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doi: 10.1242/10.1242/jcs.00032

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aPKC kinase activity is required for the asymmetric differentiation of the premature junctional complex during epithelial cell polarization

Atsushi Suzuki^*,1, Chikako Ishiyama^*,1, Katsutaka Hashiba¹, Miki Shimizu¹, Klaus Ebnet² and Shigeo Ohno^1,

¹ Department of Molecular Biology, Yokohama City University School of Medicine, 3-9 Fuku-ura, Kanazawa-ku, Yokohama 236-0004, Japan
² Institute of Cell Biology, ZMBE, University of Muenster, D-48149 Muenster, Germany

View larger version (88K): [in a new window]   Fig. 1. aPKC and PAR-3 translocate to cell-cell junctional regions after the establishment of spot-like AJs. (A,B) Confluent MTD1-A monolayers were scratched with a needle, cultured for 6 hours (left panels), 9 hours (middle panels) and 12 hours (right panels) and then doubly stained with PAR-3 pAb and ZO-1 mAb (A) or aPKC pAb and ZO-1 mAb (B), as indicated. Each panel represents a projected view of confocal optical sections (0.4 µm) collected from the apical to the basal region of the cells, although colocalization of the proteins was confirmed in a single section. (The panels in the following figures are similarly obtained unless otherwise mentioned.) Large arrowheads indicate the direction of the wounds. Note that many ZO-1-positive spot-like AJs are negative for PAR-3 as well as aPKC staining. In contrast with PAR-3, aPKC signal is often lacking on the ZO-1-positive continuous junctions, Bars, 10 µm.

View larger version (111K): [in a new window]   Fig. 2. Claudin-1 is a unique TJ component that is recruited into junctional regions as late as PAR-3 and aPKC are. The recruitment of TJ components into the cell-cell junctional regions of wound healing cells was examined by double immunostaining at intermediate stages of wound closure. In A, three TJ membrane proteins, JAM, occludin and claudin-1, were compared with ZO-1, whereas, in B, claudin-1 and PAR-3 were compared with JAM, as indicated. The combination of used antibodies (rat JAM mAb, rabbit occ pAb, rabbit claudin-1 pAb, mouse ZO-1 mAb and anti-PAR-3 pAb) is indicated in the panels, and merged views are also presented on the right. The direction of the wounds is indicated by large arrowheads, whereas ZO-1- or JAM-positive spot-like AJs, which showed negative or weak staining by corresponding other TJ proteins, are indicated by small arrows. Small arrowheads indicate occludin- or claudin-1-positive, but ZO-1 negative, granular structures sometimes observed in cytoplasm. Note that many ZO-1- or JAM-positive dot-like AJs are negative for claudin-1. Bars, 10 µm.

View larger version (83K): [in a new window]   Fig. 3. Ectopic expression of the dominant-negative mutant of aPKC (aPKCkn) inhibited the regeneration of TJ in the wound-healing area. Confluent monolayers of MTD1-A cells were infected with adenovirus vectors carrying LacZ or aPKCkn cDNA, and the ectopic protein expression was induced in the normal growth media for 18 hours. The wound-healing assay was performed as described in the legend to Fig. 1 (large arrowheads indicate the direction of the wounds) and, 10 hours (A) or 30 hours (B) after wounding, cells were fixed and doubly stained with anti-aPKC pAb and anti-ZO-1 mAb (A) or anti-ZO-1 pAb and anti-E-cadherin mAb (B). As shown in A, although the expression levels are virtually heterogenous, aPKCkn was expressed in almost 90% of the cells at levels higher than the endogenous one (under the photographic conditions used here, fluorescence signals of endogenous aPKC cannot be detected). Note that when aPKCkn was expressed, ZO-1 staining disappeared in cells burying the wound, although the completion of wound healing was confirmed by phase-contrast microscopy (data not shown) (see aPKC staining in cells burying the wound). Enlarged views in B revealed that ZO-1 is broadly localized in dot-like structures in the wound-healing region of aPKCkn-expressing cells, where E-cadherin staining also displayed rough, discontinuous appearance. Bars, 100 µm (A) or 20 µm (B).

View larger version (136K): [in a new window]   Fig. 4. Overexpression of aPKCkn inhibited the development of spot-like AJs into belt-like AJs. aPKCkn-expressing cells were subjected to wounding, fixed 30 hours after the wounding and doubly immunostained by the antibodies indicated. Large arrowheads indicate the direction of the wounds. The projected views of confocally obtained optical sections from the apical to the basal regions of the cells, except for merged views which were made from appropriate single confocal sections to strictly examine the co-localization of the two proteins (green: AJ proteins, red: ZO-1), are shown. Three AJ proteins, E-cadherin, -catenin and nectin, were colocalized to some of these persistent ZO-1-positive spot-like structures (small arrows in the merged view), suggesting that these correspond to the spot-like AJs that could not develop into belt-like AJs. The structures were located at the apex of the lateral membrane (see Fig. 5B), and E-cadherin and -catenin also distributed diffusely in the lateral membranes (small arrowheads in middle panels). Bars, 20 µm.

View larger version (86K): [in a new window]   Fig. 5. Development of the cortical bundle of F-actin was blocked in an intermediate step in aPKC-expressing cells burying the wound. aPKCkn-expressing cells burying the wound were doubly stained with rhodamine-phalloidin (red) and anti-ZO-1 antibody (green) 10 hours (A) or 30 hours (B and C) after wounding. Large arrowheads indicate the direction of the wounds. In A, the results for LacZ-expressing cells are also shown for comparison (top panels). Shown are projected views of confocally obtained optical sections covering apical regions (A, and top panels in B), single confocal section at basal, intermediate, or apical region (C) or z-sectional views crossing a spot-like incomplete junctional structure (small arrowhead; bottom panels of B). aPKCkn-expressing cells do not complete cortical bundle formation of F-actin, showing stress fiber-like F-actin bundles linking ZO-1-positive spot-like AJs. In some cells, the prototype of cortical loose bundles of F-actin was observed, from which short F-actin fibers emanate into the spot-like AJs (right panels in A). Note that this kind of F-actin organization is formed in the apical surface of cells independently of the basal stress fibers (B,C), and cells with spot-like AJs also develop in height to a level comparable to that of surrounding cells with complete TJs. Bars, 10 µm.

View larger version (143K): [in a new window]   Fig. 6. Three TJ membrane proteins were also trapped in the persistent spot-like AJ of aPKCkn-expressing cells. aPKCkn-expressing cells were subjected to wounding, fixed 30 hours after the wounding and doubly immunostained with pAbs against TJ proteins or PAR-3 and ZO-1 mAb, as indicated. Shown are enlarged views of cells burying the wound or their merged views. JAM, occludin and claudin-1 are all colocalized with ZO-1 in the persistent spot-like AJs (A). As shown in B, a substantial number of the spot-like AJs induced by aPKCkn are also positive for PAR-3, although the extent of the colocalization is relatively low (small arrows indicate difference in staining between PAR-3 and ZO-1). Bars, 10 µm.