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PATJ regulates tight junction formation and polarity in mammalian epithelial cells.

Shin K, Straight S, Margolis B - J. Cell Biol. (2005)

Bottom Line: We show using RNAi techniques that reduction in PATJ expression leads to delayed tight junction formation as well as defects in cell polarization.These effects are reversed by reintroduction of PATJ into these RNAi cells.This study provides new functional information on PATJ as a polarity protein and increases our understanding of the Crumbs-PALS1-PATJ complex function in epithelial polarity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109, USA.

ABSTRACT
Recent studies have revealed an important role for tight junction protein complexes in epithelial cell polarity. One of these complexes contains the apical transmembrane protein, Crumbs, and two PSD95/discs large/zonula occludens domain proteins, protein associated with Lin seven 1 (PALS1)/Stardust and PALS1-associated tight junction protein (PATJ). Although Crumbs and PALS1/Stardust are known to be important for cell polarization, recent studies have suggested that Drosophila PATJ is not essential and its function is unclear. Here, we find that PATJ is targeted to the apical region and tight junctions once cell polarization is initiated. We show using RNAi techniques that reduction in PATJ expression leads to delayed tight junction formation as well as defects in cell polarization. These effects are reversed by reintroduction of PATJ into these RNAi cells. This study provides new functional information on PATJ as a polarity protein and increases our understanding of the Crumbs-PALS1-PATJ complex function in epithelial polarity.

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Localization of PATJ during cell polarization. (A) Wild-type MDCKII cells grown in low calcium media were fixed, permeabilized, and immunostained for PATJ (green, left) and a marker protein for VACs, GP135 (red, right). (B–F) Wild-type MDCKII cells were subject to the calcium switch experiment at different time course (B, 0 min; C, 30 min; D, 90 min; E, 3 h; F, 6 h) followed by immunostaining for PATJ (green), E-cadherins (red), and ZO1 (blue). All panels are Z section images from confocal microscopy. Bars, 20 μm.
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fig1: Localization of PATJ during cell polarization. (A) Wild-type MDCKII cells grown in low calcium media were fixed, permeabilized, and immunostained for PATJ (green, left) and a marker protein for VACs, GP135 (red, right). (B–F) Wild-type MDCKII cells were subject to the calcium switch experiment at different time course (B, 0 min; C, 30 min; D, 90 min; E, 3 h; F, 6 h) followed by immunostaining for PATJ (green), E-cadherins (red), and ZO1 (blue). All panels are Z section images from confocal microscopy. Bars, 20 μm.

Mentions: To understand the role of PATJ in epithelial cells, we first examined the movement of PATJ in wild-type MDCKII cells during polarization. In low calcium medium, MDCK cells lost polarization and PATJ appeared to concentrate in the VAC where it colocalized with GP135 but not ZO1 (Fig. 1, A and B; Low et al., 2000; Ivanov et al., 2004). We examined the movement of PATJ to the tight junction during polarization. We fixed cellular monolayers that were in calcium-free media (Fig. 1 B), then 30 min (Fig. 1 C), 90 min (Fig. 1 D), 3 h (Fig. 1 E), and 6 h (Fig. 1 F) after readdition of calcium to reinitiate polarization. We stained the monolayers for PATJ, E-cadherin, and ZO1. We confirmed as previously reported that ZO1 is initially recruited to sites of cell–cell adhesion with E-cadherin (Yonemura et al., 1995). However, in contrast, we were surprised to find that PATJ moves to the apical region at the early stage of polarization away from the site of cell–cell contacts (Fig. 1 C). After this initial apically directed movement, PATJ begins to localize at the tight junction (Fig. 1 D) and finally is concentrated in this junction (Fig. 1, E and F).


PATJ regulates tight junction formation and polarity in mammalian epithelial cells.

Shin K, Straight S, Margolis B - J. Cell Biol. (2005)

Localization of PATJ during cell polarization. (A) Wild-type MDCKII cells grown in low calcium media were fixed, permeabilized, and immunostained for PATJ (green, left) and a marker protein for VACs, GP135 (red, right). (B–F) Wild-type MDCKII cells were subject to the calcium switch experiment at different time course (B, 0 min; C, 30 min; D, 90 min; E, 3 h; F, 6 h) followed by immunostaining for PATJ (green), E-cadherins (red), and ZO1 (blue). All panels are Z section images from confocal microscopy. Bars, 20 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171825&req=5

fig1: Localization of PATJ during cell polarization. (A) Wild-type MDCKII cells grown in low calcium media were fixed, permeabilized, and immunostained for PATJ (green, left) and a marker protein for VACs, GP135 (red, right). (B–F) Wild-type MDCKII cells were subject to the calcium switch experiment at different time course (B, 0 min; C, 30 min; D, 90 min; E, 3 h; F, 6 h) followed by immunostaining for PATJ (green), E-cadherins (red), and ZO1 (blue). All panels are Z section images from confocal microscopy. Bars, 20 μm.
Mentions: To understand the role of PATJ in epithelial cells, we first examined the movement of PATJ in wild-type MDCKII cells during polarization. In low calcium medium, MDCK cells lost polarization and PATJ appeared to concentrate in the VAC where it colocalized with GP135 but not ZO1 (Fig. 1, A and B; Low et al., 2000; Ivanov et al., 2004). We examined the movement of PATJ to the tight junction during polarization. We fixed cellular monolayers that were in calcium-free media (Fig. 1 B), then 30 min (Fig. 1 C), 90 min (Fig. 1 D), 3 h (Fig. 1 E), and 6 h (Fig. 1 F) after readdition of calcium to reinitiate polarization. We stained the monolayers for PATJ, E-cadherin, and ZO1. We confirmed as previously reported that ZO1 is initially recruited to sites of cell–cell adhesion with E-cadherin (Yonemura et al., 1995). However, in contrast, we were surprised to find that PATJ moves to the apical region at the early stage of polarization away from the site of cell–cell contacts (Fig. 1 C). After this initial apically directed movement, PATJ begins to localize at the tight junction (Fig. 1 D) and finally is concentrated in this junction (Fig. 1, E and F).

Bottom Line: We show using RNAi techniques that reduction in PATJ expression leads to delayed tight junction formation as well as defects in cell polarization.These effects are reversed by reintroduction of PATJ into these RNAi cells.This study provides new functional information on PATJ as a polarity protein and increases our understanding of the Crumbs-PALS1-PATJ complex function in epithelial polarity.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109, USA.

ABSTRACT
Recent studies have revealed an important role for tight junction protein complexes in epithelial cell polarity. One of these complexes contains the apical transmembrane protein, Crumbs, and two PSD95/discs large/zonula occludens domain proteins, protein associated with Lin seven 1 (PALS1)/Stardust and PALS1-associated tight junction protein (PATJ). Although Crumbs and PALS1/Stardust are known to be important for cell polarization, recent studies have suggested that Drosophila PATJ is not essential and its function is unclear. Here, we find that PATJ is targeted to the apical region and tight junctions once cell polarization is initiated. We show using RNAi techniques that reduction in PATJ expression leads to delayed tight junction formation as well as defects in cell polarization. These effects are reversed by reintroduction of PATJ into these RNAi cells. This study provides new functional information on PATJ as a polarity protein and increases our understanding of the Crumbs-PALS1-PATJ complex function in epithelial polarity.

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