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.