scholarly journals Myosin VI is required for sorting of AP-1B–dependent cargo to the basolateral domain in polarized MDCK cells

2007 ◽  
Vol 177 (1) ◽  
pp. 103-114 ◽  
Author(s):  
Josephine Sui-Yan Au ◽  
Claudia Puri ◽  
Gudrun Ihrke ◽  
John Kendrick-Jones ◽  
Folma Buss
Keyword(s):  
Membrane Proteins ◽  
Epithelial Cells ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Adaptor Protein ◽  
Site Directed Mutagenesis ◽  
Myosin Vi ◽  
Tail Domain ◽  
Functional Complex ◽  
Polarized Epithelial Cells

In polarized epithelial cells, newly synthesized membrane proteins are delivered on specific pathways to either the apical or basolateral domains, depending on the sorting motifs present in these proteins. Because myosin VI has been shown to facilitate secretory traffic in nonpolarized cells, we investigated its role in biosynthetic trafficking pathways in polarized MDCK cells. We observed that a specific splice isoform of myosin VI with no insert in the tail domain is required for the polarized transport of tyrosine motif containing basolateral membrane proteins. Sorting of other basolateral or apical cargo, however, does not involve myosin VI. Site-directed mutagenesis indicates that a functional complex consisting of myosin VI, optineurin, and probably the GTPase Rab8 plays a role in the basolateral delivery of membrane proteins, whose sorting is mediated by the clathrin adaptor protein complex (AP) AP-1B. Our results suggest that myosin VI is a crucial component in the AP-1B–dependent biosynthetic sorting pathway to the basolateral surface in polarized epithelial cells.

Visualization of transcytosis in MDCK cells using laser scanning confocal microscopy

1994 ◽  
Vol 52 ◽  
pp. 220-221
Author(s):  
Greg Martin ◽  
Rohit Cariappa ◽  
Ann L. Hubbard
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Epithelial Cells ◽  
Laser Scanning ◽  
Mdck Cells ◽  
Transmembrane Protein ◽  
Basolateral Membrane ◽  
Apical Plasma Membrane ◽  
Plasma Membrane Proteins ◽  
Polarized Epithelial Cells

The plasma membrane of polarized epithelial cells is composed of two structurally and functionally distinct domains -- the apical and basolateral -- that also differ in molecular composition. The routes followed by integral membrane proteins from their site of synthesis to their site of function varies between different kinds of epithelia. Madin-Darby canine kidney (MDCK) cells deliver plasma membrane proteins directly to the correct domain, while polarized hepatocytes deliver all newly synthesized plasma membrane proteins initially to the basolateral membrane, then retrieve and redirect the apical membrane proteins. We are studying the targeting signals and delivery routes of DPPIV, a single transmembrane protein whose destination is the apical domain in polarized epithelial cells.DPPIV transfected into MDCK cells is delivered to the basolateral plasma membrane after long (13hr) treatment with Brefeldin A (BFA). After BFA’s removal these molecules are retrieved from the basolateral membrane and transcytosed to the apical plasma membrane. This protocol provides a useful model for studies of the indirect route of protein sorting in polarized epithelial cells, since DPPIV at the basolateral surface can be labeled with specific antibody and then subsequently followed in living cells.

scholarly journals Vesicular Stomatitis Virus Glycoprotein Does Not Determine the Site of Virus Release in Polarized Epithelial Cells

2002 ◽  
Vol 76 (8) ◽  
pp. 4103-4107 ◽  
Author(s):  
Gert Zimmer ◽  
Klaus-Peter Zimmer ◽  
Ina Trotz ◽  
Georg Herrler
Keyword(s):  
Epithelial Cells ◽  
Vesicular Stomatitis Virus ◽  
Plasma Membranes ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Vesicular Stomatitis Virus Glycoprotein ◽  
Recombinant Viruses ◽  
Virus Glycoprotein ◽  
Vesicular Stomatitis ◽  
Polarized Epithelial Cells

ABSTRACT In polarized epithelial cells, the vesicular stomatitis virus glycoprotein is segregated to the basolateral plasma membrane, where budding of the virus takes place. We have generated recombinant viruses expressing mutant glycoproteins without the basolateral-membrane-targeting signal in the cytoplasmic domain. Though about 50% of the mutant glycoproteins were found at the apical plasma membranes of infected MDCK cells, the virus was still predominantly released at the basolateral membranes, indicating that factors other than the glycoprotein determine the site of virus budding.

scholarly journals A dileucine motif in the COOH-terminal domain of NKCC1 targets the cotransporter to the plasma membrane

2019 ◽  
Vol 316 (4) ◽  
pp. C545-C558 ◽  
Author(s):  
Rainelli Koumangoye ◽  
Salma Omer ◽  
Eric Delpire
Keyword(s):  
Epithelial Cells ◽  
Basolateral Membrane ◽  
Site Directed Mutagenesis ◽  
Heterozygous Mutation ◽  
Terminal Domain ◽  
Late Endosomes ◽  
Dileucine Motif ◽  
Polarized Epithelial Cells ◽  
Epithelial Dysfunction ◽  
Golgi Network

Na+-K+-2Cl− cotransporter-1 (NKCC1) mediates the electroneutral transport of Na+, K+, and Cl− and is normally localized to the basolateral membrane of polarized epithelial cells. We recently reported the first known solute carrier family 12 member 2 ( SLC12A2) mutation (we call NKCC1-DFX) that causes epithelial dysfunction in an undiagnosed disease program case. The heterozygous mutation leads to truncation of the COOH-terminal tail of the cotransporter, resulting in both mutant and wild-type cotransporters being mistrafficked to the apical membrane of polarized epithelial cells. Here we demonstrate by using consecutive truncations and site-directed mutagenesis of the COOH-terminal domain of NKCC1 that truncation of NKCC1 COOH domain uncouples the cotransporter from the lateral membrane. We identify a dileucine motif that, when mutated, leads to cotransporter accumulation in the cytoplasm and mistrafficking to the apical/subapical region of epithelial cells, thereby recapitulating the phenotype observed with the patient mutation. We show that truncation deletion and LL substitution mutants are trafficked out of the endoplasmic reticulum and trans-Golgi network but accumulate in early and late endosomes where they are degraded.

scholarly journals Rab8 Regulates Basolateral Secretory, But Not Recycling, Traffic at the Recycling Endosome

2008 ◽  
Vol 19 (5) ◽  
pp. 2059-2068 ◽  
Author(s):  
Lauren Henry ◽  
David R. Sheff
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Mdck Cells ◽  
Apical Surface ◽  
Recycling Endosome ◽  
Recycling Pathway ◽  
Polarized Epithelial Cells ◽  
Pass Through

Rab8 is a monomeric GTPase that regulates the delivery of newly synthesized proteins to the basolateral surface in polarized epithelial cells. Recent publications have demonstrated that basolateral proteins interacting with the μ1-B clathrin adapter subunit pass through the recycling endosome (RE) en route from the TGN to the plasma membrane. Because Rab8 interacts with these basolateral proteins, these findings raise the question of whether Rab8 acts before, at, or after the RE. We find that Rab8 overexpression during the formation of polarity in MDCK cells, disrupts polarization of the cell, explaining how Rab8 mutants can disrupt basolateral endocytic and secretory traffic. However, once cells are polarized, Rab8 mutants cause mis-sorting of newly synthesized basolateral proteins such as VSV-G to the apical surface, but do not cause mis-sorting of membrane proteins already at the cell surface or in the endocytic recycling pathway. Enzymatic ablation of the RE also prevents traffic from the TGN from reaching the RE and similarly results in mis-sorting of newly synthesized VSV-G. We conclude that Rab8 regulates biosynthetic traffic through REs to the plasma membrane, but not trafficking of endocytic cargo through the RE. The data are consistent with a model in which Rab8 functions in regulating the delivery of TGN-derived cargo to REs.

scholarly journals Membrane proteins follow multiple pathways to the basolateral cell surface in polarized epithelial cells

2009 ◽  
Vol 186 (2) ◽  
pp. 269-282 ◽  
Author(s):  
Glen A. Farr ◽  
Michael Hull ◽  
Ira Mellman ◽  
Michael J. Caplan
Keyword(s):  
Membrane Proteins ◽  
Epithelial Cells ◽  
Basolateral Membrane ◽  
Small Gtpases ◽  
Recycling Endosomes ◽  
Multiple Routes ◽  
Sorting Process ◽  
Polarized Epithelial Cells ◽  
Pass Through ◽  
Golgi Network

Newly synthesized apical and basolateral membrane proteins are sorted from one another in polarized epithelial cells. The trans-Golgi network participates in this sorting process, but some basolateral proteins travel from the Golgi to recycling endosomes (REs) before their surface delivery. Using a novel system for pulse–chase microscopy, we have visualized the postsynthetic route pursued by a newly synthesized cohort of Na,K-ATPase. We find that the basolateral delivery of newly synthesized Na,K-ATPase occurs via a pathway distinct from that pursued by the vesicular stomatitis virus G protein (VSV-G). Na,K-ATPase surface delivery occurs at a faster rate than that observed for VSV-G. The Na,K-ATPase does not pass through the RE compartment en route to the plasma membrane, and Na,K-ATPase trafficking is not regulated by the same small GTPases as other basolateral proteins. Finally, Na,K-ATPase and VSV-G travel in separate post-Golgi transport intermediates, demonstrating directly that multiple routes exist for transport from the Golgi to the basolateral membrane in polarized epithelial cells.

scholarly journals The M protein of vesicular stomatitis virus associates specifically with the basolateral membranes of polarized epithelial cells independently of the G protein.

1988 ◽  
Vol 107 (5) ◽  
pp. 1707-1715 ◽  
Author(s):  
J E Bergmann ◽  
P J Fusco
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
M Protein ◽  
Infected Cells ◽  
Vesicular Stomatitis ◽  
Polarized Epithelial Cells

Using monoclonal antibodies and indirect immunofluorescence microscopy, we investigated the distribution of the M protein in situ in vesicular stomatitis virus-(VSV) infected MDCK cells. M protein was observed free in the cytoplasm and associated with the plasma membrane. Using the ts045 mutant of VSV to uncouple the synthesis and transport of the VSV G protein we demonstrated that this distribution was not related to the presence of G protein on the cell surface. Sections of epon-embedded infected cells labeled with antibody to the M protein and processed for indirect horseradish peroxidase immunocytochemistry revealed that the M protein was associated specifically with the basolateral plasma membrane. The G and M proteins of VSV have therefore evolved features which bring them independently to the basolateral membrane of polarized epithelial cells and allow virus to bud specifically from that membrane.

scholarly journals Naked2 Acts as a Cargo Recognition and Targeting Protein to Ensure Proper Delivery and Fusion of TGF-α–containing Exocytic Vesicles at the Lower Lateral Membrane of Polarized MDCK Cells

2007 ◽  
Vol 18 (8) ◽  
pp. 3081-3093 ◽  
Author(s):  
Cunxi Li ◽  
Mingming Hao ◽  
Zheng Cao ◽  
Wei Ding ◽  
Ramona Graves-Deal ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Surface ◽  
Egf Receptor ◽  
Transforming Growth Factor ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Distinct Region ◽  
Lateral Membrane ◽  
Polarized Epithelial Cells

Transforming growth factor-α (TGF-α) is the major autocrine EGF receptor ligand in vivo. In polarized epithelial cells, proTGF-α is synthesized and then delivered to the basolateral cell surface. We previously reported that Naked2 interacts with basolateral sorting determinants in the cytoplasmic tail of a Golgi-processed form of TGF-α and that TGF-α is not detected at the basolateral surface of Madin-Darby canine kidney (MDCK) cells expressing myristoylation-deficient (G2A) Naked2. By high-resolution microscopy, we now show that wild-type, but not G2A, Naked2-associated vesicles fuse at the plasma membrane. We further demonstrate that Naked2-associated vesicles are delivered to the lower lateral membrane of polarized MDCK cells independent of μ1B adaptin. We identify a basolateral targeting segment within Naked2; residues 1-173 redirect NHERF-1 from the apical cytoplasm to the basolateral membrane, and internal deletion of residues 37-104 results in apical mislocalization of Naked2 and TGF-α. Short hairpin RNA knockdown of Naked2 leads to a dramatic reduction in the 16-kDa cell surface isoform of TGF-α and increased cytosolic TGF-α immunoreactivity. We propose that Naked2 acts as a cargo recognition and targeting (CaRT) protein to ensure proper delivery, tethering, and fusion of TGF-α–containing vesicles to a distinct region at the basolateral surface of polarized epithelial cells.

scholarly journals JAM-A interacts in cis with α3β1 integrin through CD151 to regulate collective cell migration of polarized epithelial cells

2021 ◽  
Author(s):  
Sonja Thölmann ◽  
Jochen Seebach ◽  
Tetsuhisa Otani ◽  
Luise Florin ◽  
Hans Schnittler ◽  
...  
Keyword(s):  
Cell Migration ◽  
Epithelial Cells ◽  
Mdck Cells ◽  
Collagen I ◽  
Cell Contact ◽  
Collective Cell Migration ◽  
Α3β1 Integrin ◽  
Barrier Formation ◽  
Functional Complex ◽  
Polarized Epithelial Cells

Abstract Junctional adhesion molecule (JAM)-A is a cell adhesion receptor localized at epithelial cellcell contacts with enrichment at the tight junctions. Its role during cell-cell contact formation and epithelial barrier formation has intensively been studied. In contrast, its role during collective cell migration is largely unexplored. Here we show that JAM-A regulates collective cell migration of polarized epithelial cells. Depletion of JAM-A in MDCK cells enhances the motility of singly migrating cells but reduces cell motility of cells embedded in a collective by impairing the dynamics of cryptic lamellipodia formation. This activity of JAM-A is observed in cells grown on laminin and collagen I but not on fibronectin or vitronectin. Accordingly, we find that JAM-A exists in a complex with the laminin- and collagen I-binding α3β1 integrin. We also find that JAM-A interacts with CD151, a tetraspanin that forms a stoichiometric complex with α3β1 integrin and that regulates α3β1 integrin activity in different contexts. Mapping experiments indicate that JAM-A associates with both α3β1 integrin and CD151 through its extracellular domain. Similar to depletion of JAM-A, depletion of either α3β1 integrin or CD151 in MDCK cells slows down collective cell migration. Our findings suggest that JAM-A, α3β1 integrin and CD151 exist as a functional complex to regulate collective cell migration of epithelial cells.

scholarly journals Targeting of Transmembrane Protein Shrew-1 to Adherens Junctions Is Controlled by Cytoplasmic Sorting Motifs

2006 ◽  
Vol 17 (8) ◽  
pp. 3397-3408 ◽  
Author(s):  
Viktor Jakob ◽  
Alexander Schreiner ◽  
Ritva Tikkanen ◽  
Anna Starzinski-Powitz
Keyword(s):  
Amino Acids ◽  
Epithelial Cells ◽  
Mutational Analysis ◽  
Transmembrane Protein ◽  
Adherens Junctions ◽  
Basolateral Membrane ◽  
Adaptor Protein ◽  
Sorting Signals ◽  
A Subunit ◽  
Polarized Epithelial Cells

We recently identified transmembrane protein shrew-1 and showed that it is able to target to adherens junctions in polarized epithelial cells. This suggested shrew-1 possesses specific basolateral sorting motifs, which we analyzed by mutational analysis. Systematic mutation of amino acids in putative sorting signals in the cytoplasmic domain of shrew-1 revealed three tyrosines and a dileucine motif necessary for basolateral sorting. Substitution of these amino acids leads to apical localization of shrew-1. By applying tannic acid to either the apical or basolateral part of polarized epithelial cells, thereby blocking vesicle fusion with the plasma membrane, we obtained evidence that the apically localized mutants were primarily targeted to the basolateral membrane and were then redistributed to the apical domain. Further support for a postendocytic sorting mechanism of shrew-1 was obtained by demonstrating that μ1B, a subunit of the epithelial cell-specific adaptor complex AP-1B, interacts with shrew-1. In conclusion, our data provide evidence for a scenario where shrew-1 is primarily delivered to the basolateral membrane by a so far unknown mechanism. Once there, adaptor protein complex AP-1B is involved in retaining shrew-1 at the basolateral membrane by postendocytic sorting mechanisms.

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