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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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PATJ RNAi reduces PATJ but not PALS1 or CRB3 expression. (A) Lysates derived from control and two different PATJ RNAi expressing MDCKII cell lines were resolved by SDS-PAGE, followed by Western blot analysis for PATJ, PALS1, and CRB3. Blotting for actin was used as a loading control. (B) Beads containing the COOH terminus of CRB3 were used to precipitate proteins from control and PATJ RNAi lysates, followed by Western blot for PATJ and PALS1.
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fig2: PATJ RNAi reduces PATJ but not PALS1 or CRB3 expression. (A) Lysates derived from control and two different PATJ RNAi expressing MDCKII cell lines were resolved by SDS-PAGE, followed by Western blot analysis for PATJ, PALS1, and CRB3. Blotting for actin was used as a loading control. (B) Beads containing the COOH terminus of CRB3 were used to precipitate proteins from control and PATJ RNAi lysates, followed by Western blot for PATJ and PALS1.

Mentions: To investigate the function of PATJ in epithelia, we used the cultured MDCKII cells as a model system and generated PATJ knockdown MDCKII stable cell lines using RNAi techniques. Two independent target regions were chosen to generate two different RNAi constructs (Fig. 2, PATJ RNAi #1 and #2). Using these constructs, we successfully developed independent MDCKII cell lines in which the expression of PATJ is significantly reduced (Fig. 2). In these cell lines, the expression of endogenous PATJ was markedly decreased, but the expression of CRB3 was not changed. The significant reduction of PATJ expression was also analyzed by performing precipitation assays using beads that contain a peptide from the COOH terminus of CRB3. CRB3 peptide beads interact with the PDZ domain of PALS1 and precipitate PALS1 and PATJ together (Straight et al., 2004). In control MDCKII cells, CRB3 peptide beads successfully precipitated PALS1 and PATJ. However, due to the reduction of PATJ expression, CRB3 peptide beads precipitated only a small amount of PATJ in PATJ RNAi MDCKII cells. The amount of PALS1 precipitated by CRB3 peptide beads was also decreased, likely due to a reduced expression of PALS1 in PATJ RNAi MDCKII cells (Fig. 2 B). The precipitation of the Par proteins and aPKC by the Crumbs peptide beads (Straight et al., 2004) was not affected by the loss of PATJ (unpublished data).


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

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

PATJ RNAi reduces PATJ but not PALS1 or CRB3 expression. (A) Lysates derived from control and two different PATJ RNAi expressing MDCKII cell lines were resolved by SDS-PAGE, followed by Western blot analysis for PATJ, PALS1, and CRB3. Blotting for actin was used as a loading control. (B) Beads containing the COOH terminus of CRB3 were used to precipitate proteins from control and PATJ RNAi lysates, followed by Western blot for PATJ and PALS1.
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Related In: Results  -  Collection

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fig2: PATJ RNAi reduces PATJ but not PALS1 or CRB3 expression. (A) Lysates derived from control and two different PATJ RNAi expressing MDCKII cell lines were resolved by SDS-PAGE, followed by Western blot analysis for PATJ, PALS1, and CRB3. Blotting for actin was used as a loading control. (B) Beads containing the COOH terminus of CRB3 were used to precipitate proteins from control and PATJ RNAi lysates, followed by Western blot for PATJ and PALS1.
Mentions: To investigate the function of PATJ in epithelia, we used the cultured MDCKII cells as a model system and generated PATJ knockdown MDCKII stable cell lines using RNAi techniques. Two independent target regions were chosen to generate two different RNAi constructs (Fig. 2, PATJ RNAi #1 and #2). Using these constructs, we successfully developed independent MDCKII cell lines in which the expression of PATJ is significantly reduced (Fig. 2). In these cell lines, the expression of endogenous PATJ was markedly decreased, but the expression of CRB3 was not changed. The significant reduction of PATJ expression was also analyzed by performing precipitation assays using beads that contain a peptide from the COOH terminus of CRB3. CRB3 peptide beads interact with the PDZ domain of PALS1 and precipitate PALS1 and PATJ together (Straight et al., 2004). In control MDCKII cells, CRB3 peptide beads successfully precipitated PALS1 and PATJ. However, due to the reduction of PATJ expression, CRB3 peptide beads precipitated only a small amount of PATJ in PATJ RNAi MDCKII cells. The amount of PALS1 precipitated by CRB3 peptide beads was also decreased, likely due to a reduced expression of PALS1 in PATJ RNAi MDCKII cells (Fig. 2 B). The precipitation of the Par proteins and aPKC by the Crumbs peptide beads (Straight et al., 2004) was not affected by the loss of PATJ (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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