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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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Tight junction formation is delayed in PATJ RNAi MDCKII cells and expression of EGFP-PATJ rescues the formation of tight junctions. (A) Control and PATJ RNAi MDCKII cells were subject to calcium switch experiments to assess the formation of tight junctions. At different time points after addition of calcium (0 h, 6 h, and 24 h), the cells were fixed, permeabilized, and immunostained for PATJ, PALS1, and the tight junction marker protein, ZO1. (B) Lysates were prepared from control (first lane), PATJ RNAi (second lane), EGFP rescue PATJ RNAi (third lane), and EGFP-PATJ rescue PATJ RNAi (fourth lane). Anti-PATJ (top) and anti-EGFP (bottom) antibodies were used for Western blot analysis to detect endogenous PATJ and EGFP-PATJ. (C) EGFP (top) and EGFP-PATJ (bottom) rescue PATJ RNAi MDCKII cells were fixed, permeabilized, and immunostained for a tight junction marker protein, ZO1 (red) at 6 h after calcium switch as described in Materials and methods. EGFP and EGFP-PATJ are shown in green colors. (D) TER was measured with control (blue circle, long dashed line), PATJ RNAi (gray square, dash-dot line), EGFP rescue PATJ RNAi (red triangle, short dashed line) and EGFP-PATJ rescue PATJ RNAi (black diamond, solid line) MDCKII cells. SDs are shown as error bars, n = 3. Bars, 20 μm.
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fig3: Tight junction formation is delayed in PATJ RNAi MDCKII cells and expression of EGFP-PATJ rescues the formation of tight junctions. (A) Control and PATJ RNAi MDCKII cells were subject to calcium switch experiments to assess the formation of tight junctions. At different time points after addition of calcium (0 h, 6 h, and 24 h), the cells were fixed, permeabilized, and immunostained for PATJ, PALS1, and the tight junction marker protein, ZO1. (B) Lysates were prepared from control (first lane), PATJ RNAi (second lane), EGFP rescue PATJ RNAi (third lane), and EGFP-PATJ rescue PATJ RNAi (fourth lane). Anti-PATJ (top) and anti-EGFP (bottom) antibodies were used for Western blot analysis to detect endogenous PATJ and EGFP-PATJ. (C) EGFP (top) and EGFP-PATJ (bottom) rescue PATJ RNAi MDCKII cells were fixed, permeabilized, and immunostained for a tight junction marker protein, ZO1 (red) at 6 h after calcium switch as described in Materials and methods. EGFP and EGFP-PATJ are shown in green colors. (D) TER was measured with control (blue circle, long dashed line), PATJ RNAi (gray square, dash-dot line), EGFP rescue PATJ RNAi (red triangle, short dashed line) and EGFP-PATJ rescue PATJ RNAi (black diamond, solid line) MDCKII cells. SDs are shown as error bars, n = 3. Bars, 20 μm.

Mentions: To test if PATJ has a role in the formation of tight junctions, calcium switch experiments were performed with control and PATJ RNAi MDCKII cells. Cells were incubated overnight in low calcium media to disrupt the existing junctions. Subsequently, low calcium media was replaced by normal media to initiate junctional formation and the formation of tight junctions was assessed by immunostaining for the tight junction marker protein, ZO1. As shown in Fig. 3 A, tight junctions rapidly redeveloped after calcium switch and were complete within 6 h in control MDCKII cells. ZO1 and PALS1 correctly localized to the tight junctions (Fig. 3 A, left panels). In contrast, there was a significant delay in the formation of tight junctions in PATJ RNAi MDCKII cells (Fig. 3 A, right). At 24 h, a small amount of PATJ can be seen to localize at tight junctions in the PATJ RNAi cell lines. This is consistent with the residual PATJ expression we observe in the CRB3 bead pulldown from these RNAi cell lines (Fig. 2 B). It is not clear if this small amount of PATJ is necessary for the tight junctions to eventually form. The recruitment of ZO1 to the tight junctions did not take place until 6 h after calcium switch. Interestingly, the localization of PALS1 was also affected by the suppression of PATJ (Fig. 3 A), confirming previous studies from our group that PATJ is responsible for the targeting of PALS1 to the tight junctions (Roh et al., 2002b). The disruption of tight junctions was also confirmed by immunostaining for Par3, another tight junction protein. In control MDCKII cells, Par3 and ZO1 colocalized to the tight junctions within 6 h after calcium switch. However, localization of Par3 to the tight junctions in PATJ RNAi MDCKII cells was significantly disrupted (unpublished data).


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

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

Tight junction formation is delayed in PATJ RNAi MDCKII cells and expression of EGFP-PATJ rescues the formation of tight junctions. (A) Control and PATJ RNAi MDCKII cells were subject to calcium switch experiments to assess the formation of tight junctions. At different time points after addition of calcium (0 h, 6 h, and 24 h), the cells were fixed, permeabilized, and immunostained for PATJ, PALS1, and the tight junction marker protein, ZO1. (B) Lysates were prepared from control (first lane), PATJ RNAi (second lane), EGFP rescue PATJ RNAi (third lane), and EGFP-PATJ rescue PATJ RNAi (fourth lane). Anti-PATJ (top) and anti-EGFP (bottom) antibodies were used for Western blot analysis to detect endogenous PATJ and EGFP-PATJ. (C) EGFP (top) and EGFP-PATJ (bottom) rescue PATJ RNAi MDCKII cells were fixed, permeabilized, and immunostained for a tight junction marker protein, ZO1 (red) at 6 h after calcium switch as described in Materials and methods. EGFP and EGFP-PATJ are shown in green colors. (D) TER was measured with control (blue circle, long dashed line), PATJ RNAi (gray square, dash-dot line), EGFP rescue PATJ RNAi (red triangle, short dashed line) and EGFP-PATJ rescue PATJ RNAi (black diamond, solid line) MDCKII cells. SDs are shown as error bars, n = 3. Bars, 20 μm.
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fig3: Tight junction formation is delayed in PATJ RNAi MDCKII cells and expression of EGFP-PATJ rescues the formation of tight junctions. (A) Control and PATJ RNAi MDCKII cells were subject to calcium switch experiments to assess the formation of tight junctions. At different time points after addition of calcium (0 h, 6 h, and 24 h), the cells were fixed, permeabilized, and immunostained for PATJ, PALS1, and the tight junction marker protein, ZO1. (B) Lysates were prepared from control (first lane), PATJ RNAi (second lane), EGFP rescue PATJ RNAi (third lane), and EGFP-PATJ rescue PATJ RNAi (fourth lane). Anti-PATJ (top) and anti-EGFP (bottom) antibodies were used for Western blot analysis to detect endogenous PATJ and EGFP-PATJ. (C) EGFP (top) and EGFP-PATJ (bottom) rescue PATJ RNAi MDCKII cells were fixed, permeabilized, and immunostained for a tight junction marker protein, ZO1 (red) at 6 h after calcium switch as described in Materials and methods. EGFP and EGFP-PATJ are shown in green colors. (D) TER was measured with control (blue circle, long dashed line), PATJ RNAi (gray square, dash-dot line), EGFP rescue PATJ RNAi (red triangle, short dashed line) and EGFP-PATJ rescue PATJ RNAi (black diamond, solid line) MDCKII cells. SDs are shown as error bars, n = 3. Bars, 20 μm.
Mentions: To test if PATJ has a role in the formation of tight junctions, calcium switch experiments were performed with control and PATJ RNAi MDCKII cells. Cells were incubated overnight in low calcium media to disrupt the existing junctions. Subsequently, low calcium media was replaced by normal media to initiate junctional formation and the formation of tight junctions was assessed by immunostaining for the tight junction marker protein, ZO1. As shown in Fig. 3 A, tight junctions rapidly redeveloped after calcium switch and were complete within 6 h in control MDCKII cells. ZO1 and PALS1 correctly localized to the tight junctions (Fig. 3 A, left panels). In contrast, there was a significant delay in the formation of tight junctions in PATJ RNAi MDCKII cells (Fig. 3 A, right). At 24 h, a small amount of PATJ can be seen to localize at tight junctions in the PATJ RNAi cell lines. This is consistent with the residual PATJ expression we observe in the CRB3 bead pulldown from these RNAi cell lines (Fig. 2 B). It is not clear if this small amount of PATJ is necessary for the tight junctions to eventually form. The recruitment of ZO1 to the tight junctions did not take place until 6 h after calcium switch. Interestingly, the localization of PALS1 was also affected by the suppression of PATJ (Fig. 3 A), confirming previous studies from our group that PATJ is responsible for the targeting of PALS1 to the tight junctions (Roh et al., 2002b). The disruption of tight junctions was also confirmed by immunostaining for Par3, another tight junction protein. In control MDCKII cells, Par3 and ZO1 colocalized to the tight junctions within 6 h after calcium switch. However, localization of Par3 to the tight junctions in PATJ RNAi MDCKII cells was significantly disrupted (unpublished data).

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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