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Files in this Data Supplement:
Fig. S1. Junction development and actin reorganization in control MTD1-A cells. Confluent monolayers of MTD1-A cells were cultured in LC medium for 40 hours and then subjected to a Ca2+ switch. The cells were fixed at the indicated times 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 projected views of xy confocal sections. Scale bar: 10 µm.
Fig. S2. MLC2 phosphorylation in MTD1-A cells during a Ca2+-switch-induced junctional formation. (A) MTD1-A cells infected with adenovirus vectors encoding β-galactosidase (LacZ) or aPKC kn were subjected to Ca2+ switch for the indicated times. Cells were double stained with anti-p-MLC2 (magenta in merged images) and anti-pp-MLC2 (green in merged images). Note that pp-MLC2 was only observed on the prejunctional actin belts that appeared at the final step of the junction development. (B) Total-cell extracts of MTD1-A cells were prepared at the indicated times after the Ca2+ switch, and the changes in the pp-MLC2 level were analyzed by western blotting. Scale bars: 10 µm.
Fig. S3. Circumferential actin cables in aPKCλ-kn-expressing cells sometimes exhibit hyper-contraction after a Ca2+ switch. MTD1-A cells transfected with adenovirus vectors encoding β-galactosidase (LacZ) or aPKCλ kn were subjected to a Ca2+ switch as described in the legend for Fig. 1. The cells were fixed at 6 hours after the Ca2+ switch and double stained with a mixture of anti-nonmuscle myosin heavy chain IIA and IIB antibodies (magenta in merged images) and phalloidin (green in merged images). Scale bar: 10 µm.
Fig. S4. Serum is essential for junction development by MTD1-A cells. Confluent monolayers of MTD1-A cells incubated in LC medium for 20 hours were subjected to a Ca2+ switch by adding 1.8 mM CaCl2. The cells were stained with an anti-ZO-1 antibody (magenta in merged images) and phalloidin (green in merged images) at 6 hours after the Ca2+ switch (left panels). For serum starvation, cells were incubated in serum-depleted medium for 1 hour before the Ca2+ switch (middle and right panels). Note that the Ca2+ switch could not induce junction development in the absence of serum (middle panels). LPA addition at 2 hours after the Ca2+ switch completely restores the junction development (right panels). Scale bar: 10 µm.
Fig. S5. LPA-induced circumferential actin cables in aPKCλ-kn-expressing cells shrunk into small aggregates in the very apical region. Subconfluent MTD1-A cells expressing GFP-actin together with aPKCλ 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. The images shown are still-frames (projected xy views of apical sections) from the time-lapse data at the indicated times. Scale bar: 10 µm.
Movie 1. Ca2+ depletion from the medium induces abrupt shrinkage of cortical actin bundles into small actin rings that are tethered to the plasma membrane by radial actin fibers. Confluent monolayers of MTD1-A cells expressing GFP-actin were subjected to Ca2+ depletion at time 0. Every 3 minutes, confocal z-stack images were acquired at 1-µm intervals for 20 µm. Projected images are shown. Scale bar: 10 µm.
Movie 2. Small actin rings in depolarized MTD1-A cells gradually expand during Ca2+ switch-induced epithelial cell polarization. Confluent monolayers of MTD1-A cells expressing GFP-actin and lacZ were depolarized in LC medium for 20 hours and then subjected to a Ca2+ switch at time 0. Every 4 minutes, confocal z-stack images were acquired at 1-µm intervals for 4 µm. Projected images are shown. Scale bar: 10 µm.
Movie 3. Inhibition of aPKC kinase activity compromises the expansion of the small actin rings induced by a Ca2+ switch. Confluent monolayers of MTD1-A cells expressing GFP-actin and aPKCλ kn were depolarized in LC medium for 20 hours and then subjected to a Ca2+ switch at time 0. Every 4 minutes, confocal z-stack images were acquired at 1-µm intervals for 4 µm. Projected images are shown. Scale bar: 10 µm.
Movie 4. LPA-induced development of perijunctional actin belts observed in serum-starved MTD1-A cells. Subconfluent MTD1-A cells expressing GFP-actin and lacZ were depolarized and serum-starved as described in the legend for Fig. 2. After simultaneous addition of CaCl2 and LPA (00:00), confocal z-stack images were acquired at 1-µm intervals for 4 µm every 3 minutes. The images shown are single confocal sections that clearly show the radial fiber formation. Scale bar: 10 µm.
Movie 5. aPKCλ kn does not affect radial actin fiber formation but inhibits the expansion of circumferential actin cables. Subconfluent MTD1-A cells expressing GFP-actin and aPKCλ kn were depolarized and serum-starved as described in the legend for Fig.2. After simultaneous addition of CaCl2 and LPA (00:00), confocal z-stack images were acquired at 1-µm intervals for 4 µm every 3 minutes. The images shown are single confocal sections that clearly show the radial fiber formation. Scale bar: 10 µm.
Movie 6. Time-lapse analysis of F-actin reorganization during wound healing of MTD1-A cells. Confluent monolayers of MTD1-A cells stably expressing GFP-actin were scratched with a needle (18 gauge) after a brief incubation in PBS as described in the legend for Fig. 7. Confocal single sections were acquired every 2 minutes for 58 minutes. Scale bar: 10 µm.
Movie 7. Radial actin fibers gradually become aligned and fused with the circumferential actin cables at the late stage of wound healing. The wound-healing process of MTD1-A cells stably expressing GFP-actin was recorded for a longer period than that in Movie 6 (every 6 minutes for 156 minutes) to analyze the late stage of the F-actin reorganization. Projected images are shown. Scale bar: 10 µm.
Movie 8. Wound healing of MTD1-A cells sometimes exhibits a purse-string type of F-actin reorganization. Confocal z-stack images of typical wound healing were acquired at 1-µm intervals for 20 µm every 2 minutes for 51 minutes. Projected images are shown. Scale bar: 10 µm.
Movie 9. Prolonged recording of wound healing of MTD1-A cells reveals direct transformation of the circumferential actin cables into strong cortical actin bundles. Confocal z-stack images of typical wound healing were acquired at 1-µm intervals for 20 µm every 18 minutes for 342 minutes. Projected images are shown. Scale bar: 10 µm.
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