Polarized Madin-Darby Canine Kidney Research Articles

Sonic hedgehog is basolaterally sorted from the TGN and transcytosed to the apical domain involving Dispatched-1 at Rab11-ARE.

Hedgehog proteins (Hhs) secretion from apical and/or basolateral domains occurs in different epithelial cells impacting development and tissue homeostasis. Palmitoylation and cholesteroylation attach Hhs to membranes, and Dispatched-1 (Disp-1) promotes their release. How these lipidated proteins are handled by the complex secretory and endocytic pathways of polarized epithelial cells remains unknown. We show that polarized Madin-Darby canine kidney cells address newly synthesized sonic hedgehog (Shh) from the TGN to the basolateral cell surface and then to the apical domain through a transcytosis pathway that includes Rab11-apical recycling endosomes (Rab11-ARE). Both palmitoylation and cholesteroylation contribute to this sorting behavior, otherwise Shh lacking these lipid modifications is secreted unpolarized. Disp-1 mediates first basolateral secretion from the TGN and then transcytosis from Rab11-ARE. At the steady state, Shh predominates apically and can be basolaterally transcytosed. This Shh trafficking provides several steps for regulation and variation in different epithelia, subordinating the apical to the basolateral secretion.

Canid herpesvirus 1 Preferentially Infects Polarized Madin-Darby Canine Kidney Cells from the Basolateral Surface.

Canid herpesvirus 1 (CHV-1) infects polarized canine epithelia. Herein, we present our initial work characterizing CHV-1 infection of Madin-Darby canine kidney (MDCK) cells that were polarized on trans-wells. We previously showed that infection of these cells in non-polarized cultures stimulated the formation of extensive lamellipodial membrane protrusions. Uninfected polarized MDCK cells already form extensive lamellipodial membrane protrusions on the apical surface in the absence of virus. Using scanning electron microscopy, we found that CHV-1 infection does not lead to a change in the form of the lamellipodial membrane protrusions on the apical surface of polarized MDCK cells. We found that CHV-1 was able to infect polarized cultures from either the apical or basolateral side; however, higher viral titers were produced upon infection of the basolateral side. Regardless of the side infected, titers of virus were higher in the apical compartment compared to the basal compartment; however, these differences were not statistically significant. In addition to cell-free virus that was recovered in the media, the highest amount of virus produced remained cell-associated over the course of the experiment. The efficiency of CHV-1 infection of the basolateral side of polarized epithelial cells is consistent with the pathobiology of this varicellovirus.

CD320 expression and apical membrane targeting in renal and intestinal epithelial cells

Vitamin B12 is an essential nutrient acquired via dietary intake. Receptor-mediated endocytosis is a key mechanism in vitamin B12 absorption, cellular uptake, and reabsorption. CD320 is a type I transmembrane protein responsible for cellular uptake of vitamin B12 in peripheral tissues. In this study, we examined segmental distribution and cellular expression of CD320 in mouse kidneys and intestines. We show that CD320 is expressed on the luminal surface in the small intestine and in proximal tubules in the kidney, suggesting that, in addition to its role in vitamin B12 uptake in peripheral tissues, CD320 may participate in vitamin B12 absorption in the small intestine and reabsorption in the kidney. Moreover, we show that an amino acid motif, DSSDE, in the second low-density lipoprotein receptor class A domain of CD320 is a key apical membrane targeting signal in both renal and intestinal epithelial cells. Mutations or deletion of this motif abolish the specific apical membrane expression of CD320 in polarized Madin–Darby canine kidney cells and human colon cancer-derived Caco-2 cells. In short-hairpin RNA-based gene knockdown experiments, we show that the apical membrane targeting of CD320 is mediated by a Rab11a-dependent mechanism. These results extend our knowledge regarding the cell biology of CD320 and its role in vitamin B12 metabolism.

The voltage-gated sodium channel β2 subunit associates with lipid rafts by S-palmitoylation.

The voltage-gated sodium channel is critical for cardiomyocyte function. It consists of a protein complex comprising a pore-forming α subunit and associated β subunits. In polarized Madin-Darby canine kidney cells, we show evidence by acyl-biotin exchange that β2 is S-acylated at Cys-182. Interestingly, we found that palmitoylation increases β2 association with detergent-resistant membranes. β2 localizes exclusively to the apical surface. However, depletion of plasma membrane cholesterol, or blocking intracellular cholesterol transport, caused mislocalization of β2, as well as of the non-palmitoylable C182S mutant, to the basolateral domain. Apical β2 did not undergo endocytosis and displayed limited diffusion within the plane of the membrane; such behavior suggests that, at least in part, it is cytoskeleton anchored. Upon acute cholesterol depletion, its mobility was greatly reduced, and a slight reduction was also measured as a result of lack of palmitoylation, supporting β2 association with cholesterol-rich lipid rafts. Indeed, lipid raft labeling confirmed a partial overlap with apical β2. Although β2 palmitoylation was not required to promote surface localization of the α subunit, our data suggest that it is likely implicated in lipid raft association and the polarized localization of β2.

A conserved LDL-receptor motif regulates corin and CD320 membrane targeting in polarized renal epithelial cells.

Selective protein distribution on distinct plasma membranes is important for epithelial cell function. To date, how proteins are directed to specific epithelial cell surface is not fully understood. Here we report a conserved DSSDE motif in LDL-receptor (LDLR) modules of corin (a transmembrane serine protease) and CD320 (a receptor for vitamin B12 uptake), which regulates apical membrane targeting in renal epithelial cells. Altering this motif prevents specific apical corin and CD320 expression in polarized Madin-Darby canine kidney (MDCK) cells. Mechanistic studies indicate that this DSSDE motif participates in a Rab11a-dependent mechanism that specifies apical sorting. In MDCK cells, inhibition of Rab11a, but not Rab11b, expression leads to corin and CD320 expression on both apical and basolateral membranes. Together, our results reveal a novel molecular recognition mechanism that regulates LDLR module-containing proteins in their specific apical expression in polarized renal epithelial cells.

Chlorpromazine Induces Basolateral Aquaporin-2 Accumulation via F-Actin Depolymerization and Blockade of Endocytosis in Renal Epithelial Cells.

We previously showed that in polarized Madin–Darby canine kidney (MDCK) cells, aquaporin-2 (AQP2) is continuously targeted to the basolateral plasma membrane from which it is rapidly retrieved by clathrin-mediated endocytosis. It then undertakes microtubule-dependent transcytosis toward the apical plasma membrane. In this study, we found that treatment with chlorpromazine (CPZ, an inhibitor of clathrin-mediated endocytosis) results in AQP2 accumulation in the basolateral, but not the apical plasma membrane of epithelial cells. In MDCK cells, both AQP2 and clathrin were concentrated in the basolateral plasma membrane after CPZ treatment (100 µM for 15 min), and endocytosis was reduced. Then, using rhodamine phalloidin staining, we found that basolateral, but not apical, F-actin was selectively reduced by CPZ treatment. After incubation of rat kidney slices in situ with CPZ (200 µM for 15 min), basolateral AQP2 and clathrin were increased in principal cells, which simultaneously showed a significant decrease of basolateral compared to apical F-actin staining. These results indicate that clathrin-dependent transcytosis of AQP2 is an essential part of its trafficking pathway in renal epithelial cells and that this process can be inhibited by selectively depolymerizing the basolateral actin pool using CPZ.

Influenza entry pathways in polarized MDCK cells

In non-polarized cell culture models, influenza virus has been shown to enter host cells via multiple endocytic pathways, including classical clathrin-mediated endocytic routes (CME), clathrin- and caveolae-independent routes and macropinocytosis. However, little is known about the entry route of influenza virus in differentiated epithelia, in vivo site of infection for influenza virus. Here, we show that in polarized Madin–Darby canine kidney type II (MDCK II) cells, influenza virus has a specific utilization of the clathrin-mediated endocytic pathway and requires Eps15 for host cell entry.

Myosin-1c regulates the dynamic stability of E-cadherin–based cell–cell contacts in polarized Madin–Darby canine kidney cells

Cooperation between cadherins and the actin cytoskeleton controls the formation and maintenance of cell-cell adhesions in epithelia. We find that the molecular motor protein myosin-1c (Myo1c) regulates the dynamic stability of E-cadherin-based cell-cell contacts. In Myo1c-depleted Madin-Darby canine kidney cells, E-cadherin localization was dis-organized and lateral membranes appeared less vertical with convoluted edges versus control cells. In polarized monolayers, Myo1c-knockdown (KD) cells were more sensitive to reduced calcium concentration. Myo1c separated in the same plasma membrane fractions as E-cadherin, and Myo1c KD caused a significant reduction in the amount of E-cadherin recovered in one peak fraction. Expression of green fluorescent protein (GFP)-Myo1c mutants revealed that the phosphatidylinositol-4,5-bisphosphate-binding site is necessary for its localization to cell-cell adhesions, and fluorescence recovery after photobleaching assays with GFP-Myo1c mutants revealed that motor function was important for Myo1c dynamics at these sites. At 18°C, which inhibits vesicle recycling, Myo1c-KD cells accumulated more E-cadherin-positive vesicles in their cytoplasm, suggesting that Myo1c affects E-cadherin endocytosis. Studies with photoactivatable GFP-E-cadherin showed that Myo1c KD reduced the stability of E-cadherin at cell-cell adhesions. We conclude that Myo1c stabilizes E-cadherin at adherens junctions in polarized epithelial cells and that the motor function and ability of Myo1c to bind membrane are critical.

Similar uptake but different trafficking and escape routes of reovirus virions and infectious subvirion particles imaged in polarized Madin-Darby canine kidney cells.

Polarized epithelial cells that line the digestive, respiratory, and genitourinary tracts form a barrier that many viruses must breach to infect their hosts. Current understanding of cell entry by mammalian reovirus (MRV) virions and infectious subvirion particles (ISVPs), generated from MRV virions by extracellular proteolysis in the digestive tract, are mostly derived from in vitro studies with nonpolarized cells. Recent live-cell imaging advances allow us for the first time to visualize events at the apical surface of polarized cells. In this study, we used spinning-disk confocal fluorescence microscopy with high temporal and spatial resolution to follow the uptake and trafficking dynamics of single MRV virions and ISVPs at the apical surface of live polarized Madin-Darby canine kidney cells. Both types of particles were internalized by clathrin-mediated endocytosis, but virions and ISVPs exhibited strikingly different trafficking after uptake. While virions reached early and late endosomes, ISVPs did not and instead escaped the endocytic pathway from an earlier location. This study highlights the broad advantages of using live-cell imaging combined with single-particle tracking for identifying key steps in cell entry by viruses.

Trafficking of the IKs‐Complex in MDCK Cells: Site of Subunit Assembly and Determinants of Polarized Localization

The voltage-gated potassium channel KV 7.1 is regulated by non-pore forming regulatory KCNE β-subunits. Together with KCNE1, it forms the slowly activating delayed rectifier potassium current IKs . However, where the subunits assemble and which of the subunits determines localization of the IKs -complex has not been unequivocally resolved yet. We employed trafficking-deficient KV 7.1 and KCNE1 mutants to investigate IKs trafficking using the polarized Madin-Darby Canine Kidney cell line. We find that the assembly happens early in the secretory pathway but provide three lines of evidence that it takes place in a post-endoplasmic reticulum compartment. We demonstrate that KV 7.1 targets the IKs -complex to the basolateral membrane, but that KCNE1 can redirect the complex to the apical membrane upon mutation of critical KV 7.1 basolateral targeting signals. Our data provide a possible explanation to the fact that KV 7.1 can be localized apically or basolaterally in different epithelial tissues and offer a solution to divergent literature results regarding the effect of KCNE subunits on the subcellular localization of KV 7.1/KCNE complexes.

Sialylation of N-linked glycans mediates apical delivery of endolyn in MDCK cells via a galectin-9–dependent mechanism

The sialomucin endolyn is implicated in adhesion, migration, and differentiation of various cell types. Along rat kidney tubules, endolyn is variously localized to the apical surface and endosomal/lysosomal compartments. Apical delivery of newly synthesized rat endolyn predominates over direct lysosomal delivery in polarized Madin-Darby canine kidney cells. Apical sorting depends on terminal processing of a subset of lumenal N-glycans. Here we dissect the requirements of N-glycan processing for apical targeting and investigate the underlying mechanism. Modulation of glycan branching and subsequent polylactosamine elongation by knockdown of N-acetylglucosaminyltransferase III or V had no effect on apical delivery of endolyn. In contrast, combined but not individual knockdown of sialyltransferases ST3Gal-III, ST3Gal-IV, and ST6Gal-I, which together are responsible for addition of α2,3- and α2,6-linked sialic acids on N-glycans, dramatically decreased endolyn surface polarity. Endolyn synthesized in the presence of kifunensine, which blocks terminal N-glycan processing, reduced its interaction with several recombinant canine galectins, and knockdown of galectin-9 (but not galectin-3, -4, or -8) selectively disrupted endolyn polarity. Our data suggest that sialylation enables recognition of endolyn by galectin-9 to mediate efficient apical sorting. They raise the intriguing possibility that changes in glycosyltransferase expression patterns and/or galectin-9 distribution may acutely modulate endolyn trafficking in the kidney.

Retromer Regulates Postendocytic Sorting of β‐Secretase in Polarized Madin–Darby Canine Kidney Cells

β-Amyloid (Aβ) peptides are generated from the successive proteolytic processing of the amyloid precursor protein (APP) by the β-APP cleaving enzyme (BACE or β-secretase) and the γ-secretase complex. Initial cleavage of APP by BACE leads into the amyloidogenic pathway, causing or exacerbating Alzheimer's disease. Therefore, their intracellular traffic can determine how easily and frequently BACE has access to and cleaves APP. Here, we have used polarized Madin-Darby canine kidney (MDCK) cells stably expressing APP and BACE to examine the regulation of their polarized trafficking by retromer, a protein complex previously implicated in their endosome-to-Golgi transport. Our data show that retromer interacts with BACE and regulates its postendocytic sorting in polarized MDCK cells. Depleting retromer, inhibiting retromer function, or preventing BACE interaction with retromer, alters trafficking of BACE, which thereby increases its localization in the early endocytic compartment. As a result, this slows endocytosis of apically localized BACE, promoting its recycling and apical-to-basolateral transcytosis, which increases APP/BACE interaction and subsequent cleavage of APP toward generation and secretion of Aβ peptides.

Effects of calcium oxalate monohydrate crystals on expression and function of tight junction of renal tubular epithelial cells

Tight junction has a crucial role in regulating paracellular transports (as a barrier) and in separating apical from basolateral compartments to maintain cell polarity (as a fence). Tight junction can be disrupted by various stimuli, including oxidative stress, pathogens and proinflammatory cytokines. However, association of defective tight junction with kidney stone pathogenesis remains unknown. We therefore examined whether calcium oxalate monohydrate (COM) crystals, which are the major crystalline composition in kidney stones, have any effects on expression and function of tight junction of polarized renal tubular epithelial cells. Western blot analysis revealed marked decrease in levels of occludin and zonula occludens-1 (ZO-1) in COM-treated polarized Madin-Darby canine kidney (MDCK) cells. Immunofluorescence staining revealed not only the decline of these tight junction proteins but also their redistribution and dissociation in COM-treated cells. Additionally, transepithelial resistance was significantly decreased, indicating impaired tight junction barrier and increased paracellular permeability in COM-treated cells. Subcellular fractionation followed by western blot analysis of Na+/K+-ATPase-α1 revealed that this basolateral membrane marker was also detectable in apical membrane fraction of COM-treated cells, but not in apical membrane fraction of control cells. Immunofluorescence study confirmed the translocation of Na+/K+-ATPase-α1 (from basolateral to apical membranes) in COM-treated cells, indicating impaired fence function of the tight junction. Moreover, dihydrorhodamine assay using flow cytometry revealed the significantly higher level of hydrogen peroxide in the COM-treated cells. These data provide the first evidence to demonstrate decreased expression and defective barrier and fence functions of the tight junction of renal tubular epithelial cells exposed to COM crystals that may be fundamental for subsequent renal tubulointerstitial injury, which in turn enhances the stone pathogenesis.

The Structural Requirements of Matriptase in Its Ectodomain Release in Polarized Epithelial Cells

Matriptase is a type-II transmembrane serine protease abundantly expressed in polarized epithelia. The ectodomain of matriptase is released from the cell surface. In the present study, we found that the post-translational cleavage between Gly149 and Ser150 and the existence of catalytic domain are critical for the ectodomain release of matriptase in stable transfection experiments using the polarized Madin-Darby canine kidney epithelial cell line.

MDR1 function is sensitive to the phosphorylation state of myosin regulatory light chain

Multiple drug resistance protein 1 (MDR1) is composed of two homologous halves separated by an intracellular linker region. The linker has been reported to bind myosin regulatory light chain (RLC), but it is not clear how this can occur in the context of a myosin II complex. We characterized MDR1-RLC interactions and determined that binding occurs via the amino terminal of the RLC, a domain that typically binds myosin heavy chain. MDR1-RLC interactions were sensitive to the phosphorylation state of the light chain in that phosphorylation by myosin light chain kinase (MLCK) resulted in a loss of binding in vitro. We used ML-7, a specific inhibitor of MLCK, to study the functional consequences of disrupting RLC phosphorylation in intact cells. Pretreatment of polarized Madin–Darby canine kidney cells stably expressing MDR1 with ML-7 produced a significant increase in apical to basal permeability and a corresponding decrease in the efflux ratio (threefold; p<0.01) of [3H]-digoxin, a classic MDR1 substrate. Together these data show that MDR1-mediated transport of [3H]-digoxin can be modulated by pharmacological manipulation of myosin RLC, but direct MDR1-RLC interactions are atypical and not explained by the structure of the myosin II holoenzyme.

Jaagsiekte Sheep Retrovirus Transformation in Madin-Darby Canine Kidney Epithelial Cell Three-Dimensional Culture

Jaagsiekte sheep retrovirus (JSRV) is the causative agent of a contagious lung cancer in sheep that shares similarities with human bronchioloalveolar carcinoma (BAC). JSRV is unique because the envelope gene (env) is the oncogene, as it can transform cells in culture and induce tumors in animals. The phosphatidylinositol 3-kinase (PI3K)-Akt-mTOR and H/N-Ras-MEK-mitogen-activated protein kinase (MAPK) pathways have been shown to be critical for Env transformation. However, the question still remains of how disruption of these pathways relates to tumor formation. To address this, JSRV Env transformation was studied in the context of epithelial structure, using the polarized Madin-Darby canine kidney (MDCK) epithelial cell three-dimensional (3-D) culture system. The results indicated that JSRV Env-transformed MDCK cells were larger and had full or multiple lumens, in contrast to the single lumens observed in controls. The altered phenotype was largely mediated by an increase in proliferation, in addition to overcoming the proliferative suppression signal. JSRV Env was not found to disrupt polarity or tight junctions or to inhibit lumen apoptosis. The PI3K-Akt-mTOR pathway was important for Env transformation in MDCK cells, although the mechanisms of action differed in 3-D and monolayer cultures. PI3K-dependent signaling to mTOR occurred in monolayers, while PI3K-independent signaling to mTOR occurred in 3-D culture. In contrast, the H/N-Ras-MEK-MAPK pathway was found to be inhibitory to transformation in both normal and transformed MDCK cells in 3-D culture. However, in monolayer culture, inhibition of MEK reverted the transformed phenotype, suggesting a different mechanism(s) of action in monolayer versus 3-D culture.

Polycystin-1 Interacts with Inositol 1,4,5-Trisphosphate Receptor to Modulate Intracellular Ca2+ Signaling with Implications for Polycystic Kidney Disease

The PKD1 or PKD2 genes encode polycystins (PC) 1 and 2, which are associated with polycystic kidney disease. Previously we demonstrated that PC2 interacts with the inositol 1,4,5-trisphosphate receptor (IP(3)R) to modulate Ca(2+) signaling. Here, we investigate whether PC1 also regulates IP(3)R. We generated a fragment encoding the last six transmembrane (TM) domains of PC1 and the C-terminal tail (QIF38), a section with the highest homology to PC2. Using a Xenopus oocyte Ca(2+) imaging system, we observed that expression of QIF38 significantly reduced the initial amplitude of IP(3)-induced Ca(2+) transients, whereas a mutation lacking the C-terminal tail did not. Thus, the C terminus is essential to QIF38 function. Co-immunoprecipitation assays demonstrated that through its C terminus, QIF38 associates with the IP(3)-binding domain of IP(3)R. A shorter PC1 fragment spanning only the last TM and the C-terminal tail also reduced IP(3)-induced Ca(2+) release, whereas another C-terminal fragment lacking any TM domain did not. Thus, only endoplasmic reticulum-localized PC1 can modulate IP(3)R. Finally, we show that in the polarized Madin-Darby canine kidney cells, heterologous expression of full-length PC1 resulted in a smaller IP(3)-induced Ca(2+) response. Overexpression of the IP(3)-binding domain of IP(3)R reversed the inhibitory effect of PC1, suggesting interaction of full-length PC1 (or its cleavage forms) with endogenous IP(3)R in Madin-Darby canine kidney cells. These results indicate that the behavior of full-length PC1 in mammalian cells is congruent with that of PC1 C-terminal fragments in the oocyte system. These data demonstrate that PC1 inhibits Ca(2+) release, perhaps opposing the effect of PC2, which facilitates Ca(2+) release through the IP(3)R.

RhoB‐Dependent Modulation of Postendocytic Traffic in Polarized Madin‐Darby Canine Kidney Cells

The Rho family of GTPases is implicated in the control of endocytic and biosynthetic traffic of many cell types; however, the cellular distribution of RhoB remains controversial and its function is not well understood. Using confocal microscopy, we found that endogenous RhoB and green fluorescent protein-tagged wild-type RhoB were localized to early endosomes, and to a much lesser extent to recycling endosomes, late endosomes or Golgi complex of fixed or live polarized Madin-Darby canine kidney cells. Consistent with RhoB localization to early endosomes, we observed that expression of dominant-negative RhoBN19 or dominant-active RhoBV14 altered postendocytic traffic of ligand-receptor complexes that undergo recycling, degradation or transcytosis. In vitro assays established that RhoB modulated the basolateral-to-apical transcytotic pathway by regulating cargo exit from basolateral early endosomes. Our results indicate that RhoB is localized, in part, to early endosomes where it regulates receptor egress through the early endocytic system.

FAPP2, cilium formation, and compartmentalization of the apical membrane in polarized Madin–Darby canine kidney (MDCK) cells

We have analyzed the role of the phosphatidylinositol-4-phosphate adaptor protein-2 (FAPP2), a component of the apical transport machinery, in cilium formation in polarized Madin-Darby canine kidney (MDCK) cells. We show that ciliogenesis is defective in FAPP2 knockdown cells. Furthermore, by using fluorescence recovery after photobleaching studies of domain connectivity and the generalized polarization spectra of Laurdan, we demonstrate that FAPP2 depletion impairs the formation of condensed apical membrane domains. Laurdan staining also revealed that the ciliary membrane has a highly condensed bilayer domain at its base that could function as a fence to separate the ciliary membrane from the surrounding apical membrane. These results indicate that the compartmentalization of the apical membrane in MDCK cells into the ciliary membrane and the surrounding membrane depends on the balance of raft and nonraft domains.

Rab10 is Involved in Basolateral Transport in Polarized Madin–Darby Canine Kidney Cells

The sorting of newly synthesized membrane proteins to the cell surface is an important mechanism of cell polarity. To identify more of the molecular machinery involved, we investigated the function of the small GTPase Rab10 in polarized epithelial Madin-Darby canine kidney cells. We find that GFP-tagged Rab10 localizes primarily to the Golgi during early cell polarization. Expression of an activated Rab10 mutant inhibits biosynthetic transport from the Golgi and missorts basolateral cargo to the apical membrane. Depletion of Rab10 by RNA interference has only mild effects on biosynthetic transport and epithelial polarization, but simultaneous inhibition of Rab10 and Rab8a more strongly impairs basolateral sorting. These results indicate that Rab10 functions in trafficking from the Golgi at early stages of epithelial polarization, is involved in biosynthetic transport to the basolateral membrane and may co-operate with Rab8.