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First published online 15 July 2008
doi: 10.1242/jcs.024109

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aPKC enables development of zonula adherens by antagonizing centripetal contraction of the circumferential actomyosin cables

Department of Molecular Biology, Yokohama City University Graduate School of Medical Science, 3-9 Fuku-ura, Kanazawa-ku, Yokohama, 236-0004, Japan

View larger version (92K): [in this window] [in a new window]   Fig. 1. Comparison of the effects of aPKC kn and of myosin-II inhibitors on the junction development of MTD1-A cells. (A) Myosin inhibitors and aPKC kn, a dominant-negative mutant of aPKC, block junction development similarly but affect actin reorganization differently. Confluent monolayers of MTD1-A cells transformed by adenovirus vectors encoding β-galactosidase (LacZ) or aPKC kn were cultured in LC medium for 40 hours and then subjected to a Ca2+ switch in the presence or absence of the myosin inhibitors blebbistatin (100 µM) or Y27632 (20 µM). The cells were fixed at 6 hours after the Ca2+ switch and stained with an anti-ZO-1 antibody (magenta in merged images) and phalloidin (green in merged images). The images shown are single confocal sections that were selected to demonstrate the ZO-1 staining most clearly. Enlargements of the boxed regions in the merged views are shown at the bottom. For cells overexpressing aPKC kn, two typical images are presented, in which the left and right panels show cells exhibiting tightly and loosely bundled circumferential actin cables, respectively (each represents 40% and 60% of aPKC-kn-overexpressing cells, respectively). White arrowheads, circumferential actin cables; arrows, punctate staining of F-actin on spot-like AJs; yellow arrowheads, radial actin fibers. (B) Schematic presentation of the intermediate state of F-actin organization observed in aPKC-kn-overexpressing cells. (C) RNAi knockdown of aPKC isoforms in MTD1-A cells. Subconfluent MTD1-A cells were transfected with the indicated siRNA oligonucleotide duplexes (NS, non-silencing siRNA; 3, aPKC siRNA; 1, aPKC siRNA). Top panels: western blot analyses of total extracts of cells subjected to the indicated RNAi. aPKC was specifically detected by an anti-aPKC (human aPKC) antibody, whereas both aPKC isoforms (aPKC and aPKC) were simultaneously detected by an anti-aPKC antibody (C20) that reacts with both isoforms. GAPDH was used as a loading control. The data for E-cadherin, -catenin and β-catenin indicated no significant change in the expression levels of these AJ proteins. Images shown underneath: the indicated cells were immunostained with an anti-aPKC antibody (C20). (D) aPKC knockdown suppresses junction development after a Ca2+ switch, in a similar manner to aPKC-kn overexpression. The indicated cells were subjected to a Ca2+ switch as described in A and then immunostained with an anti-ZO-1 antibody (magenta in merged images) and phalloidin (green in merged images). Scale bars: 10 µm.

View larger version (98K): [in this window] [in a new window]   Fig. 2. Suppression of aPKC activity does not affect cell-cell-contact-induced myosin-II activation. (A) MTD1-A cells transfected with adenovirus vectors encoding β-galactosidase (LacZ) or aPKC kn were subjected to a Ca2+ switch as described in the legend for Fig. 1, fixed at the indicated times after the Ca2+ switch, and double stained with a mixture of antibodies against nonmuscle myosin heavy chain IIA and IIB (magenta in merged images) and against -actinin (green in merged images). Arrows, circumferential actin cables; arrowheads, perijunctional actin belts. (B) Control MTD1A cells were fixed at 30 minutes after a Ca2+ switch and double stained for F-actin (phalloidin; green in merged images) and with antibodies against either myosin II, Ser19 monophosphorylated myosin light-chain 2 (p-MLC2) or Thr18 and Ser19 diphosphorylated MLC2 (pp-MLC2) (magenta in merged images) as indicated at the top. Note that radial actin fibers were free from myosin II. (C) MTD1-A cells were immunostained with anti-p-MLC2 antibody at the indicated times after the Ca2+ switch. Pre-treatment with Y27632 completely abolishes the staining, suggesting that ROCK is dominantly responsible for this phosphorylation. (D) Total cell extracts of MTD1-A cells were prepared at the indicated times after the Ca2+ switch, and the changes in the p-MLC2 level were analyzed by western blotting. Note that overexpression of aPKC kn does not affect the Ca2+-switch-induced increase in p-MLC2. Scale bars: 10 µm.

View larger version (75K): [in this window] [in a new window]   Fig. 3. Suppression of aPKC activity does not disrupt the polarized organization of vinculin-containing structures at regions of cell-cell and cell-substrate contact. (A) MTD1-A cells treated with the indicated adenovirus vectors or myosin inhibitors were subjected to a Ca2+ switch as described in the legend for Fig. 1, and were then immunostained with rhodamine-phalloidin (top panels) and an anti-vinculin antibody (bottom panels) at 6 hours after the Ca2+ switch. Single confocal sections at the basal planes (basal) and apical planes 2.5 µm upward from the basal planes (apical) are shown. The signal intensity of phalloidin staining in the basal planes was enhanced to a comparable level to that in the apical planes. Note that aPKC kn does not disrupt the polarized distribution of vinculin into the two distinct structures at apical cell-cell contacts (arrows) and basal cell-substrate contacts (arrowheads). By contrast, cells treated with myosin-II inhibitors exhibit vinculin staining in the same planes. (B) Reconstituted xz views of the cells demonstrated in A were presented for rhodamine-phalloidin staining. Arrowheads point to the apical and basal planes at which the confocal data were obtained. Scale bar: 10 µm.

View larger version (117K): [in this window] [in a new window]   Fig. 4. Suppression of aPKC activity induces hyper-shrinkage of the circumferential actin cables during LPA-induced junction development in serum-starved MTD1-A cells. Confluent monolayers of MTD1-A cells infected with adenovirus vectors encoding β-galactosidase (– and +LPA) or aPKC kn were incubated in LC medium for 20 hours, with serum depletion from the medium during the last 1 hour, and then subjected to a Ca2+ switch by adding CaCl2 with or without 10 µM LPA. The cells were stained with an anti-ZO-1 antibody (magenta in merged images) and phalloidin (green in merged images) at 2 hours after the Ca2+ switch. The images shown are projected views of xy confocal sections. The results did not change even when cells were stained at 6 hours after the Ca2+ switch (M.K. and A.S., unpublished). Note that serum starvation results in blockade of the development of dot-like AJs into belt-like AJs and this blockade is released by the addition of LPA. Overexpression of aPKC kn suppresses this LPA-induced junction development and induces hyper-shrinkage of F-actin. Scale bar: 10 µm.

View larger version (95K): [in this window] [in a new window]   Fig. 5. Live-cell imaging of GFP-actin reveals the presence of a centripetal force imposed on the perijunctional actin belts. (A) Confluent monolayers of MTD1-A cells transfected with an adenovirus vector encoding GFP-actin were depolarized by Ca2+ depletion (00:00). Every 3 minutes, z-stack images were acquired at 1-µm intervals by time-lapse confocal microscopy (see supplementary material Movie 1). The images shown are still frames from the time-lapse data at the indicated times. Projected xy views as well as z-sectional views (xz and yz views) are presented. Note that the perijunctional actin belts rapidly shrink and squeeze the cells at their basal regions. The resultant small actin rings remain tethered to the cell peripheries by prominent radially running actin fibers. (B) Depolarized MTD1-A cells were fixed 45 minutes after Ca2+ depletion, and were double stained with the indicated antibodies (green in merged images) and rhodamine-phalloidin (magenta in merged images). Projected xy views of confocal sections are presented. Note that E-cadherin and ZO-1 predominantly localized on small actin rings but not at the tip of radially running actin fibers to which paxillin concentrated. (C) aPKC (green) localized on the apical surface of depolarized MTD1-A cells fixed as described in B. Scale bars: 10 µm. (D) Schematic illustrating the differences between polarizing and depolarizing MTD1-A cells. Note that the localizations of E-cadherin and ZO-1 (blue), and aPKC (green) are completely different between both states. Red lines illustrate F-actin organization.

View larger version (99K): [in this window] [in a new window]   Fig. 6. Live-cell imaging of GFP-actin reveals the presence of a centrifugal force that expands the contractile circumferential actin cables in an aPKC-dependent manner during epithelial-cell polarization. (A,B) Confluent monolayers of MTD1-A cells transfected with an adenovirus vector encoding GFP-actin together with a vector encoding β-galactosidase (LacZ; A) or aPKC kn (B) were depolarized in LC medium for 20 hours and then subjected to a Ca2+ switch (00:00). z-stack images taken at 1-µm intervals for 4 µm were acquired by time-lapse confocal microscopy (see supplementary material Movies 2, 3). The images shown here are still frames from the time-lapse data at the indicated times. Projected xy views are presented. (A) Note that, in control cells, the actin-ring structures observed in depolarized cells are connected to cell-cell-contact regions and expand to form perijunctional actin belts as the cells become polarized. (B) In aPKC-kn-overexpressing cells, the rings are tethered by radial actin fibers but fail to expand and instead show hyper-shrinkage. Scale bars: 10 µm.

View larger version (79K): [in this window] [in a new window]   Fig. 7. aPKC kn does not affect the formation of the radial actin fibers that is rapidly induced by LPA in serum-depleted MTD1-A cells. (A) Subconfluent MTD1-A cells expressing GFP-actin together with β-galactosidase (LacZ) or aPKC kn were depolarized in LC medium for 48 hours and subjected to serum starvation as described in the legend for Fig. 4. CaCl2 and LPA were simultaneously added to the cells at time 0, and time-lapse images were acquired by confocal microscopy (see supplementary material Movies 4, 5). The images shown are still-frames (projected xy views of intermediate sections) from the time-lapse data at the indicated times. (B) Enlarged views corresponding to the boxed regions in A. Scale bar: 10 µm.

View larger version (86K): [in this window] [in a new window]   Fig. 8. GFP-actin dynamics during wound healing of MTD1-A cells. (A) Confluent monolayers of MTD1-A cells stably expressing GFP-actin were scratched with a needle (18 gauge) after a brief incubation in PBS. Time-lapse images of GFP-actin (single confocal sections) were acquired by confocal microscopy at an appropriate time after wounding (see supplementary material Movie 6). The images shown are still-frames from the time-lapse data at the indicated times. Note that rapid actin polymerization occurs at the tips of the leading edges immediately after the initial cell-cell contacts (arrowheads), and the resultant radial actin fibers tag the circumferential actin cables when they come into contact (00:35). (B) The images in A were subjected to kymograph analysis to monitor the changes in the distance between the two circumferential actin cables in contacting cells along the broken line. Arrow, indicates the time point when radial actin fibers reached the intracellular circumferential actin cables. (C) GFP-actin dynamics at the late stage of wound healing. Note that the radial actin fibers (traced by magenta lines in the bottom panels) are aligned and fused with the corresponding circumferential actin cables (illustrated by white lines in the bottom panels) (see supplementary material Movie 7). Scale bars: 10 µm.

View larger version (19K): [in this window] [in a new window]   Fig. 9. New model for the role of the circumferential actin cables in junction development of confluent epithelial cells. (A) Newly proposed model for the role of the contraction of the circumferential actin cables in epithelial-junction development. In this model, the centripetal contractile force of the circumferential actomyosin cable (blue arrows) is counterbalanced by an aPKC-dependent putative centrifugal force (red arrows), such that it can be used for the development of continuous junction structures. Grey lines and circles, F-actin structures; dumb-bell-like symbols, myosin II; red circles, aPKC. (B) A previously published model suggesting that actomyosin contraction works at cell-cell-contact-free regions of the cell membrane in order to be used for lateral expansion of cell-cell contact regions (Adams et al., 1998; Gloushankova et al., 1997; Krendel and Bonder, 1999; Yamada and Nelson, 2007). (C) A possible mechanism by which the radial actin fibers are integrated into the circumferential actin cables. Grey lines and circles, F-actin structures; red circles, aPKC.

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