scholarly journals The MAGUK Protein MPP7 Binds to the Polarity Protein hDlg1 and Facilitates Epithelial Tight Junction Formation

2007 ◽  
Vol 18 (5) ◽  
pp. 1744-1755 ◽  
Author(s):  
Volker M. Stucke ◽  
Evy Timmerman ◽  
Joel Vandekerckhove ◽  
Kris Gevaert ◽  
Alan Hall
Keyword(s):  
Epithelial Cell ◽  
Tight Junction ◽  
Tight Junctions ◽  
Cell Polarity ◽  
Functional Organization ◽  
Epithelial Cell Polarity ◽  
Tight Junction Formation ◽  
Evolutionarily Conserved ◽  
Junction Formation ◽  
Epithelial Tight Junction

Three groups of evolutionarily conserved proteins have been implicated in the establishment of epithelial cell polarity: the apically-localized proteins of the Par (Par3-Par6-aPKC-Cdc42) and Crumbs groups (Crb3-PALS1-PATJ) and the basolaterally localized proteins of the Dlg group (Dlg1-Scribble-Lgl). During epithelial morphogenesis, these proteins participate in a complex network of interdependent interactions that define the position and functional organization of adherens junctions and tight junctions. However, the biochemical pathways through which they control polarity are poorly understood. In this study, we identify an interaction between endogenous hDlg1 and MPP7, a previously uncharacterized MAGUK-p55 subfamily member. We find that MPP7 targets to the lateral surface of epithelial cells via its L27N domain, through an interaction with hDlg1. Loss of either hDlg1 or MPP7 from epithelial Caco-2 cells results in a significant defect in the assembly and maintenance of functional tight junctions. We conclude that the formation of a complex between hDlg1 and MPP7 promotes epithelial cell polarity and tight junction formation.

scholarly journals Shank2 Binds to aPKC and Controls Tight Junction Formation with Rap1 Signaling during Establishment of Epithelial Cell Polarity

Cell Reports ◽  
2020 ◽  
Vol 31 (1) ◽  
pp. 107407
Author(s):  
Kazunori Sasaki ◽  
Noriko Kojitani ◽  
Hiroko Hirose ◽  
Yohei Yoshihama ◽  
Hidefumi Suzuki ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Tight Junction ◽  
Cell Polarity ◽  
Epithelial Cell Polarity ◽  
Tight Junction Formation ◽  
Junction Formation

Involvement of ASIP/PAR-3 in the promotion of epithelial tight junction formation

2002 ◽  
Vol 115 (12) ◽  
pp. 2485-2495 ◽  
Author(s):  
Tomonori Hirose ◽  
Yasushi Izumi ◽  
Yoji Nagashima ◽  
Yoko Tamai-Nagai ◽  
Hidetake Kurihara ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Tight Junction ◽  
Tight Junctions ◽  
Mdck Cells ◽  
C Elegans ◽  
Tight Junction Formation ◽  
Junction Formation ◽  
Epithelial Tight Junction ◽  
Binding Sequence ◽  
Cell Cell

The mammalian protein ASIP/PAR-3 interacts with atypical protein kinase C isotypes (aPKC) and shows overall sequence similarity to the invertebrate proteins C. elegans PAR-3 and Drosophila Bazooka, which are crucial for the establishment of polarity in various cells. The physical interaction between ASIP/PAR-3 and aPKC is also conserved in C. elegans PAR-3 and PKC-3 and in Drosophila Bazooka and DaPKC. In mammals, ASIP/PAR-3 colocalizes with aPKC and concentrates at the tight junctions of epithelial cells, but the biological meaning of ASIP/PAR-3 in tight junctions remains to be clarified. In the present study, we show that ASIP/PAR-3 staining distributes to the subapical domain of epithelial cell-cell junctions, including epithelial cells with less-developed tight junctions, in clear contrast with ZO-1, another tight-junction-associated protein, the staining of which is stronger in cells with well-developed tight junctions. Consistently, immunogold electron microscopy revealed that ASIP/PAR-3 concentrates at the apical edge of tight junctions, whereas ZO-1 distributes alongside tight junctions. To clarify the meaning of this characteristic localization of ASIP, we analyzed the effects of overexpressed ASIP/PAR-3 on tight junction formation in cultured epithelial MDCK cells. The induced overexpression of ASIP/PAR-3, but not its deletion mutant lacking the aPKC-binding sequence, promotes cell-cell contact-induced tight junction formation in MDCK cells when evaluated on the basis of transepithelial electrical resistance and occludin insolubilization. The significance of the aPKC-binding sequence in tight junction formation is also supported by the finding that the conserved PKC-phosphorylation site within this sequence,ASIP-Ser827, is phosphorylated at the most apical tip of cell-cell contacts during the initial phase of tight junction formation in MDCK cells. Together,our present data suggest that ASIP/PAR-3 regulates epithelial tight junction formation positively through interaction with aPKC.

Effect of temperature on the assembly of tight junctions and on the mobility of lipids in membranes of HT29 cells

1990 ◽  
Vol 97 (1) ◽  
pp. 119-125
Author(s):  
E. Cohen ◽  
I. Ophir ◽  
Y.I. Henis ◽  
A. Bacher ◽  
Y. Ben Shaul
Keyword(s):  
Plasma Membrane ◽  
Tight Junction ◽  
Temperature Dependence ◽  
Tight Junctions ◽  
Membrane Lipids ◽  
Effect Of Temperature ◽  
Proteolytic Activation ◽  
Tight Junction Formation ◽  
Threshold Temperatures ◽  
Junction Formation

In the human colon adenocarcinoma cell line HT29, tight junctions can be induced by treatment with appropriate proteases or salt solutions. The temperature dependence of induced tight junction formation is characterized by a marked sigmoidal behavior. The different methods of induction used in this study were characterized by threshold temperatures ranging from 15 to 32 degrees C. Fluorescence photobleaching recovery measurements of the lateral diffusion of a fluorescent phospholipid probe in the cellular plasma membrane gave no evidence for a phase transition or for alteration in the organization of membrane lipids in lateral domains in the temperature range between 0 and 37 degrees C. Moreover, dynamic parameters of the probe in the plasma membrane did not change substantially on mild treatment with trypsin. Thus, the temperature dependence of tight junction formation is not dictated by the bulk properties of the cytoplasmic membrane lipids. The observed temperature dependence suggests that the assembly of tight junctions is a cooperative process, which may involve conformational rearrangement in a protein precursor subsequent to its proteolytic activation.

Epithelial cell polarity: what flies can teach us about cancer

2012 ◽  
Vol 53 ◽  
pp. 129-140 ◽  
Author(s):  
Daniel T. Bergstralh ◽  
Daniel St Johnston
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Cell Polarity ◽  
Human Cancer ◽  
Fruit Fly ◽  
Model Organisms ◽  
Epithelial Cell Polarity ◽  
Energetic Stress ◽  
Evolutionarily Conserved ◽  
Cellular Machinery

Epithelial cells are polarized along their apical–basal axis. Much of the cellular machinery that goes into establishing and maintaining epithelial cell polarity is evolutionarily conserved. Model organisms, including the fruit fly, Drosophila melanogaster, are thus particularly useful for the study of cell polarity. Work in Drosophila has identified several important components of the polarity machinery and has also established the surprising existence of a secondary cell polarity pathway required only under conditions of energetic stress. This work has important implications for the understanding of human cancer. Most cancers are epithelial in origin, and the loss of cell polarity is a critical step towards malignancy. Thus a better understanding of how polarity is established and maintained in epithelial cells will help us to understand the process of malignant transformation and may lead to improved therapies. In the present chapter we discuss the current understanding of how epithelial cell polarity is regulated and the known associations between polarity factors and cancer.

scholarly journals Ric-8A, an activator protein of Gαi, controls mammalian epithelial cell polarity for tight junction assembly and cystogenesis

2017 ◽  
Vol 22 (3) ◽  
pp. 293-309 ◽  
Author(s):  
Kanako Chishiki ◽  
Sachiko Kamakura ◽  
Junya Hayase ◽  
Hideki Sumimoto
Keyword(s):  
Epithelial Cell ◽  
Tight Junction ◽  
Cell Polarity ◽  
Epithelial Cell Polarity ◽  
Activator Protein

scholarly journals Possible Involvement of Phosphorylation of Occludin in Tight Junction Formation

1997 ◽  
Vol 137 (6) ◽  
pp. 1393-1401 ◽  
Author(s):  
Akira Sakakibara ◽  
Mikio Furuse ◽  
Mitinori Saitou ◽  
Yuhko Ando-Akatsuka ◽  
Shoichiro Tsukita
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Immunofluorescence Microscopy ◽  
Insoluble Fraction ◽  
Band Shift ◽  
Basolateral Membranes ◽  
Tight Junction Formation ◽  
Phosphorylated Form ◽  
Upward Shift ◽  
Junction Formation

Occludin is an integral membrane protein localizing at tight junctions in epithelial and endothelial cells. Occludin from confluent culture MDCK I cells resolved as several (>10) bands between 62 and 82 kD in SDS-PAGE, of which two or three bands of the lowest Mr were predominant. Among these bands, the lower predominant bands were essentially extracted with 1% NP-40, whereas the other higher Mr bands were selectively recovered in the NP-40–insoluble fraction. Alkaline phosphatase treatment converged these bands of occludin both in NP-40–soluble and -insoluble fractions into the lowest Mr band, and phosphoamino acid analyses identified phosphoserine (and phosphothreonine weakly) in the higher Mr bands of occludin. These findings indicated that phosphorylation causes an upward shift of occludin bands and that highly phosphorylated occludin resists NP-40 extraction. When cells were grown in low Ca medium, almost all occludin was NP-40 soluble. Switching from low to normal Ca medium increased the amount of NP-40–insoluble occludin within 10 min, followed by gradual upward shift of bands. This insolubilization and the band shift correlated temporally with tight junction formation detected by immunofluorescence microscopy. Furthermore, we found that the anti–chicken occludin mAb, Oc-3, did not recognize the predominant lower Mr bands of occludin (non- or less phosphorylated form) but was specific to the higher Mr bands (phosphorylated form) on immunoblotting. Immunofluorescence microscopy revealed that this mAb mainly stained the tight junction proper of intestinal epithelial cells, whereas other anti-occludin mAbs, which can recognize the predominant lower Mr bands, labeled their basolateral membranes (and the cytoplasm) as well as tight junctions. Therefore, we conclude that non- or less phosphorylated occludin is distributed on the basolateral membranes and that highly phosphorylated occludin is selectively concentrated at tight juctions as the NP-40–insoluble form. These findings suggest that the phosphorylation of occludin is a key step in tight junction assembly.

scholarly journals PALS1 Regulates E-Cadherin Trafficking in Mammalian Epithelial Cells

2007 ◽  
Vol 18 (3) ◽  
pp. 874-885 ◽  
Author(s):  
Qian Wang ◽  
Xiao-Wei Chen ◽  
Ben Margolis
Keyword(s):  
Tight Junction ◽  
Adherens Junction ◽  
Cell Periphery ◽  
Madin Darby Canine Kidney ◽  
Tight Junction Formation ◽  
Junction Protein ◽  
Evolutionarily Conserved ◽  
Junction Formation ◽  
Darby Canine Kidney ◽  
E Cadherin

Protein Associated with Lin Seven 1 (PALS1) is an evolutionarily conserved scaffold protein that targets to the tight junction in mammalian epithelia. Prior work in our laboratory demonstrated that the knockdown of PALS1 in Madin Darby canine kidney cells leads to tight junction and polarity defects. We have created new PALS1 stable knockdown cell lines with more profound reduction of PALS1 expression, and a more severe defect in tight junction formation was observed. Unexpectedly, we also observed a severe adherens junction defect, and both defects were corrected when PALS1 wild type and certain PALS1 mutants were expressed in the knockdown cells. We found that the adherens junction structural component E-cadherin was not effectively delivered to the cell surface in the PALS1 knockdown cells, and E-cadherin puncta accumulated in the cell periphery. The exocyst complex was also found to be mislocalized in PALS1 knockdown cells, potentially explaining why E-cadherin trafficking is disrupted. Our results suggest a broad and evolutionarily conserved role for the tight junction protein PALS1 in the biogenesis of adherens junction.

A spectroscopic method for assessing confluence of epithelial cell cultures

1991 ◽  
Vol 261 (6) ◽  
pp. C1196-C1203 ◽  
Author(s):  
B. Jovov ◽  
N. K. Wills ◽  
S. A. Lewis
Keyword(s):  
Epithelial Cell ◽  
Tight Junction ◽  
Culture Media ◽  
Spectroscopic Method ◽  
Isosbestic Point ◽  
Epithelial Cell Line ◽  
Phenol Red ◽  
Renal Epithelial Cell ◽  
Tight Junction Formation ◽  
Junction Formation

We describe a convenient nonelectrophysiological technique for assessing cell proliferation and subsequent tight junction formation for epithelial monolayers grown on permeable supports. The method involves the use of phenol red (PR), a standard pH indicator in most cell culture media. In addition, we report a systematic error in a commercially available system for measuring transepithelial electrical properties. Briefly, the flux of PR across the epithelium was measured from the serosal solution into the mucosal solution. The mucosal solution was first replaced with a PR-free solution and then collected at timed intervals. The PR concentration was measured using a spectrophotometer set at the isosbestic point for PR (479 nm). PR flux was then calculated and used as an index of the permeability of the epithelium to PR. This method was tested using the renal epithelial cell line A6. After cell seeding, PR flux decreased in two phases: an initial large decrease, associated with cell growth and monolayer confluence, and a second decrease associated with tight junction formation [assessed by measuring transepithelial conductance (Gt)]. In addition to monitoring tight junction formation, PR flux measurements were also used to estimate the net movement of solution by the epithelial cells between the mucosal and serosal compartments. For convenience, Gt was initially measured in culture dishes using a commercially available “chopstick” electrode system. However, the chopstick system yielded Gt values that were on average 51% lower than values for the same preparations when measured in standard Ussing-type chambers. The discrepancy was due to a nonuniform current field produced by the chopstick electrodes.

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