Fig. 1. Inducible tMLCK expression causes MLC phosphorylation and actin reorganization in differentiated Caco-2 monolayers. (A) tMLCK mRNA is readily detected by RT-PCR in Caco-2 cell monolayers after, but not before, induction. Digestion with NsiI results in a doublet, verifying the identity of the PCR product. Results are representative of three or more experiments. (B) Increased tMLCK transcription is accompanied by a 4.7±0.4-fold increase in MLC kinase activity, assayed using an in vitro kinase assay. In the assay shown, which was performed with exogenous calmodulin and Ca²⁺, the activity in lysates of cells without tMLCK expression represents endogenous MLC kinase. Results are representative of three or more independent experiments with this clone and at least two each with five independently generated clones. (C) Immunoblots using antisera specific for phosphorylated MLC show that induction of tMLCK expression causes progressive increases in endogenous MLC phosphorylation. Control immunoblots show that total MLC content did not change. Data from densitometric analysis of samples from the same experiment are shown in the graph. Data shown are means ± s.e.m. of triplicate samples and are representative of three or more independent experiments. Similar results were obtained with five independently generated clones. (D) tMLCK induction (open circles) caused increases in phalloidin binding that were not seen in uninduced (closed circles) monolayers. Total actin content, as assessed on western blots, was not changed. Data shown are means ± s.e.m. of quadruplicate samples and are representative of four independent experiments. (E) F-actin distribution was assessed by labeling with fluorescent phalloidin conjugates. The three-dimensional projection demonstrates typical perijunctional actin rings with focal mild intensifications within the ring and prominent apical microvillus cores. (F) Four hours after tMLCK induction, phalloidin-labeled F-actin shows perijunctional rings to be irregularly intensified, with brighter foci alternating with areas of reduced or unchanged labeling intensity. Bars, 10 µm.