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First published online July 2, 2007
doi: 10.1242/10.1242/jcs.03470

Journal of Cell Science 120, 2352-2365 (2007)
Published by The Company of Biologists 2007
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Cooperative roles of Par-3 and afadin in the formation of adherens and tight junctions

Takako Ooshio1, Naoyuki Fujita1, Akio Yamada1, Tatsuhiro Sato1, Yuichi Kitagawa1, Ryoko Okamoto1, Shinsuke Nakata1, Ayaka Miki1, Kenji Irie1,* and Yoshimi Takai1,2,{ddagger}

1 Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Osaka, Japan
2 Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine/Faculty of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Hyogo, Japan


Figure 1
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  		Fig. 1. Necessity of Par-3 for the formation of AJs and TJs. (Aa) Reduction in the amount of Par-3 by RNAi in MDCK cells. The cell lysates were subjected to SDS-PAGE, followed by western blotting with the indicated Abs. (Ab) Reduction in the immunofluorescence signal for Par-3 at cell-cell adhesion sites between two Par-3-knockdown MDCK cells. After Ca2+ switch, the cells were fixed and stained with the anti-Par-3 pAb. (Ac) Reduction in the immunofluorescence signals for E-cadherin and occludin at cell-cell adhesion sites between two Par-3-knockdown MDCK cells. After Ca2+ switch, the cells were fixed and stained with various combinations of the indicated Abs. Bars, 10 µm. (Ba) Re-concentration of the immunofluorescence signals for E-cadherin and occludin at cell-cell adhesion sites in the Par-3-knockdown MDCK cells that re-expressed GFP–Par-3. After Ca2+ switch, the cells were fixed and stained with various combinations of the indicated Abs. Bars, 10 µm. (Bb) Exogenous expression of GFP–Par-3 in Par-3-knockdown MDCK cells. MDCK cells were co-transfected with pBS-H1-Par-3 and pEGFP-Par-3. The cell lysates were subjected to SDS-PAGE, followed by Western blotting with anti-Par-3 pAb. (Ca) Weak cell-cell adhesion activity of Par-3-knockdown MDCK cells. After Ca2+ switch, the wild-type MDCK cells transfected with pBS-H1-Par-3 or pBS-H1-scramble as a control were trypsinized in the presence of 1 mM CaCl2 (TC treatment) or 1 mM EGTA (TE treatment) for 1 hour and dissociated through pipetting ten times. The extent of the cell dissociation was represented by the index NTC/NTE, where NTC and NTE were the total particle numbers after the TC and TE treatments, respectively. The results shown are representative of three independent experiments. (Cb) The paracellular diffusion of FITC-conjugated dextran (average 40 kDa) in Par-3-knockdown MDCK cells. Data are expressed as the means±s.d. of three independent experiments.

 

Figure 2
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  		Fig. 2. No requirement of Par-3 for nectin-based cell-cell adhesion. After Ca2+ switch, Par-3-knockdown nectin-1-MDCK cells were fixed and stained with various combinations of the indicated Abs. Bars, 10 µm.

 

Figure 3
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  		Fig. 3. Requirement of Par-3 for co-localization of afadin with nectin. (A) Reduction in the immunofluorescence signal for afadin at cell-cell adhesion sites in Par-3-knockdown MDCK cells. After Ca2+ switch, the cells were fixed and stained with various combinations of the indicated Abs. Bars, 10 µm. (B) Reduction in the immunofluorescence signal for afadin, but not that for nectin-1, at the nectin-based cell-cell adhesion sites in Par-3-knockdown nectin-1-MDCK cells. After Ca2+ switch, the cells were fixed and stained with various combinations of the indicated Abs. Bars, 10 µm.

 

Figure 4
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  		Fig. 4. No requirement of afadin for co-localization of Par-3 with nectin. (A) Reduction in the amount of afadin by RNAi. The lysates of afadin-knockdown nectin-1-MDCK cells were subjected to western blotting with the indicated Abs. (B) Concentration of the immunofluorescence signals for Par-3 and nectin-1 at nectin-based cell-cell adhesion sites in afadin-knockdown nectin-1-MDCK cells. After Ca2+ switch, the cells were fixed and stained with various combinations of the indicated Abs. Bars, 10 µm.

 

Figure 5
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  		Fig. 5. Similar phenotypes between Par-3- and afadin-knockdown MDCK cells. (A) Reduction in the immunofluorescence signals for claudin-1, JAM-A and ZO-1, as well as those for E-cadherin and afadin, but not those for p120ctn, beta-catenin, and {alpha}-catenin, at cell-cell adhesion sites in Par-3-knockdown MDCK cells. After Ca2+ switch, the cells were fixed and stained with various combinations of the indicated Abs. Bars, 10 µm. (B) Re-concentration of the immunofluorescence signals for claudin-1, JAM-A and ZO-1, as well as that for afadin at cell-cell adhesion sites in the Par-3-knockdown MDCK cells that re-expressed GFP–Par-3. After Ca2+ switch, the cells were fixed and stained with various combinations of the indicated Abs. Bars, 10 µm.

 

Figure 6
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  		Fig. 6. Requirement of Par-6beta for co-localization of afadin with nectin. (A) Reduction in the amount of Par-6beta by RNAi. The lysates of Par-6beta-knockdown MDCK cells were subjected to SDS-PAGE, followed by western blotting with the indicated Abs. (B) Reduction in the immunofluorescence signals for E-cadherin, afadin, claudin-1, occludin, JAM-A, Par-3 and ZO-1 at cell-cell adhesion sites in Par-6beta-knockdown MDCK cells. After Ca2+ switch, the cells were fixed and stained with various combinations of the indicated Abs. (C) Reduction in the immunofluorescence signal for afadin, but not that for nectin-1, at the nectin-based cell-cell adhesion sites in Par-6beta-knockdown nectin-1-MDCK cells. After Ca2+ switch, the cells were fixed and stained with various combinations of the indicated Abs. Bars, 10 µm.

 

Figure 7
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  		Fig. 7. Requirement of aPKC for the association of afadin with nectin. (A) Reduction in the amount of aPKC by RNAi. The lysates of aPKC-knockdown MDCK cells were subjected to SDS-PAGE, followed by western blotting with the indicated Abs. (B) Reduction in the immunofluorescence signals for E-cadherin, afadin, claudin-1, occludin, JAM-A, Par-3 and ZO-1 at cell-cell adhesion sites in aPKC-knockdown MDCK cells. After Ca2+ switch, the cells were fixed and stained with various combinations of the indicated Abs. (C) Reduction in the immunofluorescence signal for afadin, but not that for nectin-1, at the nectin-based cell-cell adhesion sites in aPKC-knockdown nectin-1-MDCK cells. After Ca2+ switch, the cells were fixed and stained with various combinations of the indicated Abs. Bars, 10 µm.

 

Figure 8
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  		Fig. 8. Cooperative actions of Par-3 and afadin in the formation of AJs and TJs. After Ca2+ switch, the Par-3-knockdown MDCK cells that expressed GFP-afadin were fixed and stained with various combinations of the indicated Abs. Bars, 10 µm. Asterisks indicate the Par-3-knockdown MDCK cells that expressed GFP-afadin.

 

Figure 9
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  		Fig. 9. Involvement of Par-3 in the trans-interacting E-cadherin-induced, but not nectin-induced, activation of Rac. (A) Reduction in the activation of Rac1 in Par-3-knockdown MDCK cells. After Ca2+ switch, the cell lysates were subjected to pull-down assay using GST-PAK-CRIB, followed by western blotting using the anti-Rac1 mAb. The results shown are representative of three independent experiments. (B) No effect of Par-3 knockdown on nectin-induced cell-spreading. The nectin-1-MDCK cells co-transfected either with pBS-H1-Par-3 and pEGFP or with pBS-H1-scramble and pEGFP as a control were cultured on the Nef-3- or IgG-coated coverslips for 2 hours. The cells were fixed and stained for F-actin with rhodamine-phalloidin. Bars, 10 µm. Bars in the graph represent the percentage of spreading cells with lamellipodial cell protrusions of the 100 GFP-positive cells counted, and are expressed as means±s.d. of three independent experiments. (C) Reduction of E-cadherin-induced cell-spreading by Par-3 knockdown. The E-cadherin-MDCK cells co-transfected either with pBS-H1-Par-3 and pDsRed, with pBS-H1-Par-3, pEGFP-V12Rac1, and pDsRed, or with pBS-H1-scramble and pDsRed as a control were cultured on the Cef- or IgG-coated coverslips for 2 hours. The cells were fixed and stained for F-actin with Cy5-phalloidin. Bars, 10 µm. Bars in the graph represent the percentage of spreading cells with lamellipodial cell protrusions of the 100 DsRed-positive cells counted, and are expressed as means±s.d. of three independent experiments. (D) No effect of afadin knockdown on E-cadherin-induced cell-spreading. The E-cadherin-MDCK cells co-transfected either with pBS-H1-afadin and pDsRed or with pBS-H1-scramble and pDsRed as a control were cultured on the Cef- or IgG-coated coverslips for 2 hours. The cells were fixed and stained for F-actin with Cy5-phalloidin. Bars, 10 µm. Bars in the graph represent the percentage of spreading cells with lamellipodial cell protrusions of the 100 DsRed-positive cells counted, and are expressed as means±s.d. of three independent experiments. (Ea) Reduction in the immunofluorescence signals for E-cadherin and occludin, but not that for GFP-V12Rac1, at cell-cell adhesion sites in the Par-3-knockdown MDCK cells that expressed GFP-V12Rac1. After Ca2+ switch, the cells were fixed and stained with various combinations of the indicated Abs. Bars, 10 µm. (Eb) Reduction in the immunofluorescence signals for E-cadherin and occludin, but not that for GFP-V12Rac1 and FLAG-afadin, at cell-cell adhesion sites in the Par-3-knockdown MDCK cells that expressed GFP-V12Rac1 and FLAG-afadin. After Ca2+ switch, the cells were fixed and stained with various combinations of the indicated Abs. Bars, 10 µm.

 

Figure 10
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  		Fig. 10. The mode of action of Par-3 in the process of the formation of AJs and TJs. Par-3 regulates at least three sites of the junctional formation of epithelial cells: (1) the association of afadin with nectin; (2) the E-cadherin-induced activation of Rac; and (3) a site other than these first two sites.

 

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© The Company of Biologists Ltd 2007