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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 MDCK II cells do not polarize correctly in collagen gels and expression of EGFP-PATJ rescues the development of cysts in PATJ RNAi MDCKII cells. (A) Control MDCKII cells grown in collagen gels form normal cysts with a single lumen. A single MDCKII cell was grown in a collagen gel for 10 d. Cysts were immunostained for actin (red), GP135 (green) as an apical marker, and E-cadherin (gray) as a lateral marker as described in Materials and methods. (B) PATJ knockdown MDCKII cells are mispolarized without an apical lumen. Cysts were immunostained as described in A. (C) EGFP rescue PATJ RNAi MDCKII cells were grown in collagen gels for 10 d. Cysts were immunostained for actin (red) and GP135 (gray) as described in Materials and methods. (D) EGFP-PATJ rescue PATJ RNAi MDCKII cells were grown in collagen gels for 10 d. Cysts were immunostained for actin (red) and GP135 (gray) as described in C. (E) Quantification of cysts containing correct lumens. Cysts containing lumens were counted in wild-type, EGFP rescue, and EGFP-PATJ rescue PATJ RNAi MDCKII cells. Cysts which have contiguous lumen were counted as positive. Cysts containing no lumen were counted as negative. SDs are shown as error bars, n = 3. The number of lumen containing cysts was increased from 2.0% (EGFP rescue MDCKII) to 24% (EGFP-PATJ rescue MDCKII), P < 0.001, unpaired t test. Bars, 20 μm.
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fig4: PATJ RNAi MDCK II cells do not polarize correctly in collagen gels and expression of EGFP-PATJ rescues the development of cysts in PATJ RNAi MDCKII cells. (A) Control MDCKII cells grown in collagen gels form normal cysts with a single lumen. A single MDCKII cell was grown in a collagen gel for 10 d. Cysts were immunostained for actin (red), GP135 (green) as an apical marker, and E-cadherin (gray) as a lateral marker as described in Materials and methods. (B) PATJ knockdown MDCKII cells are mispolarized without an apical lumen. Cysts were immunostained as described in A. (C) EGFP rescue PATJ RNAi MDCKII cells were grown in collagen gels for 10 d. Cysts were immunostained for actin (red) and GP135 (gray) as described in Materials and methods. (D) EGFP-PATJ rescue PATJ RNAi MDCKII cells were grown in collagen gels for 10 d. Cysts were immunostained for actin (red) and GP135 (gray) as described in C. (E) Quantification of cysts containing correct lumens. Cysts containing lumens were counted in wild-type, EGFP rescue, and EGFP-PATJ rescue PATJ RNAi MDCKII cells. Cysts which have contiguous lumen were counted as positive. Cysts containing no lumen were counted as negative. SDs are shown as error bars, n = 3. The number of lumen containing cysts was increased from 2.0% (EGFP rescue MDCKII) to 24% (EGFP-PATJ rescue MDCKII), P < 0.001, unpaired t test. Bars, 20 μm.

Mentions: To investigate if PATJ controls the correct development of apico–basal polarity in epithelial cells, we used three dimensional cell culture assays in which single cells from control and PATJ RNAi MDCKII cells were grown in collagen gels for up to 10 d until they developed into cysts. The structure of cysts was closely examined by immunostaining experiments to assess if the correct polarity was established (Fig. 4). Control MDCKII cells developed into the perfect cysts with a single lumen, demonstrating well defined apico–basal polarity. In these cysts, epithelial cells surround a lumen and the apical membranes face the cyst lumen as demonstrated by staining for the apical membrane protein, GP135 (Fig. 4 A, green). In Fig. 4, lateral membranes are marked by E-cadherin (gray), whereas cortical actin staining is red. In contrast, PATJ RNAi MDCKII cells failed to develop into the normal cysts (Fig. 4 B). These cysts showed severe defects in development of a lumen displaying aberrant GP135 staining (Fig. 4 B, green). These data demonstrated that PATJ plays a critical role in the establishment of apico–basal polarity during the formation of cysts.


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

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

PATJ RNAi MDCK II cells do not polarize correctly in collagen gels and expression of EGFP-PATJ rescues the development of cysts in PATJ RNAi MDCKII cells. (A) Control MDCKII cells grown in collagen gels form normal cysts with a single lumen. A single MDCKII cell was grown in a collagen gel for 10 d. Cysts were immunostained for actin (red), GP135 (green) as an apical marker, and E-cadherin (gray) as a lateral marker as described in Materials and methods. (B) PATJ knockdown MDCKII cells are mispolarized without an apical lumen. Cysts were immunostained as described in A. (C) EGFP rescue PATJ RNAi MDCKII cells were grown in collagen gels for 10 d. Cysts were immunostained for actin (red) and GP135 (gray) as described in Materials and methods. (D) EGFP-PATJ rescue PATJ RNAi MDCKII cells were grown in collagen gels for 10 d. Cysts were immunostained for actin (red) and GP135 (gray) as described in C. (E) Quantification of cysts containing correct lumens. Cysts containing lumens were counted in wild-type, EGFP rescue, and EGFP-PATJ rescue PATJ RNAi MDCKII cells. Cysts which have contiguous lumen were counted as positive. Cysts containing no lumen were counted as negative. SDs are shown as error bars, n = 3. The number of lumen containing cysts was increased from 2.0% (EGFP rescue MDCKII) to 24% (EGFP-PATJ rescue MDCKII), P < 0.001, unpaired t test. Bars, 20 μm.
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Related In: Results  -  Collection

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fig4: PATJ RNAi MDCK II cells do not polarize correctly in collagen gels and expression of EGFP-PATJ rescues the development of cysts in PATJ RNAi MDCKII cells. (A) Control MDCKII cells grown in collagen gels form normal cysts with a single lumen. A single MDCKII cell was grown in a collagen gel for 10 d. Cysts were immunostained for actin (red), GP135 (green) as an apical marker, and E-cadherin (gray) as a lateral marker as described in Materials and methods. (B) PATJ knockdown MDCKII cells are mispolarized without an apical lumen. Cysts were immunostained as described in A. (C) EGFP rescue PATJ RNAi MDCKII cells were grown in collagen gels for 10 d. Cysts were immunostained for actin (red) and GP135 (gray) as described in Materials and methods. (D) EGFP-PATJ rescue PATJ RNAi MDCKII cells were grown in collagen gels for 10 d. Cysts were immunostained for actin (red) and GP135 (gray) as described in C. (E) Quantification of cysts containing correct lumens. Cysts containing lumens were counted in wild-type, EGFP rescue, and EGFP-PATJ rescue PATJ RNAi MDCKII cells. Cysts which have contiguous lumen were counted as positive. Cysts containing no lumen were counted as negative. SDs are shown as error bars, n = 3. The number of lumen containing cysts was increased from 2.0% (EGFP rescue MDCKII) to 24% (EGFP-PATJ rescue MDCKII), P < 0.001, unpaired t test. Bars, 20 μm.
Mentions: To investigate if PATJ controls the correct development of apico–basal polarity in epithelial cells, we used three dimensional cell culture assays in which single cells from control and PATJ RNAi MDCKII cells were grown in collagen gels for up to 10 d until they developed into cysts. The structure of cysts was closely examined by immunostaining experiments to assess if the correct polarity was established (Fig. 4). Control MDCKII cells developed into the perfect cysts with a single lumen, demonstrating well defined apico–basal polarity. In these cysts, epithelial cells surround a lumen and the apical membranes face the cyst lumen as demonstrated by staining for the apical membrane protein, GP135 (Fig. 4 A, green). In Fig. 4, lateral membranes are marked by E-cadherin (gray), whereas cortical actin staining is red. In contrast, PATJ RNAi MDCKII cells failed to develop into the normal cysts (Fig. 4 B). These cysts showed severe defects in development of a lumen displaying aberrant GP135 staining (Fig. 4 B, green). These data demonstrated that PATJ plays a critical role in the establishment of apico–basal polarity during the formation of cysts.

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.

Show MeSH
Related in: MedlinePlus