scholarly journals Interaction with mLin-7 Alters the Targeting of Endocytosed Transmembrane Proteins in Mammalian Epithelial Cells

2001 ◽  
Vol 12 (5) ◽  
pp. 1329-1340 ◽  
Author(s):  
Samuel W. Straight ◽  
Liguang Chen ◽  
David Karnak ◽  
Ben Margolis
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Protein Targeting ◽  
Mdck Cells ◽  
Pdz Domain ◽  
Receptor Protein ◽  
Membrane Domain ◽  
Basolateral Plasma Membrane ◽  
Carboxyl Terminal ◽  
Plasma Membrane Domain

To investigate the targeting mechanism for proteins bound to the mammalian Lin-7 (mLin-7) PDZ domain, we created receptor protein chimeras composed of the carboxyl-terminal amino acids of LET-23 fused to truncated nerve growth factor receptor/P75. mLin-7 bound to the chimera with a wild-type LET-23 carboxyl-terminal tail (P75t-Let23WT), but not a mutant tail (P75t-Let23MUT). In Madin-Darby canine kidney (MDCK) cells, P75t-Let23WT localized to the basolateral plasma membrane domain, whereas P75t-Let23MUT remained apical. Furthermore, mutant mLin-7 constructs acted as dominant interfering proteins and inhibited the basolateral localization of P75t-Let23WT. The mechanisms for this differential localization were examined further, and, initially, we found that P75t-Let23WT and P75t-Let23MUT were delivered equally to the apical and basolateral plasma membrane domains. Although basolateral retention of P75t-Let23WT, but not P75t-Let23MUT, was observed, the greatest difference in receptor localization was seen in the rapid trafficking of P75t-Let23WT to the basolateral plasma membrane domain after endocytosis, whereas P75t-Let23MUT was degraded in lysosomes, indicating that mLin-7 binding can alter the fate of endocytosed proteins. Altogether, these data support a model for basolateral protein targeting in mammalian epithelial cells dependent on protein–protein interactions with mLin-7, and also suggest a dynamic role for mLin-7 in endosomal sorting.

Selective localization of the polytopic membrane protein prominin in microvilli of epithelial cells - a combination of apical sorting and retention in plasma membrane protrusions

1999 ◽  
Vol 112 (7) ◽  
pp. 1023-1033 ◽  
Author(s):  
D. Corbeil ◽  
K. Roper ◽  
M.J. Hannah ◽  
A. Hellwig ◽  
W.B. Huttner
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Mdck Cells ◽  
Apical Plasma Membrane ◽  
Membrane Domain ◽  
Membrane Protrusions ◽  
Selective Retention ◽  
Polytopic Membrane Protein ◽  
Plasma Membrane Domain

Prominin is a recently identified polytopic membrane protein expressed in various epithelial cells, where it is selectively associated with microvilli. When expressed in non-epithelial cells, prominin is enriched in plasma membrane protrusions. This raises the question of whether the selective association of prominin with microvilli in epithelial cells is solely due to its preference for, and stabilization in, plasma membrane protrusions, or is due to both sorting to the apical plasma membrane domain and subsequent enrichment in plasma membrane protrusions. To investigate this question, we have generated stably transfected MDCK cells expressing either full-length or C-terminally truncated forms of mouse prominin. Confocal immunofluorescence and domain-selective cell surface biotinylation experiments on transfected MDCK cells grown on permeable supports demonstrated the virtually exclusive apical localization of prominin at steady state. Pulse-chase experiments in combination with domain-selective cell surface biotinylation showed that newly synthesized prominin was directly targeted to the apical plasma membrane domain. Immunoelectron microscopy revealed that prominin was confined to microvilli rather than the planar region of the apical plasma membrane. Truncation of the cytoplasmic C-terminal tail of prominin impaired neither its apical cell surface expression nor its selective retention in microvilli. Both the apical-specific localization of prominin and its selective retention in microvilli were maintained when MDCK cells were cultured in low-calcium medium, i.e. in the absence of tight junctions. Taken together, our results show that: (i) prominin contains dual targeting information, for direct delivery to the apical plasma membrane domain and for the enrichment in the microvillar subdomain; and (ii) this dual targeting does not require the cytoplasmic C-terminal tail of prominin and still occurs in the absence of tight junctions. The latter observation suggests that entry into, and retention in, plasma membrane protrusions may play an important role in the establishment and maintenance of the apical-basal polarity of epithelial cells.

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

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 1057
Author(s):  
Richard Bouley ◽  
Naofumi Yui ◽  
Abby Terlouw ◽  
Pui W. Cheung ◽  
Dennis Brown
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Mdck Cells ◽  
Rat Kidney ◽  
Apical Plasma Membrane ◽  
Rhodamine Phalloidin ◽  
Renal Epithelial Cells ◽  
Aquaporin 2 ◽  
Basolateral Plasma Membrane

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.

scholarly journals Expression of macrophage-lymphocyte Fc receptors in Madin-Darby canine kidney cells: polarity and transcytosis differ for isoforms with or without coated pit localization domains.

1989 ◽  
Vol 109 (6) ◽  
pp. 3291-3302 ◽  
Author(s):  
W Hunziker ◽  
I Mellman
Keyword(s):  
Plasma Membrane ◽  
Mdck Cells ◽  
Apical Plasma Membrane ◽  
Madin Darby Canine Kidney ◽  
Coated Pits ◽  
Receptor Isoforms ◽  
Basolateral Plasma Membrane ◽  
Plasma Membrane Domain ◽  
Darby Canine Kidney ◽  
Canine Kidney

Many cells of the immune system and certain epithelia express receptors for the Fc domain of IgG (FcR). On mouse macrophages and lymphocytes, two distinct receptor isoforms have been identified, designated FcRII-B1 and FcRII-B2. The isoforms are identical except for an in-frame insertion of 47 amino acids in the cytoplasmic tail of FcRII-B1 that blocks its ability to be internalized by clathrin-coated pits. We have recently found that at least one IgG-transporting epithelium, namely placental syncytial trophoblasts, expresses transcripts encoding a receptor similar or identical to macrophage-lymphocyte FcRII. To determine whether FcRII of hematopoietic cells might also function as a transcytotic receptor if expressed in epithelial cells, FcRII-B1 and -B2 were transfected into Madin-Darby canine kidney (MDCK) cells and grown on permeable filter units. The two FcRII isoforms exhibited different patterns of polarized expression: FcRII-B1 was localized mainly to the apical plasma membrane domain, whereas FcRII-B2 was found predominantly on the basolateral surface. As expected for FcR in placenta, FcRII-B2 and to a lesser extent FcRII-B1 mediated transcellular transport of IgG-complexes from the apical to the basolateral plasma membrane. Neither receptor mediated transcytosis in the opposite direction, although FcRII-B2 also delivered ligand to lysosomes when internalized from either the basolateral or apical domains. Furthermore, FcRII-B2 was capable of transporting monovalent antireceptor antibody Fab fragments across the cell, suggesting that transcytosis was not dependent on receptor cross-linking. These findings suggest the possibility that FcRII can mediate transepithelial IgG transport when expressed in placental syncytial trophoblasts in addition to its "classical" endocytic and signaling activities when expressed in macrophages. Because FcRII-B1 and -B2 are expressed with distinct polarities, the results also suggest that interactions with clathrin-coated pits may play a role in generating the polarized distribution of at least some plasma membrane proteins in MDCK cells.

scholarly journals Trace Amine-Associated Receptor 1 Localization at the Apical Plasma Membrane Domain of Fisher Rat Thyroid Epithelial Cells Is Confined to Cilia

2015 ◽  
Vol 4 (1) ◽  
pp. 30-41 ◽  
Author(s):  
Joanna Szumska ◽  
Maria Qatato ◽  
Maren Rehders ◽  
Dagmar F�hrer ◽  
Heike Biebermann ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Apical Plasma Membrane ◽  
Membrane Domain ◽  
Trace Amine ◽  
Rat Thyroid ◽  
Plasma Membrane Domain

scholarly journals Segregation of Glucosylceramide and Sphingomyelin Occurs in the Apical to Basolateral Transcytotic Route in HepG2 Cells

1997 ◽  
Vol 137 (2) ◽  
pp. 347-357 ◽  
Author(s):  
Sven C.D. van IJzendoorn ◽  
Mirjam M.P. Zegers ◽  
Jan Willem Kok ◽  
Dick Hoekstra
Keyword(s):  
Plasma Membrane ◽  
Hepg2 Cells ◽  
Accurate Estimation ◽  
Apical Plasma Membrane ◽  
Membrane Domain ◽  
Dibutyryl Camp ◽  
Preferential Localization ◽  
Basolateral Plasma Membrane ◽  
Plasma Membrane Domain ◽  
Fluorescent Lipid

HepG2 cells are highly differentiated hepatoma cells that have retained an apical, bile canalicular (BC) plasma membrane polarity. We investigated the dynamics of two BC-associated sphingolipids, glucosylceramide (GlcCer) and sphingomyelin (SM). For this, the cells were labeled with fluorescent acyl chainlabeled 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)- amino]hexanoic acid (C6-NBD) derivatives of either GlcCer (C6-NBD-GlcCer) or SM (C6-NBD-SM). The pool of the fluorescent lipid analogues present in the basolateral plasma membrane domain was subsequently depleted and the apically located C6-NBD-lipid was chased at 37°C. By using fluorescence microscopical analysis and a new assay that allows an accurate estimation of the fluorescent lipid pool in the apical membrane, qualitative and quantitative insight was obtained concerning kinetics, extent and (intra)cellular sites of the redistribution of apically located C6-NBD-GlcCer and C6-NBD-SM. It is demonstrated that both lipids display a preferential localization, C6-NBD-GlcCer in the apical and C6-NBD-SM in the basolateral area. Such a preference is expressed during transcytosis of both sphingolipids from the apical to the basolateral plasma membrane domain, a novel lipid trafficking route in HepG2 cells. Whereas the vast majority of the apically derived C6-NBD-SM was rapidly transcytosed to the basolateral surface, most of the apically internalized C6-NBD-GlcCer was efficiently redirected to the BC. The redirection of C6-NBD-GlcCer did not involve trafficking via the Golgi apparatus. Evidence is provided which suggests the involvement of vesicular compartments, located subjacent to the apical plasma membrane. Interestingly, the observed difference in preferential localization of C6-NBD-GlcCer and C6NBD-SM was perturbed by treatment of the cells with dibutyryl cAMP, a stable cAMP analogue. While the preferential apical localization of C6-NBD-GlcCer was amplified, dibutyryl cAMP-treatment caused apically retrieved C6-NBD-SM to be processed via a similar pathway as that of C6-NBD-GlcCer. The data unambiguously demonstrate that segregation of GlcCer and SM occurs in the reverse transcytotic route, i.e., during apical to basolateral transport, which results in the preferential localization of GlcCer and SM in the apical and basolateral region of the cells, respectively. A role for non-Golgi–related, sub-apical vesicular compartments in the sorting of GlcCer and SM is proposed.

scholarly journals The Receptor Recycling Pathway Contains Two Distinct Populations of Early Endosomes with Different Sorting Functions

1999 ◽  
Vol 145 (1) ◽  
pp. 123-139 ◽  
Author(s):  
David R. Sheff ◽  
Elizabeth A. Daro ◽  
Michael Hull ◽  
Ira Mellman
Keyword(s):  
Plasma Membrane ◽  
Transferrin Receptor ◽  
Receptor Recycling ◽  
Membrane Domain ◽  
Recycling Endosomes ◽  
Early Endosomes ◽  
Basolateral Plasma Membrane ◽  
Recycling Pathway ◽  
Plasma Membrane Domain ◽  
Two Populations

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

A SCL4A10 gene product maps selectively to the basolateral plasma membrane of choroid plexus epithelial cells

2004 ◽  
Vol 286 (3) ◽  
pp. C601-C610 ◽  
Author(s):  
J. Praetorius ◽  
L. N. Nejsum ◽  
S. Nielsen
Keyword(s):  
Cerebrospinal Fluid ◽  
Plasma Membrane ◽  
Choroid Plexus ◽  
Gene Product ◽  
Pcr Analysis ◽  
Membrane Domain ◽  
Choroid Plexus Epithelium ◽  
Basolateral Plasma Membrane ◽  
Plasma Membrane Domain ◽  
The Brain

The choroid plexus epithelium of the brain ventricular system produces the majority of the cerebrospinal fluid and thereby defines the ionic composition of the interstitial fluid in the brain. The transepithelial movement of Na+ and water in the choroid plexus depend on a yet-unidentified basolateral stilbene-sensitive [Formula: see text]-[Formula: see text] uptake protein. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed the expression in the choroid plexus of SLC4A10 mRNA, which encodes a stilbene-sensitive [Formula: see text]-[Formula: see text] transporter. Anti-COOH-terminal antibodies were developed to determine the specific expression and localization of this [Formula: see text]-[Formula: see text] transport protein. Immunoblotting demonstrated antibody binding to a 180-kDa protein band from mouse and rat brain preparations enriched with choroid plexus. The immunoreactive band migrated as a 140-kDa protein after N-deglycosylation, consistent with the predicted molecular size of the SLC4A10 gene product. Bright-field immunohistochemistry and immunoelectron microscopy demonstrated strong labeling confined to the basolateral plasma membrane domain of the choroid plexus epithelium. Furthermore, the stilbene-insensitive [Formula: see text]-[Formula: see text] cotransporter, NBCn1, was also localized to the basolateral plasma membrane domain of the choroid plexus epithelium. Hence, we propose that the SLC4A10 gene product and NBCn1 both function as basolateral [Formula: see text] entry pathways and that the SLC4A10 gene product may be responsible for the stilbene-sensitive [Formula: see text]-[Formula: see text] uptake that is essential for cerebrospinal fluid production.

scholarly journals Dynamics of membrane-skeleton (fodrin) organization during development of polarity in Madin-Darby canine kidney epithelial cells.

1986 ◽  
Vol 103 (5) ◽  
pp. 1751-1765 ◽  
Author(s):  
W J Nelson ◽  
P J Veshnock
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Mdck Cells ◽  
Membrane Skeleton ◽  
Cell Contact ◽  
Cell Periphery ◽  
Madin Darby Canine Kidney ◽  
Basolateral Plasma Membrane ◽  
Darby Canine Kidney ◽  
Canine Kidney

Madin-Darby canine kidney (MDCK) epithelial cells exhibit a polarized distribution of membrane proteins between the apical and basolateral domains of the plasma membrane. We have initiated studies to investigate whether the spectrin-based membrane skeleton plays a role in the establishment and maintenance of these membrane domains. MDCK cells express an isoform of spectrin composed of two subunits, Mr 240,000 (alpha-subunit) and Mr 235,000 (gamma-subunit). This isoform is immunologically and structurally related to fodrin in lens and brain cells, which is a functional and structural analog of alpha beta-spectrin, the major component of the erythrocyte membrane skeleton. Analysis of fodrin in MDCK cells by immunoblotting, immunofluorescence, and metabolic labeling revealed significant changes in the biophysical properties, subcellular distribution, steady-state levels, and turnover of the protein during development of a continuous monolayer of cells. The changes in the cellular organization of fodrin did not appear to coincide with the distributions of microfilaments, microtubules, or intermediate filaments. These changes result in the formation of a highly insoluble, relatively dense and stable layer of fodrin which appears to be localized to the cell periphery and predominantly in the region of the basolateral plasma membrane of MDCK cells in continuous monolayers. The formation of this structure coincides temporally and spatially with extensive cell-cell contact, and with the development of the polarized distribution of the Na+, K+-ATPase, a marker protein of the basolateral plasma membrane.

scholarly journals A membrane-cytoskeletal complex containing Na+,K+-ATPase, ankyrin, and fodrin in Madin-Darby canine kidney (MDCK) cells: implications for the biogenesis of epithelial cell polarity.

1989 ◽  
Vol 108 (3) ◽  
pp. 893-902 ◽  
Author(s):  
W J Nelson ◽  
R W Hammerton
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Sucrose Gradient ◽  
Mdck Cells ◽  
Beta Subunits ◽  
Madin Darby Canine Kidney ◽  
Electrophoretic Mobilities ◽  
Basolateral Plasma Membrane ◽  
Darby Canine Kidney ◽  
Canine Kidney

In polarized Madin-Darby canine kidney (MDCK) epithelial cells, ankyrin, and the alpha- and beta-subunits of fodrin are components of the basolateral membrane-cytoskeleton and are colocalized with the Na+,K+-ATPase, a marker protein of the basolateral plasma membrane. Recently, we showed with purified proteins that the Na+,K+-ATPase is competent to bind ankyrin with high affinity and specificity (Nelson, W. J., and P. J. Veshnock. 1987. Nature (Lond.). 328:533-536). In the present study we have sought biochemical evidence for interactions between these proteins in MDCK cells. Proteins were solubilized from MDCK cells with an isotonic buffer containing Triton X-100 and fractionated rapidly in sucrose density gradients. Complexes of cosedimenting proteins were detected by analysis of sucrose gradient fractions in nondenaturing polyacrylamide gels. The results showed that ankyrin and fodrin cosedimented in sucrose gradient. Analysis of the proteins from the sucrose gradient in nondenaturing polyacrylamide gels revealed two distinct ankyrin:fodrin complexes that differed in their relative electrophoretic mobilities; both complexes had electrophoretic mobilities slower than that of purified spectrin heterotetramers. Parallel analysis of the distribution of solubilized Na+,K+-ATPase in sucrose gradients showed that there was a significant overlap with the distribution of ankyrin and fodrin. Analysis by nondenaturing polyacrylamide gel electrophoresis showed that the alpha- and beta-subunits of the Na+,K+-ATPase colocalized with the slower migrating of the two ankyrin:fodrin complexes. The faster migrating ankyrin:fodrin complex did not contain Na+,K+-ATPase. These results indicate strongly that the Na+,K+-ATPase, ankyrin, and fodrin are coextracted from whole MDCK cells as a protein complex. We suggest that the solubilized complex containing these proteins reflects the interaction of the Na+,K+-ATPase, ankyrin, and fodrin in the cell. This interaction may play an important role in the spatial organization of the Na+,K+-ATPase to the basolateral plasma membrane in polarized epithelial cells.

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