scholarly journals The Cytoplasmic Tail of Rhodopsin Acts as a Novel Apical Sorting Signal in Polarized MDCK Cells

1998 ◽  
Vol 142 (5) ◽  
pp. 1245-1256 ◽  
Author(s):  
Jen-Zen Chuang ◽  
Ching-Hwa Sung
Keyword(s):  
Secretory Pathway ◽  
Apical Membrane ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Brefeldin A ◽  
Cytoplasmic Tail ◽  
Apical Plasma Membrane ◽  
Glutathione S Transferase ◽  
Sorting Signals ◽  
Apical Sorting

All basolateral sorting signals described to date reside in the cytoplasmic domain of proteins, whereas apical targeting motifs have been found to be lumenal. In this report, we demonstrate that wild-type rhodopsin is targeted to the apical plasma membrane via the TGN upon expression in polarized epithelial MDCK cells. Truncated rhodopsin with a deletion of 32 COOH-terminal residues shows a nonpolar steady-state distribution. Addition of the COOH-terminal 39 residues of rhodopsin redirects the basolateral membrane protein CD7 to the apical membrane. Fusion of rhodopsin's cytoplasmic tail to a cytosolic protein glutathione S-transferase (GST) also targets this fusion protein (GST–Rho39Tr) to the apical membrane. The targeting of GST–Rho39Tr requires both the terminal 39 amino acids and the palmitoylation membrane anchor signal provided by the rhodopsin sequence. The apical transport of GST–Rho39Tr can be reversibly blocked at the Golgi complex by low temperature and can be altered by brefeldin A treatment. This indicates that the membrane-associated GST–Rho39Tr protein may be sorted along a yet unidentified pathway that is similar to the secretory pathway in polarized MDCK cells. We conclude that the COOH-terminal tail of rhodopsin contains a novel cytoplasmic apical sorting determinant. This finding further indicates that cytoplasmic sorting machinery may exist in MDCK cells for some apically targeted proteins, analogous to that described for basolaterally targeted proteins.

scholarly journals A specific sorting signal is not required for the polarized secretion of newly synthesized proteins from cultured intestinal epithelial cells.

1988 ◽  
Vol 107 (2) ◽  
pp. 471-479 ◽  
Author(s):  
M J Rindler ◽  
M G Traber
Keyword(s):  
Epithelial Cells ◽  
Secretory Pathway ◽  
Intestinal Epithelial Cells ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Apical Cell ◽  
Apical Plasma Membrane ◽  
Intestinal Epithelial ◽  
Polarized Secretion ◽  
Sorting Signals

Caco-2 cells, derived from human colon, have the morphological, functional, and biochemical properties of small intestinal epithelial cells. After infection with enveloped viruses, influenza virions assembled at the apical plasma membrane while vesicular stomatitis virus (VSV) particles appeared exclusively at the basolateral membrane, similar to the pattern observed in virus-infected Madin-Darby canine kidney (MDCK). When grown in Millicell filter chamber devices and labeled with [35S]methionine, Caco-2 monolayers released all of their radiolabeled secretory products preferentially into the basal chamber. Among the proteins identified were apolipoproteins AI and E, transferrin, and alpha-fetoprotein. No proteins were observed to be secreted preferentially from the apical cell surface. The lysosomal enzyme beta-hexosaminidase was also secreted primarily from the basolateral surface of the cells in the presence or absence of lysosomotropic drugs or tunicamycin, which inhibit the targetting of lysosomal enzymes to lysosomes. Neither of these drug treatments significantly affected the polarized secretion of other nonlysosomal proteins. In addition, growth hormone (GH), which is released in a nonpolar fashion from MDCK cells, was secreted exclusively from the basolateral membrane after transfection of Caco-2 cells with GH cDNA in a pSV2-based expression vector. Similar results were obtained in transient expression experiments and after selection of permanently transformed Caco-2 cells expressing GH. Since both beta-hexosaminidase and GH would be expected to lack sorting signals for polarized exocytosis in epithelial cells, these results indicate that in intestinal cells, proteins transported via the basolateral secretory pathway need not have specific sorting signals.

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.

Brefeldin A rapidly disrupts plasma membrane polarity by blocking polar sorting in common endosomes of MDCK cells

2001 ◽  
Vol 114 (18) ◽  
pp. 3309-3321 ◽  
Author(s):  
Exing Wang ◽  
Janice G. Pennington ◽  
James R. Goldenring ◽  
Walter Hunziker ◽  
Kenneth W. Dunn
Keyword(s):  
Plasma Membrane ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Brefeldin A ◽  
Apical Plasma Membrane ◽  
Transferrin Receptors ◽  
Recycling Endosome ◽  
Polarized Cells ◽  
Endocytic Sorting ◽  
On Line

Recent studies showing thorough intermixing of apical and basolateral endosomes have demonstrated that endocytic sorting is critical to maintaining the plasma membrane polarity of epithelial cells. Our studies of living, polarized cells show that disrupting endocytosis with brefeldin-A rapidly destroys the polarity of transferrin receptors in MDCK cells while having no effect on tight junctions. Brefeldin-A treatment induces tubulation of endosomes, but the sequential compartments and transport steps of the transcytotic pathway remain intact. Transferrin is sorted from LDL, but is then missorted from common endosomes to the apical recycling endosome, as identified by its nearly neutral pH, and association with GFP chimeras of Rabs 11a and 25. From the apical recycling endosome, transferrin is then directed to the apical plasma membrane. These data are consistent with a model in which polarized sorting of basolateral membrane proteins occurs via a brefeldin-A-sensitive process of segregation into basolateral recycling vesicles. Although disruption of polar sorting correlates with dissociation of γ-adaptin from endosomes, γ-adaptin does not appear to be specifically involved in sorting into recycling vesicles, as we find it associated with the transcytotic pathway, and particularly to the post-sorting transcytotic apical recycling endosome. Movies available on-line

scholarly journals Selective Alterations in Biosynthetic and Endocytic Protein Traffic in Madin-Darby Canine Kidney Epithelial Cells Expressing Mutants of the Small GTPase Rac1

2000 ◽  
Vol 11 (1) ◽  
pp. 287-304 ◽  
Author(s):  
Tzuu-Shuh Jou ◽  
Som-Ming Leung ◽  
Linette M. Fung ◽  
Wily G. Ruiz ◽  
W. James Nelson ◽  
...  
Keyword(s):  
Apical Membrane ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Membrane Traffic ◽  
Small Gtpase ◽  
Apical Plasma Membrane ◽  
Membrane Domains ◽  
Madin Darby Canine Kidney ◽  
Darby Canine Kidney ◽  
Canine Kidney

Madin-Darby canine kidney (MDCK) cells expressing constitutively active Rac1 (Rac1V12) accumulate a large central aggregate of membranes beneath the apical membrane that contains filamentous actin, Rac1V12, rab11, and the resident apical membrane protein GP-135. To examine the roles of Rac1 in membrane traffic and the formation of this aggregate, we analyzed endocytic and biosynthetic trafficking pathways in MDCK cells expressing Rac1V12 and dominant inactive Rac1 (Rac1N17). Rac1V12 expression decreased the rates of apical and basolateral endocytosis, whereas Rac1N17 expression increased those rates from both membrane domains. Basolateral-to-apical transcytosis of immunoglobulin A (IgA) (a ligand for the polymeric immunoglobulin receptor [pIgR]), apical recycling of pIgR-IgA, and accumulation of newly synthesized GP-135 at the apical plasma membrane were all decreased in cells expressing Rac1V12. These effects of Rac1V12 on trafficking pathways to the apical membrane were the result of the delivery and trapping of these proteins in the central aggregate. In contrast to abnormalities in apical trafficking events, basolateral recycling of transferrin, degradation of EGF internalized from the basolateral membrane, and delivery of newly synthesized pIgR from the Golgi to the basolateral membrane were all relatively unaffected by Rac1V12 expression. Rac1N17 expression had little or no effect on these postendocytic or biosynthetic trafficking pathways. These results show that in polarized MDCK cells activated Rac1 may regulate the rate of endocytosis from both membrane domains and that expression of dominant active Rac1V12 specifically alters postendocytic and biosynthetic membrane traffic directed to the apical, but not the basolateral, membrane.

Identification of an apical sorting determinant in the cytoplasmic tail of megalin

2003 ◽  
Vol 284 (5) ◽  
pp. C1105-C1113 ◽  
Author(s):  
Tetsuro Takeda ◽  
Hajime Yamazaki ◽  
Marilyn G. Farquhar
Keyword(s):  
Mdck Cells ◽  
Receptor Gene ◽  
Low Density Lipoprotein ◽  
Ldl Receptor ◽  
Density Lipoprotein ◽  
Cytoplasmic Tail ◽  
Apical Plasma Membrane ◽  
Binding Motif ◽  
Cell Surface Biotinylation ◽  
Apical Sorting

Megalin is the main endocytic receptor of the proximal tubule and is responsible for reabsorption of many filtered proteins. In contrast to other members of the low-density lipoprotein (LDL) receptor gene family, it is expressed on the apical plasma membrane (PM) of polarized epithelial cells. To identify megalin's apical sorting signal, we generated deletion mutants and chimeric minireceptors composed of complementary regions of megalin and LDL receptor-related protein (LRP) and assessed the distribution of the mutants in Madin-Darby canine kidney (MDCK) cells by immunofluorescence and cell surface biotinylation. Megalin and LRP minireceptors are correctly targeted to the apical and basolateral PM, respectively, of MDCK cells. We found that the information that directs apical sorting is present in the cytoplasmic tail (CT) of megalin, which contains three NPXY motifs, YXXØ, SH3, and dileucine motifs, and a PDZ-binding motif at its COOH terminus. Deletion analysis established that amino acids 107–136 of the megalin-CT containing the second NPXY-like motif are critical for apical sorting and targeting, whereas the regions containing the first and third NPXY motifs are required for efficient endocytosis. We conclude that the megalin-CT contains a novel apical sorting determinant and that cytoplasmic sorting machinery exists in MDCK cells for some apical transmembrane proteins.

PBA increases CFTR expression but at high doses inhibits Cl−secretion in Calu-3 airway epithelial cells

1999 ◽  
Vol 277 (4) ◽  
pp. L700-L708 ◽  
Author(s):  
Johannes Loffing ◽  
Bryan D. Moyer ◽  
Donna Reynolds ◽  
Bruce A. Stanton
Keyword(s):  
Protein Expression ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Airway Epithelial Cells ◽  
Cell Phenotype ◽  
Apical Plasma Membrane ◽  
Airway Epithelial ◽  
High Concentration ◽  
Cycle Enzyme ◽  
Cf Transmembrane Conductance Regulator

Sodium 4-phenylbutyrate (PBA), a short-chain fatty acid, has been approved to treat patients with urea cycle enzyme deficiencies and is being evaluated in the management of sickle cell disease, thalassemia, cancer, and cystic fibrosis (CF). Because relatively little is known about the effects of PBA on the expression and function of the wild-type CF transmembrane conductance regulator (wt CFTR), the goal of this study was to examine the effects of PBA and related compounds on wt CFTR-mediated Cl−secretion. To this end, we studied Calu-3 cells, a human airway cell line that expresses endogenous wt CFTR and has a serous cell phenotype. We report that chronic treatment of Calu-3 cells with a high concentration (5 mM) of PBA, sodium butyrate, or sodium valproate but not of sodium acetate reduced basal and 8-(4-chlorophenylthio)-cAMP-stimulated Cl−secretion. Paradoxically, PBA enhanced CFTR protein expression 6- to 10-fold and increased the intensity of CFTR staining in the apical plasma membrane. PBA also increased protein expression of Na+-K+-ATPase. PBA reduced CFTR Cl−currents across the apical membrane but had no effect on Na+-K+-ATPase activity in the basolateral membrane. Thus a high concentration of PBA (5 mM) reduces Cl−secretion by inhibiting CFTR Cl−currents across the apical membrane. In contrast, lower therapeutic concentrations of PBA (0.05–2 mM) had no effect on cAMP-stimulated Cl−secretion across Calu-3 cells. We conclude that PBA concentrations in the therapeutic range are unlikely to have a negative effect on Cl−secretion. However, concentrations >5 mM might reduce transepithelial Cl−secretion by serous cells in submucosal glands in individuals expressing wt CFTR.

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

2005 ◽  
Vol 168 (2) ◽  
pp. 303-313 ◽  
Author(s):  
Doris Meder ◽  
Anna Shevchenko ◽  
Kai Simons ◽  
Joachim Füllekrug
Keyword(s):  
Free Surface ◽  
Apical Membrane ◽  
Mdck Cells ◽  
Single Cells ◽  
Basolateral Membrane ◽  
Pdz Domain ◽  
Binding Motif ◽  
Membrane Domains ◽  
Regulatory Factor ◽  
Pdz Protein

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

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.

Arginine vasopressin and forskolin regulate apical cell surface expression of epithelial Na+ channels in A6 cells

1994 ◽  
Vol 266 (3) ◽  
pp. F506-F511 ◽  
Author(s):  
T. R. Kleyman ◽  
S. A. Ernst ◽  
B. Coupaye-Gerard
Keyword(s):  
Cell Surface ◽  
Apical Membrane ◽  
Apical Cell ◽  
Brefeldin A ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
Apical Plasma Membrane ◽  
Na Channels ◽  
Na Transport ◽  
A6 Cells

Both arginine vasopressin (AVP) and forskolin regulate vectorial Na+ transport across high-resistance epithelia by increasing the Na+ conductance of the apical membrane mediated by amiloride-sensitive Na+ channels. Pretreatment of A6 cells with brefeldin A partially inhibited the increase in Na+ transport in response to forskolin, suggesting recruitment of Na+ channels from an intracellular pool. The activation of Cl- secretion was not affected. Apical cell surface expression of Na+ channels was examined following activation of transepithelial Na+ transport across the epithelial cell line A6 by AVP or forskolin. Apical cell surface radioiodinated Na+ channels were immunoprecipitated to quantify the biochemical pool of Na+ channels at the apical plasma membrane and to determine whether an increment in the biochemical pool of Na+ channels expressed at the apical cell surface is a potential mechanism by which AVP and forskolin increase apical membrane Na+ conductance. The activation of Na+ transport across A6 cells by AVP was accompanied by a significant increase in the biochemical pool of Na+ channels at the apical plasma membrane within 5 min after addition of hormone, which was sustained for at least 30 min. The increase in apical cell surface expression of Na+ channels was also observed 30 min after application of forskolin. No changes in the oligomeric subunit composition of the channel were noted. Brefeldin A inhibited the forskolin-stimulated increase in apical cell surface expression of Na+ channels. These results suggest that AVP and forskolin regulate Na+ transport, in part, via rapid recruitment of Na+ channels to the cell surface, perhaps from a pool of channels in the subapical cytoplasm.

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.

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