Fig. 3. Disrupted cell contacts activate SRF via monomeric actin. (A) MDCK cells were transfected with the SRF reporter p3DA-Luc (1.2 µg), pRL-TK (1 µg) and, if indicated, with 2.5 µg of wild-type actin (wt.) or the non-polymerisable actin mutant R62D and reseeded to form a confluent monolayer. Latrunculin B (LatB, 1 µM) was added 30 minutes before medium exchange. Shown is the mean luciferase activity, measured 7 hours after Ca²⁺ withdrawal. Error bars indicate s.e.m. (n=4). (B) Junction assembly and disassembly in cells expressing Flag-tagged actins. Cells were transfected with Flag-tagged actin wt., R62D or G15S, and seeded to form a confluent monolayer. 24 hours later, the medium was exchanged as indicated; after 7 hours incubation, cells were fixed and stained for Flag and E-cadherin (DECMA-1). Immunofluorescence micrographs are shown from cells maintained in physiological (upper panels) or low (lower panels) Ca²⁺. Transfected cells are marked by stars. (C) Correlation of F-actin reorganisation and Ca²⁺ level. Medium containing the indicated Ca²⁺ concentration was exchanged on a confluent monolayer of MDCK cells. After 7 hours, cells were fixed and F-actin was visualised with rhodamin-phalloidin. Mock, medium exchange with 1.8 mM Ca²⁺; –Ca²⁺, medium exchange with 0.02 mM Ca²⁺. Bars, 50 µm.