scholarly journals FAPP2 is involved in the transport of apical cargo in polarized MDCK cells

2005 ◽  
Vol 170 (4) ◽  
pp. 521-526 ◽  
Author(s):  
Otilia V. Vieira ◽  
Paul Verkade ◽  
Aki Manninen ◽  
Kai Simons
Keyword(s):  
Fluorescent Protein ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Adaptor Protein ◽  
Pleckstrin Homology Domain ◽  
Transport Pathways ◽  
Protein Encoding ◽  
Phosphatidylinositol 4 ◽  
Golgi Network ◽  
Darby Canine Kidney

Phosphatidylinositol-4-phosphate (PI(4)P) is the main phosphoinositide in the Golgi complex and has been reported to play a pleiotropic role in transport of cargo from the trans-Golgi network to the plasma membrane (PM) in polarized Madin–Darby canine kidney (MDCK) cells. Overexpression of the chimeric fluorescent protein encoding the pleckstrin homology domain, which is specific for PI(4)P, inhibited both apical and basolateral transport pathways. The transport of apical cargo from the Golgi was shown to be specifically decreased by adenovirus-mediated RNA interference directed against PI(4)P adaptor protein (FAPP) 2. FAPP1 depletion had no effect on transport. On the other hand, FAPP2 was not involved in the Golgi-to-PM transport of cargo that was targeted to the basolateral membrane domain. Thus, we conclude that FAPP2 plays a specific role in apical transport in MDCK cells.

scholarly journals GPI-anchored proteins are directly targeted to the apical surface in fully polarized MDCK cells

2006 ◽  
Vol 172 (7) ◽  
pp. 1023-1034 ◽  
Author(s):  
Simona Paladino ◽  
Thomas Pocard ◽  
Maria Agata Catino ◽  
Chiara Zurzolo
Keyword(s):  
Plasma Membrane ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Apical Surface ◽  
Madin Darby Canine Kidney ◽  
Biochemical Assays ◽  
Intracellular Sorting ◽  
Apical Sorting ◽  
Golgi Network ◽  
Darby Canine Kidney

The polarity of epithelial cells is dependent on their ability to target proteins and lipids in a directional fashion. The trans-Golgi network, the endosomal compartment, and the plasma membrane act as sorting stations for proteins and lipids. The site of intracellular sorting and pathways used for the apical delivery of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) are largely unclear. Using biochemical assays and confocal and video microscopy in living cells, we show that newly synthesized GPI-APs are directly delivered to the apical surface of fully polarized Madin–Darby canine kidney cells. Impairment of basolateral membrane fusion by treatment with tannic acid does not affect the direct apical delivery of GPI-APs, but it does affect the organization of tight junctions and the integrity of the monolayer. Our data clearly demonstrate that GPI-APs are directly sorted to the apical surface without passing through the basolateral membrane. They also reinforce the hypothesis that apical sorting of GPI-APs occurs intracellularly before arrival at the plasma membrane.

FAPP2 is required for aquaporin-2 apical sorting at trans-Golgi network in polarized MDCK cells

2009 ◽  
Vol 297 (6) ◽  
pp. C1389-C1396 ◽  
Author(s):  
Naofumi Yui ◽  
Rie Okutsu ◽  
Eisei Sohara ◽  
Tatemitsu Rai ◽  
Akihito Ohta ◽  
...  
Keyword(s):  
Cell Lines ◽  
Plasma Membranes ◽  
Mdck Cells ◽  
Adaptor Protein ◽  
Trans Golgi Network ◽  
Aquaporin 2 ◽  
Phosphatidylinositol 4 ◽  
Apical Membranes ◽  
Apical Sorting ◽  
Golgi Network

FAPP2 is an adaptor protein of phosphatidylinositol-4-phosphate and is involved in the transport of some apical cargos from the trans-Golgi network (TGN). To investigate whether the regulated apical transport of aquaporin-2 (AQP2) is involved in the FAPP2-dependent apical protein-sorting machinery, we measured apical sorting of AQP2 in Madin-Darby canine kidney (MDCK) cells with or without FAPP2 knockdown. We established MDCK cell lines that stably express rat AQP2 without any tag sequence. Then, FAPP2-deficient stable cell lines were established from the AQP2-expressing cell lines by a retrovirus-mediated RNA interference system. In the established cell lines, AQP2 was detected in both apical and basolateral membranes. Forskolin increased only the apical localization of AQP2, which was not affected by basolateral treatment with 0.5% tannic acid, indicating that the forskolin-induced apical transport of AQP2 did not include the transcytotic pathway from basolateral to apical membranes but is a direct transport from TGN to the apical membranes. Using these cell lines, we tested the effect of FAPP2 knockdown on the polarized AQP2 transport to plasma membranes and found that the forskolin-induced apical transport of AQP2 was completely abolished by FAPP2 knockdown. By contrast, the basolateral localization of AQP2 was not affected by FAPP2 knockdown. AQP2 phosphorylation by forskolin was also impaired in FAPP2 knockdown MDCK cells. These results suggest that FAPP2 is necessary to generate AQP2-bearing vesicles at trans-Golgi that will undergo phosphorylation by PKA in subapical regions.

scholarly journals The MAL Proteolipid Is Necessary for Normal Apical Transport and Accurate Sorting of the Influenza Virus Hemagglutinin in Madin-Darby Canine Kidney Cells

1999 ◽  
Vol 145 (1) ◽  
pp. 141-151 ◽  
Author(s):  
Rosa Puertollano ◽  
Fernando Martín-Belmonte ◽  
Jaime Millán ◽  
María del Carmen de Marco ◽  
Juan P. Albar ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Ectopic Expression ◽  
Apical Surface ◽  
Madin Darby Canine Kidney ◽  
Transport Pathway ◽  
Influenza Virus Hemagglutinin ◽  
Darby Canine Kidney ◽  
Canine Kidney

The MAL (MAL/VIP17) proteolipid is a nonglycosylated integral membrane protein expressed in a restricted pattern of cell types, including T lymphocytes, myelin-forming cells, and polarized epithelial cells. Transport of the influenza virus hemagglutinin (HA) to the apical surface of epithelial Madin-Darby canine kidney (MDCK) cells appears to be mediated by a pathway involving glycolipid- and cholesterol- enriched membranes (GEMs). In MDCK cells, MAL has been proposed previously as being an element of the protein machinery for the GEM-dependent apical transport pathway. Using an antisense oligonucleotide-based strategy and a newly generated monoclonal antibody to canine MAL, herein we have approached the effect of MAL depletion on HA transport in MDCK cells. We have found that MAL depletion diminishes the presence of HA in GEMs, reduces the rate of HA transport to the cell surface, inhibits the delivery of HA to the apical surface, and produces partial missorting of HA to the basolateral membrane. These effects were corrected by ectopic expression of MAL in MDCK cells whose endogenous MAL protein was depleted. Our results indicate that MAL is necessary for both normal apical transport and accurate sorting of HA.

scholarly journals Antimicrotubule drugs inhibit the polarized insertion of an intracellular glycoprotein pool into the apical membrane of Madin-Darby canine kidney (MDCK) cells

1992 ◽  
Vol 103 (3) ◽  
pp. 677-687 ◽  
Author(s):  
G.K. Ojakian ◽  
R. Schwimmer
Keyword(s):  
Apical Membrane ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Immunogold Electron Microscopy ◽  
Apical Plasma Membrane ◽  
Apical Surface ◽  
Transport Pathways ◽  
Intracellular Targeting ◽  
Polarized Delivery ◽  
Membrane Recognition

Previous experiments on MDCK cells have demonstrated that the polarized appearance of a 135 kDa glycoprotein (gp135) on the apical plasma membrane can occur through the insertion of both newly synthesized gp135 as well as a pre-existing gp135 intracellular pool. In this study, anticytoskeletal drugs were utilized to determine the role of the cytoskeleton in the polarized delivery of gp135. Colchicine and nocodazole produced a 15–20% inhibition in the apical surface accumulation of newly synthesized gp135 and inhibited the appearance of the gp135 pool by approximately 33%, while cytochalasin D had no affect on the apical accumulation of either newly synthesized gp135 or the gp135 pool. These results indicate that microtubules, but not microfilaments, are involved in the intracellular targeting of gp135. Quantitative immunogold electron microscopy of nocodazole-treated cells demonstrated that gp135 was not mistargeted to the basolateral membrane, suggesting the possibility that some vesicles containing gp135 did not fuse with the apical membrane and remained in the cells. These experiments demonstrate that microtubules are an important component of gp135 insertion into the apical membrane. They also suggest that gp135 resides within vesicles which have an apical membrane recognition signal and cannot fuse with the basolateral membrane. The possibility that these data, and those of others, could support a hypothesis for the presence of two constitutive apical transport pathways is discussed.

Riboflavin uptake transporter Slc52a2 (RFVT2) is upregulated in the mouse mammary gland during lactation

2016 ◽  
Vol 310 (7) ◽  
pp. R578-R585 ◽  
Author(s):  
Alex Man Lai Wu ◽  
Liana Dedina ◽  
Pooja Dalvi ◽  
Mingdong Yang ◽  
John Leon-Cheon ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Breast Milk ◽  
Milk Fat ◽  
Fluorescent Protein ◽  
Mammary Epithelial Cells ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Mouse Mammary Gland ◽  
Transport Systems ◽  
Human Breast Milk

While it is well recognized that riboflavin accumulates in breast milk as an essential vitamin for neonates, transport mechanisms for its milk excretion are not well characterized. The multidrug efflux transporter ABCG2 in the apical membrane of milk-producing mammary epithelial cells (MECs) is involved with riboflavin excretion. However, it is not clear whether MECs possess other riboflavin transport systems, which may facilitate its basolateral uptake into MECs. We report here that transcripts encoding the second ( SLC52A2) and third ( SLC52A3) member of the recently discovered family of SLC52A riboflavin uptake transporters are expressed in milk fat globules from human breast milk. Furthermore, Slc52a2 and Slc52a3 mRNA are upregulated in the mouse mammary gland during lactation. Importantly, the induction of Slc52a2, which was the major Slc52a riboflavin transporter in the lactating mammary gland, was also observed at the protein level. Subcellular localization studies showed that green fluorescent protein-tagged mouse SLC52A2 mainly localized to the cell membrane, with no preferential distribution to the apical or basolateral membrane in polarized kidney MDCK cells. These results strongly implicate a potential role for SLC52A2 in riboflavin uptake by milk-producing MECs, a critical step in the transfer of riboflavin into breast milk.

scholarly journals A versatile nanobody-based toolkit to analyze retrograde transport from the cell surface

2018 ◽  
Vol 115 (27) ◽  
pp. E6227-E6236 ◽  
Author(s):  
Dominik P. Buser ◽  
Kai D. Schleicher ◽  
Cristina Prescianotto-Baschong ◽  
Martin Spiess
Keyword(s):  
Electron Microscopy ◽  
Cell Surface ◽  
Adaptor Protein ◽  
Retrograde Transport ◽  
Transport Pathways ◽  
Consensus Sequences ◽  
Retrograde Traffic ◽  
Biochemical Detection ◽  
Golgi Network ◽  
Kinetics Of

Retrograde transport of membranes and proteins from the cell surface to the Golgi and beyond is essential to maintain homeostasis, compartment identity, and physiological functions. To study retrograde traffic biochemically, by live-cell imaging or by electron microscopy, we engineered functionalized anti-GFP nanobodies (camelid VHH antibody domains) to be bacterially expressed and purified. Tyrosine sulfation consensus sequences were fused to the nanobody for biochemical detection of trans-Golgi arrival, fluorophores for fluorescence microscopy and live imaging, and APEX2 (ascorbate peroxidase 2) for electron microscopy and compartment ablation. These functionalized nanobodies are specifically captured by GFP-modified reporter proteins at the cell surface and transported piggyback to the reporters’ homing compartments. As an application of this tool, we have used it to determine the contribution of adaptor protein-1/clathrin in retrograde transport kinetics of the mannose-6-phosphate receptors from endosomes back to the trans-Golgi network. Our experiments establish functionalized nanobodies as a powerful tool to demonstrate and quantify retrograde transport pathways.

Expression of 5-HT1C receptors in transfected MDCK cells is functionally and anatomically asymmetric

1993 ◽  
Vol 265 (1) ◽  
pp. C193-C200 ◽  
Author(s):  
H. Luo ◽  
A. Tesfaye ◽  
I. Schieren ◽  
H. S. Chase
Keyword(s):  
Binding Sites ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Lysergic Acid ◽  
Madin Darby Canine Kidney ◽  
High Affinity ◽  
G418 Selection ◽  
Selection Medium ◽  
Darby Canine Kidney ◽  
Canine Kidney

Madin-Darby canine kidney (MDCK) cells were transfected with the cDNA for the rat 5-HT1C receptor (pMV7-SR1c) using electroporation. Cells that survived G418 selection medium were loaded with indo-1 and run through a fluorescence-activated cell sorter (FACS); 10% responded to serotonin (5-HT) with an increase in intracellular Ca2+ concentration ([Ca2+]i). Responding cells were separated with the FACS, grown to confluence, and resorted two more times until a clone of 100% respondents was obtained (SR-MDCK). In SR-MDCK cells grown on porous filters, [Ca2+]i increased only when 5-HT was applied to the basolateral membrane (change in [Ca2+]i = 190 +/- 43 nM); there was no response of [Ca2+]i to apical application of 5-HT. The asymmetric response to 5-HT was likely due to targeting of 5-HT1C receptors exclusively to the basolateral membrane of SR-MDCK cells; 125I-labeled lysergic acid diethylamide binding sites, a marker of high-affinity 5-HT receptors, were located only in the basolateral membrane. These experiments demonstrate that epithelial cells can be stably transfected to express G protein-linked, calcium-mobilizing receptors and that the receptors may be targeted asymmetrically to specific domains of the plasma membrane.

Dissociation of spectrin-ankyrin complex as a basis for loss of Na-K-ATPase polarity after ischemia

2003 ◽  
Vol 284 (2) ◽  
pp. F358-F364 ◽  
Author(s):  
Robert Woroniecki ◽  
Jean R. Ferdinand ◽  
Jon S. Morrow ◽  
Prasad Devarajan
Keyword(s):  
Mdck Cells ◽  
Basolateral Membrane ◽  
Ischemic Injury ◽  
Atp Depletion ◽  
Dependent Manner ◽  
Binding Assays ◽  
Glutathione S Transferase ◽  
Time Points ◽  
Partial Overlap ◽  
Darby Canine Kidney

The polarized distribution of Na-K-ATPase at the basolateral membranes of renal tubule epithelial cells is maintained via a tethering interaction with the underlying spectrin-ankyrin cytoskeleton. In this study, we have explored the mechanism underlying the loss of Na-K-ATPase polarity after ischemic injury in Madin-Darby canine kidney (MDCK) cells, utilizing a novel antibody raised against a recently described kidney-specific isoform of ankyrin. In control MDCK cells, ankyrin was colocalized with Na-K-ATPase at the basolateral membrane. ATP depletion resulted in a duration-dependent mislocation of Na-K-ATPase and ankyrin throughout the cytoplasm. Colocalization studies showed a partial overlap between the distribution of ankyrin and Na-K-ATPase at all periods after ATP depletion. By immunoprecipitation with anti-ankyrin antibody, the mislocated Na-K-ATPase remained bound to ankyrin at all time points after ATP depletion. However, the interaction between ankyrin and spectrin was markedly diminished within 3 h of ATP depletion and was completely lost after 6 h. In solution binding assays using a fusion peptide of glutathione S-transferase with the ankyrin binding domain of Na-K-ATPase, a complex with ankyrin was detected at all time points after ATP depletion, but spectrin was lost from the complex in a duration-dependent manner. The loss of spectrin binding was not attributable to spectrin degradation but was associated with hyperphosphorylation of ankyrin. The results suggest that a dissociation of the membrane-cytoskeleton complex at the spectrin-ankyrin interface may contribute to the loss of Na-K-ATPase polarity after ischemic injury and reaffirm a critical adapter role for ankyrin in the normal maintenance of Na-K-ATPase polarity.

scholarly journals A basolateral lactate/H+ co-transporter in Madin-Darby Canine Kidney (MDCK) cells

1993 ◽  
Vol 289 (1) ◽  
pp. 263-268 ◽  
Author(s):  
S O Rosenberg ◽  
T Fadil ◽  
V L Schuster
Keyword(s):  
Apical Membrane ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Ionic Diffusion ◽  
Madin Darby Canine Kidney ◽  
Apical Compartment ◽  
Disulphonic Acid ◽  
Component Carrier ◽  
Darby Canine Kidney ◽  
Canine Kidney

Monolayers of Madin-Darby Canine Kidney (MDCK) cells grown on permeable filters generated lactate aerobically and accumulated it preferentially in the basolateral compartment, suggesting the presence of a lactate carrier. The mechanism of lactate transport across apical and basolateral membranes was examined by determining intracellular pH (pHi) microspectrofluorimetrically after addition of lactate to the extracellular solutions and by measuring uptake of [14C]lactate. Addition of 20 mM lactate to the apical compartment produced no change in pHi, whereas lactate added to the basolateral compartment rapidly and reversibly lowered pHi. Pyruvate produced similar results. Inhibitors of lactate/H+ co-transporters, alpha-cyano-4-hydroxycinnamate (CnCN) and quercetin, partially inhibited the fall in pHi produced by basolateral lactate. In contrast, the disulphonic stilbene. DIDS (4,4′-di-isothiocyanostilbene-2,2′-disulphonic acid) produced no inhibition at 0.5 mM. Kinetic analysis was performed by applying basolateral lactate at various concentrations and measuring the rate of entry (delta pHi/min) in the presence and absence of CnCN. Lactate flux was shown to occur by both non-ionic diffusion and a alpha-cyano-4-hydroxycinnamate-sensitive component (carrier). The latter has a Km of approximately 7 mM for the lactate anion. Propionate, but not formate, lowered pHi to the same degree as did equimolar lactate, but the propionate effect was not inhibited by CnCN. Influx of [14C]lactate was substantially greater across the basolateral membrane than across the apical membrane and occurred in the absence of Na+. We conclude that MDCK cells grown on permeable filters generate lactate aerobically and transport it across the basolateral membrane by way of a lactate/H+ cotransporter.

scholarly journals EpsinR

2003 ◽  
Vol 160 (2) ◽  
pp. 213-222 ◽  
Author(s):  
Ian G. Mills ◽  
Gerrit J.K. Praefcke ◽  
Yvonne Vallis ◽  
Brian J. Peter ◽  
Lene E. Olesen ◽  
...  
Keyword(s):  
Cathepsin D ◽  
Adaptor Protein ◽  
Biological Data ◽  
Coated Vesicle ◽  
Binding Motif ◽  
Binding Partner ◽  
High Affinity ◽  
Ef Hand ◽  
Phosphatidylinositol 4 ◽  
Golgi Network

EpsinR is a clathrin-coated vesicle (CCV) enriched 70-kD protein that binds to phosphatidylinositol-4-phosphate, clathrin, and the gamma appendage domain of the adaptor protein complex 1 (AP1). In cells, its distribution overlaps with the perinuclear pool of clathrin and AP1 adaptors. Overexpression disrupts the CCV-dependent trafficking of cathepsin D from the trans-Golgi network to lysosomes and the incorporation of mannose-6-phosphate receptors into CCVs. These biochemical and cell biological data point to a role for epsinR in AP1/clathrin budding events in the cell, just as epsin1 is involved in the budding of AP2 CCVs. Furthermore, we show that two gamma appendage domains can simultaneously bind to epsinR with affinities of 0.7 and 45 μM, respectively. Thus, potentially, two AP1 complexes can bind to one epsinR. This high affinity binding allowed us to identify a consensus binding motif of the form DFxDF, which we also find in γ-synergin and use to predict that an uncharacterized EF-hand–containing protein will be a new gamma binding partner.

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