartments. Apical recycling represents the rate-limiting component of endocytic traffic, suggesting that this step has the largest impact in determining the endocytic capacity of PT cells. Adaptation of our model to the S1 segment of mouse PT using colocalization data obtained in kidney sections confirms basic aspects of our model and suggests that our OK cell model largely recapitulates in vivo membrane trafficking kinetics. We provide a downloadable application that can be used to adapt our working parameters to further study how endocytic capacity of PT cells may be altered under normal and disease conditions.

GRP75 as a functional element of cholix transcytosis

ABSTRACT Cholix (Chx) is secreted by non-pandemic strains of Vibrio cholerae in the intestinal lumen. For this exotoxin to induce cell death in non-polarized cells in the intestinal lamina propria, it must traverse the epithelium in the fully intact form. We identified host cell elements in polarized enterocytes associated with Chx endocytosis and apical to basal (A→B) vesicular transcytosis. This pathway overcomes endogenous mechanisms of apical vesicle recycling and lysosomal targeting by interacting with several host cell proteins that include the 75 kDa glucose-regulated protein (GRP75). Apical endocytosis of Chx appears to involve the single membrane spanning protein TMEM132A, and interaction with furin before it engages GRP75 in apical vesicular structures. Sorting within these apical vesicles results in Chx being trafficked to the basal region of cells in association with the Lectin, Mannose Binding 1 protein LMAN1. In this location, Chx interacts with the basement membrane-specific heparan sulfate proteoglycan perlecan in recycling endosomes prior to its release from this basal vesicular compartment to enter the underlying lamina propria. While the furin and LMAN1 elements of this Chx transcytosis pathway undergo cellular redistribution that are reflective of the polarity shifts noted for coatamer complexes COPI and COPII, GRP75 and perlecan fail to show these dramatic rearrangements. Together, these data define essential steps in the A→B transcytosis pathway accessed by Chx to reach the intestinal lamina propria where it can engage and intoxicate certain non-polarized cells.

Membrane Traffic in Aspergillus oryzae and Related Filamentous Fungi.

The industrially important filamentous fungus Aspergillus oryzae, known as the yellow Koji mold and also designated the Japanese National fungus, has been investigated for understanding the intracellular membrane trafficking machinery due to the great ability of valuable enzyme production. The underlying molecular mechanisms of the secretory pathway delineate the main secretion route from the hyphal tip via the vesicle cluster Spitzenkörper, but also there is a growing body of evidence that septum-directed and unconventional secretion occurs in A. oryzae hyphal cells. Moreover, not only the secretory pathway but also the endocytic pathway is crucial for protein secretion, especially having a role in apical endocytic recycling. As a hallmark of multicellular filamentous fungal cells, endocytic organelles early endosome and vacuole are quite dynamic: the former exhibits constant long-range motility through the hyphal cells and the latter displays pleiomorphic structures in each hyphal region. These characteristics are thought to have physiological roles, such as supporting protein secretion and transporting nutrients. This review summarizes molecular and physiological mechanisms of membrane traffic, i.e., secretory and endocytic pathways, in A. oryzae and related filamentous fungi and describes the further potential for industrial applications.

Localization of NPFxD motif-containing proteins in Aspergillus nidulans

During growth, filamentous fungi produce polarized cells called hyphae. It is generally presumed that polarization of hyphae is dependent upon secretion through the Spitzenkörper, as well as a mechanism called apical recycling, which maintains a balance between the tightly coupled processes of endocytosis and exocytosis. Endocytosis predominates in an annular domain called the sub-apical endocytic collar, which is located in the region of plasma membrane 1–5 μm distal to the Spitzenkörper. It has previously been proposed that one function of the sub-apical endocytic collar is to maintain the apical localization of polarization proteins. These proteins mark areas of polarization at the apices of hyphae. However, as hyphae grow, these proteins are displaced along the membrane and some must then be removed at the sub-apical endocytic collar in order to maintain the hyphoid shape. While endocytosis is fairly well characterized in yeast, comparatively little is known about the process in filamentous fungi. Here, a bioinformatics approach was utilized to identify 39 Aspergillus nidulans proteins that are predicted to be cargo of endocytosis based on the presence of an NPFxD peptide motif. This motif is a necessary endocytic signal sequence first established in Saccharomyces cerevisiae, where it marks proteins for endocytosis through an interaction with the adapter protein Sla1p. It is hypothesized that some proteins that contain this NPFxD peptide sequence in A. nidulans will be potential targets for endocytosis, and therefore will localize either to the endocytic collar or to more proximal polarized regions of the cell, e.g. the apical dome or the Spitzenkörper. To test this, a subset of the motif-containing proteins in A. nidulans was tagged with GFP and the dynamic localization was evaluated. The documented localization patterns support the hypothesis that the motif marks proteins for localization to the polarized cell apex in growing hyphae.

The Physiology of the Gastric Parietal Cell.

Parietal cells are responsible for gastric acid secretion, which aids in the digestion of food, absorption of minerals, and control of harmful bacteria. However, a fine balance of activators and inhibitors of parietal cell-mediated acid secretion is required to ensure proper digestion of food, while preventing damage to the gastric and duodenal mucosa. As a result, parietal cell secretion is highly regulated through numerous mechanisms including the vagus nerve, gastrin, histamine, ghrelin, somatostatin, glucagon-like peptide 1, and other agonists and antagonists. The tight regulation of parietal cells ensures the proper secretion of HCl. The H+-K+-ATPase enzyme expressed in parietal cells regulates the exchange of cytoplasmic H+ for extracellular K+. The H+ secreted into the gastric lumen by the H+-K+-ATPase combines with luminal Cl- to form gastric acid, HCl. Inhibition of the H+-K+-ATPase is the most efficacious method of preventing harmful gastric acid secretion. Proton pump inhibitors and potassium competitive acid blockers are widely used therapeutically to inhibit acid secretion. Stimulated delivery of the H+-K+-ATPase to the parietal cell apical surface requires the fusion of intracellular tubulovesicles with the overlying secretory canaliculus, a process that represents the most prominent example of apical membrane recycling. In addition to their unique ability to secrete gastric acid, parietal cells also play an important role in gastric mucosal homeostasis through the secretion of multiple growth factor molecules. The gastric parietal cell therefore plays multiple roles in gastric secretion and protection as well as coordination of physiological repair.

Clathrin and clathrin adaptor AP-1 control apical trafficking of megalin in the biosynthetic and recycling routes.

Megalin (gp330, LRP-2) is a protein structurally related to the low-density lipoprotein receptor family that displays a large luminal domain with multiligand binding properties. Megalin localizes to the apical surface of multiple epithelia, where it participates in endocytosis of a variety of ligands performing roles important for development or homeostasis. We recently described the apical recycling pathway of megalin in Madin–Darby canine kidney (MDCK) cells and found that it is a long-lived, fast recycling receptor with a recycling turnover of 15 min and a half-life of 4.8 h. Previous work implicated clathrin and clathrin adaptors in the polarized trafficking of fast recycling basolateral receptors. Hence, here we study the role of clathrin and clathrin adaptors in megalin’s apical localization and trafficking. Targeted silencing of clathrin or the γ1 subunit of clathrin adaptor AP-1 by RNA interference in MDCK cells disrupted apical localization of megalin, causing its redistribution to the basolateral membrane. In contrast, silencing of the γ2 subunit of AP-1 had no effect on megalin polarity. Trafficking assays we developed using FM4-HA-miniMegalin-GFP, a reversible conditional endoplasmic reticulum–retained chimera, revealed that clathrin and AP-1 silencing disrupted apical sorting of megalin in both biosynthetic and recycling routes. Our experiments demonstrate that clathrin and AP-1 control the sorting of an apical transmembrane protein.

Transcytosis maintains CFTR apical polarity in the face of constitutive and mutation-induced basolateral missorting.

Apical polarity of cystic fibrosis transmembrane conductance regulator (CFTR) is essential for solute and water transport in secretory epithelia and can be impaired in human diseases. Maintenance of apical polarity in the face of CFTR non-polarized delivery and inefficient apical retention of mutant CFTRs lacking PDZ-domain protein (NHERF1, also known as SLC9A3R1) interaction, remains enigmatic. Here, we show that basolateral CFTR delivery originates from biosynthetic (∼35%) and endocytic (∼65%) recycling missorting. Basolateral channels are retrieved via basolateral-to-apical transcytosis (hereafter denoted apical transcytosis), enhancing CFTR apical expression by two-fold and suppressing its degradation. In airway epithelia, CFTR transcytosis is microtubule-dependent but independent of Myo5B, Rab11 proteins and NHERF1 binding to its C-terminal DTRL motif. Increased basolateral delivery due to compromised apical recycling and accelerated internalization upon impaired NHERF1-CFTR association is largely counterbalanced by efficient CFTR basolateral internalization and apical transcytosis. Thus, transcytosis represents a previously unrecognized, but indispensable, mechanism for maintaining CFTR apical polarity that acts by attenuating its constitutive and mutation-induced basolateral missorting.

Establishment of Microvillus Inclusion Disease (MVID) human models using small intestinal enteroids and embryonic stem cells

Microvillus Inclusion Disease (MVID) is a severe genetic disease resulting from loss‐of‐function mutations in the apical recycling associated actin motor, myosin Vb (Myo5B). MVID affects newborns (early‐stage) or infants (late‐stage), resulting in uncontrolled secretory diarrhea, and death. The diagnostic and pathognomonic features are found in enterocytes of the small intestine, specifically in the duodenum. Our laboratory investigates how apical ion transporter trafficking and signaling in the intestine results in MVID diarrhea. Treatment is unavailable due to limited understanding of MVID pathogenesis, and, access to human MVID intestinal tissues for research is not feasible due to the severity of illness in affected children. Therefore, generating appropriate research models is critical to understanding the disease and development of therapies.Our laboratory generated cultured intestinal cell models lacking Myo5B (lentivirus shRNA knockdown) for MVID studies (Kravtsov DV et al. 2016). Although cancer‐derived cell lines remain valuable, superior cutting‐edge technologies and models, such as enteroids/organoids and CRISPR/Cas 9 gene editing, are now available. Since MVID is an epithelial disease resulting in ion transporter defects in the proximal small intestine, we hypothesized that successful development of a human small intestinal model of MVID will provide a superior model for studies of MVID pathogenesis and treatment. Our goal is to develop complementary human intestinal models of MVID by impeding Myo5B function in both human duodenal enteroids (hDEs) and human embryonic stem cells (hESCs) that will be differentiated into small intestinal organoids.First, Myo5B will be silenced in 2D hDEs using lentiviral transfection as before. Immunoblot analysis will confirm Myo5B knockdown and enteroids will be characterized for expression, location, and function of ion transporters and proteins involved in MVID pathogenesis. A complementary MVID model will employ H9 hESCs for gene editing of the Myo5B single nucleotide polymorphism, G1125A, using CRISPR/Cas 9 technology. Clones will be sequenced to confirm successful gene editing of Myo5B. Once confirmed, H9 hESCs will be differentiated into small intestine and cultured into 3D intestinal organoids and characterized for MVID.To date, we have cultured undifferentiated (immature) and differentiated (mature) hDEs replicating crypt and villus enterocytes of the duodenum. For H9 hESC studies, the donor DNA guide, forward and reverse single guide RNAs were designed (http://crispr.mit.edu). Guide RNAs and donor DNA were transfected into H9 hESCs and clones sequenced to confirm Myo5B G1125A gene editing. H9 Myo5B G1125A cells will be differentiated into small intestine using an established protocol by McCracken KW et al. 2013. Successful establishment of these models will facilitate studies of ion transporter trafficking and signaling, and screening of compounds for treatment of MVID using physiologically relevant human intestinal models.Support or Funding InformationFunding: R01 DK077065This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

SAC-1 ensures epithelial endocytic recycling by restricting ARF-6 activity.

Arf6/ARF-6 is a crucial regulator of the endosomal phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) pool in endocytic recycling. To further characterize ARF-6 regulation, we performed an ARF-6 interactor screen in Caenorhabditis elegans and identified SAC-1, the homologue of the phosphoinositide phosphatase Sac1p in yeast, as a novel ARF-6 partner. In the absence of ARF-6, basolateral endosomes show a loss of SAC-1 staining in epithelial cells. Steady-state cargo distribution assays revealed that loss of SAC-1 specifically affected apical secretory delivery and basolateral recycling. PI(4,5)P2 levels and the endosomal labeling of the ARF-6 effector UNC-16 were significantly elevated in sac-1 mutants, suggesting that SAC-1 functions as a negative regulator of ARF-6. Further analyses revealed an interaction between SAC-1 and the ARF-6-GEF BRIS-1. This interaction outcompeted ARF-6(guanosine diphosphate [GDP]) for binding to BRIS-1 in a concentration-dependent manner. Consequently, loss of SAC-1 promotes the intracellular overlap between ARF-6 and BRIS-1. BRIS-1 knockdown resulted in a significant reduction in PI(4,5)P2 levels in SAC-1-depleted cells. Interestingly, the action of SAC-1 in sequestering BRIS-1 is independent of SAC-1's catalytic activity. Our results suggest that the interaction of SAC-1 with ARF-6 curbs ARF-6 activity by limiting the access of ARF-6(GDP) to its guanine nucleotide exchange factor, BRIS-1.

Clathrin and AP1 are required for apical sorting of glycosyl phosphatidyl inositol-anchored proteins in biosynthetic and recycling routes in Madin-Darby canine kidney cells.

Recently, studies in animal models demonstrate potential roles for clathrin and AP1 in apical protein sorting in epithelial tissue. However, the precise functions of these proteins in apical protein transport remain unclear. Here, we reveal mistargeting of endogenous glycosyl phosphatidyl inositol-anchored proteins (GPI-APs) and soluble secretory proteins in Madin-Darby canine kidney (MDCK) cells upon clathrin heavy chain or AP1 subunit knockdown (KD). Using a novel directional endocytosis and recycling assay, we found that these KD cells are not only affected for apical sorting of GPI-APs in biosynthetic pathway but also for their apical recycling and basal-to-apical transcytosis routes. The apical distribution of the t-SNARE syntaxin 3, which is known to be responsible for selective targeting of various apical-destined cargo proteins in both biosynthetic and endocytic routes, is compromised suggesting a molecular explanation for the phenotype in KD cells. Our results demonstrate the importance of biosynthetic and endocytic routes for establishment and maintenance of apical localization of GPI-APs in polarized MDCK cells.

Extracellular Adenosine Stimulates Vacuolar ATPase-Dependent Proton Secretion in Medullary Intercalated Cells.

Acidosis is an important complication of AKI and CKD. Renal intercalated cells (ICs) express the proton pumping vacuolar H+-ATPase (V-ATPase) and are extensively involved in acid-base homeostasis. H+ secretion in type A intercalated cells (A-ICs) is regulated by apical vesicle recycling and stimulated by cAMP. In other cell types, cAMP is increased by extracellular agonists, including adenosine, through purinergic receptors. Adenosine is a Food and Drug Administration-approved drug, but very little is known about the effect of adenosine on IC function. Therefore, we investigated the role of adenosine in the regulation of V-ATPase in ICs. Intravenous treatment of mice with adenosine or agonists of ADORA2A and ADORA2B purinergic P1 receptors induced V-ATPase apical membrane accumulation in medullary A-ICs but not in cortical A-ICs or other IC subtypes. Both receptors are located in A-IC apical membranes, and adenosine injection increased urine adenosine concentration and decreased urine pH. Cell fractionation showed that adenosine or an ADORA2A or ADORA2B agonist induced V-ATPase translocation from vesicles to the plasma membrane and increased protein kinase A (PKA)-dependent protein phosphorylation in purified medullary ICs that were isolated from mice. Either ADORA2A or ADORA2B antagonists or the PKA inhibitor mPKI blocked these effects. Finally, a fluorescence pH assay showed that adenosine activates V-ATPase in isolated medullary ICs. Our study shows that medullary A-ICs respond to luminal adenosine through ADORA2A and ADORA2B receptors in a cAMP/PKA pathway-dependent mechanism to induce V-ATPase-dependent H+ secretion.

Trafficking Ion Transporters to the Apical Membrane of Polarized Intestinal Enterocytes.

Epithelial cells lining the gastrointestinal tract require distinct apical and basolateral domains to function properly. Trafficking and insertion of enzymes and transporters into the apical brush border of intestinal epithelial cells is essential for effective digestion and absorption of nutrients. Specific critical ion transporters are delivered to the apical brush border to facilitate fluid and electrolyte uptake. Maintenance of these apical transporters requires both targeted delivery and regulated membrane recycling. Examination of altered apical trafficking in patients with Microvillus Inclusion disease caused by inactivating mutations in MYO5B has led to insights into the regulation of apical trafficking by elements of the apical recycling system. Modeling of MYO5B loss in cell culture and animal models has led to recognition of Rab11a and Rab8a as critical regulators of apical brush border function. All of these studies show the importance of apical membrane trafficking dynamics in maintenance of polarized epithelial cell function.

A targeted RNAi screen identifies factors affecting diverse stages of receptor-mediated transcytosis.

Endosome transport by transcytosis is the primary mechanism by which proteins and other large cargo traverse epithelial barriers in normal tissue. Transcytosis is also essential for establishing and maintaining membrane polarity in epithelia and other polarized cells. To identify novel components of this pathway, we conducted a high-throughput RNA interference screen for factors necessary for the bidirectional transcytosis of IgG by the Fcγ receptor FcRn. This screen identified 23 genes whose suppression resulted in a reproducible decrease in FcRn-mediated transcytosis. Pulse-chase kinetic transport assays on four of the top-ranking genes (EXOC2, EXOC7, PARD6B, and LEPROT) revealed distinct effects on the apical and basolateral recycling and transcytotic pathways, demonstrating that these pathways are genetically separable. We also found a strong dependence on PARD6B for apical, but not basolateral, recycling, implicating this cell polarity gene in assembly or maintenance of the apical endosomal system. This dataset yields insights into how vesicular transport is adapted to the specialized functions of differentiated cell types and opens new research avenues into epithelial trafficking.

The fast-recycling receptor Megalin defines the apical recycling pathway of epithelial cells.

The basolateral recycling and transcytotic pathways of epithelial cells were previously defined using markers such as transferrin (TfR) and polymeric IgA (pIgR) receptors. In contrast, our knowledge of the apical recycling pathway remains fragmentary. Here we utilize quantitative live-imaging and mathematical modelling to outline the recycling pathway of Megalin (LRP-2), an apical receptor with key developmental and renal functions, in MDCK cells. We show that, like TfR, Megalin is a long-lived and fast-recycling receptor. Megalin enters polarized MDCK cells through segregated apical sorting endosomes and subsequently intersects the TfR and pIgR pathways at a perinuclear Rab11-negative compartment termed common recycling endosomes (CRE). Whereas TfR recycles to the basolateral membrane from CRE, Megalin, like pIgR, traffics to subapical Rab11-positive apical recycling endosomes (ARE) and reaches the apical membrane in a microtubule- and Rab11-dependent manner. Hence, Megalin defines the apical recycling pathway of epithelia, with CRE as its apical sorting station.

Nuclear fallout provides a new link between aPKC and polarized cell trafficking.

BackgroundCell polarity, essential for cell physiology and tissue coherence, emerges as a consequence of asymmetric localization of protein complexes and directional trafficking of cellular components. Although molecules required in both processes are well known their relationship is still poorly understood.ResultsHere we show a molecular link between Nuclear Fallout (Nuf), an adaptor of Rab11-GTPase to the microtubule motor proteins during Recycling Endosome (RE) trafficking, and aPKC, a pivotal kinase in the regulation of cell polarity. We demonstrate that aPKC phosphorylates Nuf modifying its subcellular distribution. Accordingly, in aPKC mutants Nuf and Rab11 accumulate apically indicating altered RE delivery. We show that aPKC localization in the apico-lateral cortex is dynamic. When we block exocytosis, by means of exocyst-sec mutants, aPKC accumulates inside the cells. Moreover, apical aPKC concentration is reduced in nuf mutants, suggesting aPKC levels are maintained by recycling.ConclusionsWe demonstrate that active aPKC interacts with Nuf, phosphorylating it and, as a result, modifying its subcellular distribution. We propose a regulatory loop by which Nuf promotes aPKC apical recycling until sufficient levels of active aPKC are reached. Thus, we provide a novel link between cell polarity regulation and traffic control in epithelia.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-016-0253-6) contains supplementary material, which is available to authorized users.

Recycling of galectin-3 in epithelial cells

Galectins, a family of β-galactoside binding proteins, do not possess a signalling sequence to enter the endoplasmic reticulum as a starting point for the classical secretory pathway. They use a so-called unconventional secretion mechanism for translocation across the plasma membrane and/or into the lumen of transport vesicles. The β-galactoside binding protein galectin-3 is highly expressed in a variety of epithelial cell lines. Polarized MDCK cells secrete this lectin predominantly into the apical medium. The lectin re-enters the cell by non-clathrin mediated endocytosis and passages through endosomal organelles. This internalized galectin-3 plays an important role in apical protein trafficking by directing the subcellular targeting of apical glycoproteins via oligomerization into high molecular weight clusters, a process that can be fine-tuned by changes in the environmental pH. Following release at the apical plasma membrane, the lectin can reenter the cell for another round of recycling and apical protein sorting. This review will briefly address galectin-3-functions in epithelia and focus on distinct phases in apical recycling of the lectin.

Tu1401 Differential Intracellular Calcium Inhibition of NHE3 Activity

BACKGROUND: Elevation of intracellular calcium ([Ca]i) in intestinal epithelial cells occurs as part of normal digestion resulting in a transient decrease of NaCl absorption and increase in anion secretion. Under pathophysiological conditions, particularly rotavirus (RV) infection, elevation of [Ca]i is responsible for decreased Na+ absorption, increased Clsecretion and diarrhea. We have shown that elevated [Ca]i by either carbachol (CCH) or RV decreases NHE3 activity by stimulating rapid endocytosis; however, the differential signaling mechanisms responsible for the severe water loss due to RV infection are unclear. PURPOSE: Since NHE3 contributes to RV diarrhea, the current goal was to compare how elevated [Ca]i signaling differs under physiological (CCH) and pathophysiological (RV) conditions and determine their effects on NHE3. METHODS: Caco-2/BBe cells were grown on filters and transiently infected with a 3HA-NHE3 adenovirus construct and studied 48 h later. NHE3 activity was measured by fluorimetry using pH-sensitive dye, BCECF, in cells treated with vehicle, BAPTA-AM, latrunculin, methyl-β-cylcodextrin (MβCD), or inhibitors to PLD1 (EVJ), PLD2 (JWJ), Cdc42 (Pirl1), or acid sphingomyelinase (ASM; imipramine), in the presence or absence of CCHor simian rotavirus, RRV.NHE3 trafficking and degradation were determined by surface biotinylation. RESULTS: Both CCH and RV inhibited NHE3 activity by ~60% in a Ca2+ dependent manner. These effects were correlated with >50% decrease in NHE3 surface expression due to increased NHE3 endocytosis. NHE3 exocytosis was not different after CCH compared to control; however, NHE3 exocytosis was greatly reduced after RV infection. In addition, RV, but not CCH, treatment decreased NHE3 halflife. Pretreatment with either Pirl1, MβCD, both PLD inhibitors, imipramine, or latrunculin prevented CCH-mediated inhibition of NHE3 activity. In contrast, only MβCD and JWJ prevented RV inhibition of NHE3 while latrunculin had additive effects on NHE3 (>90% inhibition) after RV infection.CONCLUSIONS: (1) Physiologic and pathophysiologic [Ca]i elevation has differential effects on NHE3 regulation. (2) While both CCH and RV inhibit apical NHE3 activity through changes in trafficking due to elevated [Ca]i, RV also inhibits NHE3 activity by decreasing protein amount possibly due to a block in apical recycling since RV prevented NHE3 exocytosis. (3) These effects involve lipid rafts since cholesterol depletion and ceramide production by ASM prevent NHE3 inhibition; however, actin remodeling involving Cdc42 was not required for RV inhibition of NHE3 suggesting that RV infection stimulates a different endocytosis pathway for NHE3 compared to CCH resulting in increased NHE3 protein degradation. (4) Rotavirus infection is a new model to dissect mechanisms that regulate NHE3 activity in intestinal epithelial cells.

Copper directs ATP7B to the apical domain of hepatic cells via basolateral endosomes.

Physiologic Cu levels regulate the intracellular location of the Cu ATPase ATP7B. Here, we determined the routes of Cu-directed trafficking of endogenous ATP7B in the polarized hepatic cell line WIF-B and in the liver in vivo. Copper (10 µm) caused ATP7B to exit the trans-Golgi network (TGN) in vesicles, which trafficked via large basolateral endosomes to the apical domain within 1 h. Although perturbants of luminal acidification had little effect on the TGN localization of ATP7B in low Cu, they blocked delivery to the apical membrane in elevated Cu. If the vesicular proton-pump inhibitor bafilomycin-A1 (Baf) was present with Cu, ATP7B still exited the TGN, but accumulated in large endosomes located near the coverslip, in the basolateral region. Baf washout restored ATP7B trafficking to the apical domain. If ATP7B was staged apically in high Cu, Baf addition promoted the accumulation of ATP7B in subapical endosomes, indicating a blockade of apical recycling, with concomitant loss of ATP7B at the apical membrane. The retrograde pathway to the TGN, induced by Cu removal, was far less affected by Baf than the anterograde (Cu-stimulated) case. Overall, loss of acidification-impaired Cu-regulated trafficking of ATP7B at two main sites: (i) sorting and exit from large basolateral endosomes and (ii) recycling via endosomes near the apical membrane.