First published online 28 April 2009
doi: 10.1242/jcs.043174
Journal of Cell Science 122, 1595-1606 (2009)
Published by The Company of Biologists 2009
Interaction between PAR-3 and the aPKC–PAR-6 complex is indispensable for apical domain development of epithelial cells
Yosuke Horikoshi1,*,
Atsushi Suzuki1,
Tomoyuki Yamanaka1,
,
Kazunori Sasaki1,
Keiko Mizuno1,
Hajime Sawada2,
Shigenobu Yonemura3 and
Shigeo Ohno1,
1 Department of Molecular Biology, Yokohama City University Graduate School of Medical Science, 3-9 Fuku-ura, Kanazawa-ku, Yokohama 236-0004, Japan
2 Department of Histology and Cell Biology, Yokohama City University Graduate School of Medical Science, 3-9 Fuku-ura, Kanazawa-ku, Yokohama 236-0004, Japan
3 Electron Microscope Laboratory, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan
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Fig. 1. PAR-3 knockdown retards apical membrane domain development in the early phase of cell polarization. (A,B) Control or PAR-3 knockdown stable MDCK clones (clones 11-10 and 13-32, respectively) were cultured on filter supports and subjected to a calcium switch (CS). At 3 and 24 hours after the CS, the cells were immunostained for ezrin and ZO-1 (A) or gp135 and F-actin (B). Projected views of confocal sections are presented with z-sectional views. Arrowheads indicate intercellular lumens. (C) Transmission electron micrograph of PAR-3 knockdown stable clone (13-32) cells cultured on a filter support and subjected to a CS. At 3 hours after the CS, the cells were fixed and sectioned perpendicularly to the substratum. Arrow indicates vacuolar apical compartments (VACs). (D) Time courses of VAC exocytosis induced by a CS (time 0) were quantified for control (clone 11-10, white circles; clone 1-5, white triangles) and PAR-3 knockdown (clone 13-32, black circles; clone 25a, black triangles) stable MDCK clones. VACs are identified as large intracellular structures strongly stained for gp135 and F-actin. (E) The data for the PAR-3 knockdown cells shown in the bottom panels in A were supplemented with E-cadherin staining (green) and magnified. The arrow and horizontal black line indicate the position at which the z-sectional view shown at the bottom was reconstituted. Arrowheads indicate intercellular lumens. (F) Original z-stack images of the data in E presented in a gallery from the apical domain to basal domain at 1-µm intervals. Arrowheads indicate intercellular lumens. Scale bars: 20 µm in A,B,E,F; 0.5 µm in C.
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Fig. 2. PAR-3 knockdown MDCK cells fail to form normal cysts with an integrated single lumen. (A,B) Control or PAR-3 knockdown (2) cells were embedded in type I collagen gels and cultured for 7 days. The resulting cysts were fixed and immunostained for gp135, E-cadherin and ZO-1 (A) or GM130, F-actin and nuclei (TOPRO3) (B) as indicated. Confocal single sections are presented. Note that PAR-3 knockdown cells are polarized, but the apical domain of each cell is not integrated as a single lumen. (C) Cysts containing single or multiple lumens were counted for control and PAR-3 knockdown (#1 and #2) cells. Amorphous-shaped cysts with no lumen were categorized into `others'. *P<0.001, **P<0.05 versus control cells by Student's t-test. The error bars indicate the s.d. (n=3). Scale bars: 20 µm.
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Fig. 3. Defects in apical domain integration are observed at a very early stage of cystogenesis in PAR-3 knockdown cells. (A) Confocal serial sections of a multiple-lumen cyst derived from PAR-3 knockdown cells (#2). Note that each cell identified by TOPRO3 staining (white) inevitably faces one lumen identified by F-actin staining (red). (B) Developmental process of luminal cysts (control and PAR-3 knockdown #2 cells) examined at the indicated days after cell embedding. Single confocal sections of typical cysts are presented. Cysts were stained for E-cadherin (red), F-actin (green) and nuclei (blue). (C) Early-stage cysts derived from a PAR-3 knockdown stable clone (13-32) and the corresponding control clone (11-10). Each photograph represents a single confocal section. Scale bars: 20 µm.
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Fig. 4. PAR-3 S827/829A restores VAC exocytosis in PAR-3 knockdown cells less efficiently than wild-type PAR-3. (A) The PAR-3 isoforms and their S827/829A point mutants used in this study. CR3 is the aPKC-binding region. Red asterisks indicate the positions of mutated serine residues. (B) PAR-3 knockdown stable clone (25a) cells cultured on filter supports were infected with adenovirus expression vectors encoding β-galactosidase, T7-tagged wild-type PAR-3 or T7-tagged PAR-3 S827/829A. After 2 days, the cells were subjected to a CS. At 3 hours after the CS, the cells were immunostained for the indicated components to examine both apical domain re-establishment and junction reassembly. Projected views of confocal sections are presented. Equal expression levels of wild-type PAR-3 and its mutant are confirmed by the immunostaining as well as by western blotting (supplementary material Fig. S3) with an anti-T7 antibody. Arrowheads indicate VACs associated with continuous ZO-1 staining. Scale bars: 20 µm. (C) Quantification of the results shown in B. (Left) The average fluorescence intensity of ZO-1 staining (density) at cell-cell borders per cell was quantified as described in the Materials and Methods. Bars indicate the mean ± s.d. of four independent experiments. *P<0.01. The difference between wild-type and mutant PAR-3-expressing cells is not significant (P=0.38). (Right) The ratios of VAC-containing cells were estimated for cells expressing the indicated exogenous proteins. Bars indicate the mean ± s.d. of four independent experiments. **P<0.001. (D) PAR-3 knockdown stable clone (25a) cells stably expressing EGFP, T7-tagged wild-type PAR-3 or T7-tagged PAR-3 S827/829A under the control of tetracycline-inducible transactivation were established using the pOSTet14 expression vector. Cysts of each cell type were analyzed as described in the legend for Fig. 2. Expression of the exogenous proteins was induced by adding doxycycline (200 ng/ml) to the culture medium, and equal expression levels of wild-type PAR-3 and its mutant were confirmed by western blotting with an anti-PAR-3 antibody (supplementary material Fig. S3). Data represent the mean ± s.d. of three independent experiments. ***P<0.05.
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Fig. 5. sPAR-3 S827/829A cannot restore correct apical domain development in PAR-3 knockdown cells. (A) PAR-3 knockdown stable clone (25a) cells stably expressing EGFP, T7-tagged wild-type sPAR-3 or T7-tagged sPAR-3 S827/829A under the control of tetracycline-inducible transactivation were cultured on filter supports for 2 days in the presence of doxycycline (20 ng/ml). The cells were then subjected to a CS. At 20 hours after the CS, the cells were fixed and triply stained for gp135 (red in the merged images), tag (EGFP or T7) and F-actin (green in the merged images). Projected views of confocal sections are presented. Note that PAR-3 knockdown cells expressing wild-type sPAR-3 do not develop lateral lumens, whereas those expressing sPAR-3 S827/829A retain extensive lateral lumens. (B) Quantification of the results in A. The ratios of cells exhibiting intercellular lumens on their lateral membrane were estimated for cells expressing the individual exogenous proteins. The data represent the mean ± s.d. of three independent experiments. *P<0.001. (C) Cysts derived from PAR-3 knockdown stable clone (25a) cells stably overexpressing EGFP, T7-tagged wild-type sPAR-3 or T7-tagged sPAR-3 S827/829A were stained for EGFP or T7 (green), F-actin (red) and nuclei (blue). Each photograph represents a single confocal section. Expression of the exogenous proteins was induced by adding doxycycline (20 ng/ml) to the culture medium. (D) Quantification of the results in C. The ratios of cysts exhibiting multiple lumens were estimated for cysts expressing the individual exogenous proteins. The rescue efficiencies were quantified as described in the legend for Fig. 2. Data represent the mean ± s.d. of five independent experiments. *P<0.001. Scale bars: 20 µm.
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Fig. 6. Formation of the PAR-3–aPKC–PAR-6 complex and aPKC kinase activity are essential for normal apical domain development. (A) MDCK cells cultured on filter supports were infected with adenovirus expression vectors encoding HA-mLgl2. After 2 days, the cells were subjected to a CS. At 10 hours after the CS, the cells were immunostained for F-actin and gp135. Projected views of confocal sections are presented with z-sectional views. (B) Cysts derived from MDCK cells overexpressing HA-mLgl2 or HA-aPKC KN were stained for F-actin (red), HA tag (green) and nuclei (blue). (C) Quantification of the effects of HA-mLgl2, HA-aPKC WT, HA-aPKC KN, T7-PAR-6β WT and T7-PAR-6β M235W (MW) overexpression on cyst formation. The ratios of cysts exhibiting multiple lumens were estimated for cysts expressing the individual exogenous proteins. Bars indicate the mean ± s.d. of three to five independent experiments. (D) Overexpression of a dominant-negative mutant of aPKC (aPKC KN), but not wild-type aPKC (aPKC WT), induces the formation of lateral lumens in 2D-cultured MDCK cells. MDCK cells cultured on filter supports were infected with adenovirus expression vectors encoding the indicated proteins. After 2 days the cells were subjected to a CS. At 10 hours after the CS, the cells were immunostained for aPKC, F-actin and ZO-1. Single confocal sections are presented with z-sectional views. Scale bars: 20 µm.
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Fig. 8. A model for the mechanism by which aPKC, PAR-6 and PAR-3 regulate apical domain development. See Discussion for details.
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© The Company of Biologists Ltd 2009
). Projected views of confocal sections are presented with z-sectional views. (C) PAR-3 knockdown stable clone (13-32) cells extensively depolarized in low-calcium medium were subjected to a CS. At 3 hours after the CS, the cells were immunostained for PAR-3, aPKC and E-cadherin. Single confocal sections at the apical side are presented. (D) z-sectional views of cells prepared similarly to in C and immunostained for PAR-3, aPKC and E-cadherin (top) or PAR-3, PAR-6β and gp135 (bottom). Scale bars: 20 µm.