scholarly journals The WD40 protein Morg1 facilitates Par6–aPKC binding to Crb3 for apical identity in epithelial cells

2013 ◽  
Vol 200 (5) ◽  
pp. 635-650 ◽  
Author(s):  
Junya Hayase ◽  
Sachiko Kamakura ◽  
Yuko Iwakiri ◽  
Yoshihiro Yamaguchi ◽  
Tomoko Izaki ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Epithelial Cells ◽  
Tight Junction ◽  
Apical Membrane ◽  
Transmembrane Protein ◽  
Cyst Formation ◽  
Cell Polarization ◽  
Small Gtpase ◽  
Apical Surface ◽  
And Function

Formation of apico-basal polarity in epithelial cells is crucial for both morphogenesis (e.g., cyst formation) and function (e.g., tight junction development). Atypical protein kinase C (aPKC), complexed with Par6, is considered to translocate to the apical membrane and function in epithelial cell polarization. However, the mechanism for translocation of the Par6–aPKC complex has remained largely unknown. Here, we show that the WD40 protein Morg1 (mitogen-activated protein kinase organizer 1) directly binds to Par6 and thus facilitates apical targeting of Par6–aPKC in Madin-Darby canine kidney epithelial cells. Morg1 also interacts with the apical transmembrane protein Crumbs3 to promote Par6–aPKC binding to Crumbs3, which is reinforced with the apically localized small GTPase Cdc42. Depletion of Morg1 disrupted both tight junction development in monolayer culture and cyst formation in three-dimensional culture; apico-basal polarity was notably restored by forced targeting of aPKC to the apical surface. Thus, Par6–aPKC recruitment to the premature apical membrane appears to be required for definition of apical identity of epithelial cells.

scholarly journals Rab27 effector Slp2-a transports the apical signaling molecule podocalyxin to the apical surface of MDCK II cells and regulates claudin-2 expression

2012 ◽  
Vol 23 (16) ◽  
pp. 3229-3239 ◽  
Author(s):  
Takao Yasuda ◽  
Chika Saegusa ◽  
Sachiko Kamakura ◽  
Hideki Sumimoto ◽  
Mitsunori Fukuda
Keyword(s):  
Apical Membrane ◽  
Basolateral Membrane ◽  
Cell Polarization ◽  
Small Gtpase ◽  
Apical Surface ◽  
Dependent Manner ◽  
Signaling Molecule ◽  
Downstream Target ◽  
Junction Protein ◽  
Polarized Trafficking

Most cells in tissues are polarized and usually have two distinct plasma membrane domains—an apical membrane and a basolateral membrane, which are the result of polarized trafficking of proteins and lipids. However, the mechanism underlying the cell polarization is not fully understood. In this study, we investigated the involvement of synaptotagmin-like protein 2-a (Slp2-a), an effector molecule for the small GTPase Rab27, in polarized trafficking by using Madin–Darby canine kidney II cells as a model of polarized cells. The results show that the level of Slp2-a expression in MDCK II cells increases greatly as the cells become polarized and that its expression is specifically localized at the apical membrane. The results also reveal that Slp2-a is required for targeting of the signaling molecule podocalyxin to the apical membrane in a Rab27A-dependent manner. In addition, ezrin, a downstream target of podocalyxin, and ERK1/2 are activated in Slp2-a–knockdown cells, and their activation results in a dramatic reduction in the amount of the tight junction protein claudin-2. Because both Slp2-a and claudin-2 are highly expressed in mouse renal proximal tubules, Slp2-a is likely to regulate claudin-2 expression through trafficking of podocalyxin to the apical surface in mouse renal tubule epithelial cells.

Transport and function of syntaxin 3 in human epithelial intestinal cells

2000 ◽  
Vol 279 (4) ◽  
pp. C1239-C1248 ◽  
Author(s):  
Lionel Breuza ◽  
Jack Fransen ◽  
André Le Bivic
Keyword(s):  
Potential Role ◽  
Apical Membrane ◽  
Transmembrane Protein ◽  
Intestinal Cells ◽  
Apical Surface ◽  
Human Transferrin Receptor ◽  
Efficient Delivery ◽  
And Function ◽  
Syntaxin 3

To follow the transport of human syntaxin (Syn) 3 to the apical surface of intestinal cells, we produced and expressed in Caco-2 cells a chimera made of the entire Syn3 coding sequence and the extracellular domain of the human transferrin receptor (TfR). This chimera (Syn3TfR) was localized to the apical membrane and was transported along the direct apical pathway, suggesting that this is also the case for endogenous Syn3. To test the potential role of Syn3 in apical transport, we overexpressed it in Caco-2 cells and measured the efficiency of apical and basolateral delivery of several endogenous markers. We observed a strong inhibition of apical delivery of sucrase-isomaltase (SI), an apical transmembrane protein, and of α-glucosidase, an apically secreted protein. No effect was observed on the basolateral delivery of Ag525, a basolateral antigen, strongly suggesting that Syn3 is necessary for efficient delivery of proteins to the apical surface of intestinal cells.

scholarly journals Rab17 Regulates Membrane Trafficking through Apical Recycling Endosomes in Polarized Epithelial Cells

1998 ◽  
Vol 140 (5) ◽  
pp. 1039-1053 ◽  
Author(s):  
Paola Zacchi ◽  
Harald Stenmark ◽  
Robert G. Parton ◽  
Donata Orioli ◽  
Filip Lim ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Membrane Trafficking ◽  
Intracellular Transport ◽  
Cell Polarization ◽  
Small Gtpase ◽  
Recycling Endosome ◽  
Mutant Proteins ◽  
Confocal Immunofluorescence Microscopy ◽  
Polarized Epithelial Cells ◽  
And Function

A key feature of polarized epithelial cells is the ability to maintain the specific biochemical composition of the apical and basolateral plasma membrane domains while selectively allowing transport of proteins and lipids from one pole to the opposite by transcytosis. The small GTPase, rab17, a member of the rab family of regulators of intracellular transport, is specifically induced during cell polarization in the developing kidney. We here examined its intracellular distribution and function in both nonpolarized and polarized cells. By confocal immunofluorescence microscopy, rab17 colocalized with internalized transferrin in the perinuclear recycling endosome of BHK-21 cells. In polarized Eph4 cells, rab17 associated with the apical recycling endosome that has been implicated in recycling and transcytosis. The localization of rab17, therefore, strengthens the proposed homology between this compartment and the recycling endosome of nonpolarized cells. Basolateral to apical transport of two membrane-bound markers, the transferrin receptor and the FcLR 5-27 chimeric receptor, was specifically increased in Eph4 cells expressing rab17 mutants defective in either GTP binding or hydrolysis. Furthermore, the mutant proteins stimulated apical recycling of FcLR 5-27. These results support a role for rab17 in regulating traffic through the apical recycling endosome, suggesting a function in polarized sorting in epithelial cells.

scholarly journals The Small GTPase Rab13 Regulates Assembly of Functional Tight Junctions in Epithelial Cells

2002 ◽  
Vol 13 (6) ◽  
pp. 1819-1831 ◽  
Author(s):  
Anne-Marie Marzesco ◽  
Irene Dunia ◽  
Rudy Pandjaitan ◽  
Michel Recouvreur ◽  
Daniel Dauzonne ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Surface ◽  
Tight Junctions ◽  
Mdck Cells ◽  
Transmembrane Protein ◽  
Freeze Fracture ◽  
Small Gtpase ◽  
Cell Contact ◽  
Junctional Complexes ◽  
And Function

Junctional complexes such as tight junctions (TJ) and adherens junctions are required for maintaining cell surface asymmetry and polarized transport in epithelial cells. We have shown that Rab13 is recruited to junctional complexes from a cytosolic pool after cell–cell contact formation. In this study, we investigate the role of Rab13 in modulating TJ structure and functions in epithelial MDCK cells. We generate stable MDCK cell lines expressing inactive (T22N mutant) and constitutively active (Q67L mutant) Rab13 as GFP-Rab13 chimeras. Expression of GFP-Rab13Q67L delayed the formation of electrically tight epithelial monolayers as monitored by transepithelial electrical resistance (TER) and induced the leakage of small nonionic tracers from the apical domain. It also disrupted the TJ fence diffusion barrier. Freeze-fracture EM analysis revealed that tight junctional structures did not form a continuous belt but rather a discontinuous series of stranded clusters. Immunofluorescence studies showed that the expression of Rab13Q67L delayed the localization of the TJ transmembrane protein, claudin1, at the cell surface. In contrast, the inactive Rab13T22N mutant did not disrupt TJ functions, TJ strand architecture nor claudin1 localization. Our data revealed that Rab13 plays an important role in regulating both the structure and function of tight junctions.

CFTR channel insertion to the apical surface in rat duodenal villus epithelial cells is upregulated by VIP in vivo

1999 ◽  
Vol 112 (6) ◽  
pp. 887-894
Author(s):  
N.A. Ameen ◽  
B. Martensson ◽  
L. Bourguinon ◽  
C. Marino ◽  
J. Isenberg ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Apical Membrane ◽  
Surface Expression ◽  
Apical Plasma Membrane ◽  
Intracellular Camp ◽  
Apical Surface ◽  
C Terminus ◽  
Confocal Immunofluorescence ◽  
Small Intestinal Villus

cAMP activated insertion of the cystic fibrosis transmembrane conductance regulator (CFTR) channels from endosomes to the apical plasma membrane has been hypothesized to regulate surface expression and CFTR function although the physiologic relevance of this remains unclear. We previously identified a subpopulation of small intestinal villus epithelial cells or CFTR high expressor (CHE) cells possessing very high levels of apical membrane CFTR in association with a prominent subapical vesicular pool of CFTR. We have examined the subcellular redistribution of CFTR in duodenal CHE cells in vivo in response to the cAMP activated secretagogue vasoactive intestinal peptide (VIP). Using anti-CFTR antibodies against the C terminus of rodent CFTR and indirect immunofluorescence, we show by quantitative confocal microscopy that CFTR rapidly redistributes from the cytoplasm to the apical surface upon cAMP stimulation by VIP and returns to the cytoplasm upon removal of VIP stimulation of intracellular cAMP levels. Using ultrastructural and confocal immunofluorescence examination in the presence or absence of cycloheximide, we also show that redistribution was not dependent on new protein synthesis, changes in endocytosis, or rearrangement of the apical cytoskeleton. These observations suggest that physiologic cAMP activated apical membrane insertion and recycling of CFTR channels in normal CFTR expressing epithelia contributes to the in vivo regulation of CFTR mediated anion transport.

Na+-sugar cotransport system as a polarization marker during organization of epithelial membrane

1988 ◽  
Vol 255 (6) ◽  
pp. C745-C753 ◽  
Author(s):  
C. Lasheras ◽  
J. A. Scott ◽  
C. A. Rabito
Keyword(s):  
Electrical Resistance ◽  
Apical Membrane ◽  
Sugar Transport ◽  
Cell Polarization ◽  
Transepithelial Electrical Resistance ◽  
Apical Surface ◽  
Binding Studies ◽  
Occluding Junctions ◽  
Basolateral Side ◽  
Epithelial Membranes

The present study analyzed the changes in Na+-dependent sugar transport and transepithelial electrical resistance as LLC-PK1 cells reorganize into epithelial membranes. Sugar influx increased to reach a maximum 9 h after plating. The increase in the transepithelial electrical resistance, however, showed a significant delay, reaching steady state 15 h after plating. No changes in the electrochemical Na+ gradient were observed during the reorganization of the epithelial membranes. Kinetic analysis and [3H]phlorizin-binding studies showed that the increase in sugar influx resulted from an increase in the number of carriers. Unidirectional sugar influx measurements indicated that the sugar transporters were primarily located at the apical surface of the epithelial cells. These observations are consistent with the hypothesis that the sorting of native proteins occurs intracellularly before their insertion in the apical membrane, or as an alternative that they are randomly inserted, but then immediately sorted such as any carrier could be detected in the basolateral side during the reorganization process. In addition, the results suggest that the functional development of the apical membrane may occur before the complete sealing of the intercellular space during the development of the occluding junctions. Furthermore, development of the sugar transport system and occluding junctions was inhibited by cycloheximide and puromycin but not by actinomycin D, suggesting that the expression of epithelial cell polarization is probably a posttranslational event in the protein synthesis.

scholarly journals Move your microvilli

2014 ◽  
Vol 207 (1) ◽  
pp. 9-11 ◽  
Author(s):  
Robert S. Fischer
Keyword(s):  
Wound Healing ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Polarity ◽  
Apical Membrane ◽  
Apical Surface ◽  
Epithelial Cell Polarity ◽  
Cell Biol ◽  
Polarized Epithelial Cells

Polarized epithelial cells create tightly packed arrays of microvilli in their apical membrane, but the fate of these microvilli is relatively unknown when epithelial cell polarity is lost during wound healing. In this issue, Klingner et al. (2014. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201402037) show that, when epithelial cells become subconfluent, actomyosin contractions locally within the apical cortex cause their microvilli to become motile over the dorsal/apical surface. Their unexpected observations may have implications for epithelial responses in wound healing and disease.

scholarly journals Trafficking of Shigella Lipopolysaccharide in Polarized Intestinal Epithelial Cells

1999 ◽  
Vol 145 (4) ◽  
pp. 689-698 ◽  
Author(s):  
Wandy L. Beatty ◽  
Stéphane Méresse ◽  
Pierre Gounon ◽  
Jean Davoust ◽  
Joëlle Mounier ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Tight Junctions ◽  
Apical Membrane ◽  
Intestinal Epithelial Cells ◽  
Fluid Phase ◽  
Apical Surface ◽  
Intestinal Epithelial ◽  
Electron Microscopic ◽  
Basolateral Side ◽  
Endocytic Compartments

Bacterial lipopolysaccharide (LPS) at the apical surface of polarized intestinal epithelial cells was previously shown to be transported from the apical to the basolateral pole of the epithelium (Beatty, W.L., and P.J. Sansonetti. 1997. Infect. Immun. 65:4395–4404). The present study was designed to elucidate the transcytotic pathway of LPS and to characterize the endocytic compartments involved in this process. Confocal and electron microscopic analyses revealed that LPS internalized at the apical surface became rapidly distributed within endosomal compartments accessible to basolaterally internalized transferrin. This compartment largely excluded fluid-phase markers added at either pole. Access to the basolateral side of the epithelium subsequent to trafficking to basolateral endosomes occurred via exocytosis into the paracellular space beneath the intercellular tight junctions. LPS appeared to exploit other endocytic routes with much of the internalized LPS recycled to the original apical membrane. In addition, analysis of LPS in association with markers of the endocytic network revealed that some LPS was sent to late endosomal and lysosomal compartments.

scholarly journals Caenorhabditis elegans chaperonin CCT/TRiC is required for actin and tubulin biogenesis and microvillus formation in intestinal epithelial cells

2014 ◽  
Vol 25 (20) ◽  
pp. 3095-3104 ◽  
Author(s):  
Keiko Saegusa ◽  
Miyuki Sato ◽  
Katsuya Sato ◽  
Junko Nakajima-Shimada ◽  
Akihiro Harada ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Epithelial Cells ◽  
Apical Membrane ◽  
Intestinal Epithelial Cells ◽  
Intestinal Cells ◽  
Intermediate Filament Protein ◽  
Apical Surface ◽  
Intestinal Epithelial ◽  
Microtubule Network

Intestinal epithelial cells have unique apical membrane structures, known as microvilli, that contain bundles of actin microfilaments. In this study, we report that Caenorhabditis elegans cytosolic chaperonin containing TCP-1 (CCT) is essential for proper formation of microvilli in intestinal cells. In intestinal cells of cct-5(RNAi) animals, a substantial amount of actin is lost from the apical area, forming large aggregates in the cytoplasm, and the apical membrane is deformed into abnormal, bubble-like structures. The length of the intestinal microvilli is decreased in these animals. However, the overall actin protein levels remain relatively unchanged when CCT is depleted. We also found that CCT depletion causes a reduction in the tubulin levels and disorganization of the microtubule network. In contrast, the stability and localization of intermediate filament protein IFB-2, which forms a dense filamentous network underneath the apical surface, appears to be superficially normal in CCT-deficient cells, suggesting substrate specificity of CCT in the folding of filamentous cytoskeletons in vivo. Our findings demonstrate physiological functions of CCT in epithelial cell morphogenesis using whole animals.

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