Fig. 7.

Role of cof and its phosphorylation in the regulation of basal F-actin levels and osmotically induced changes in F-actin content. A: cells were transfected with 20 nM nonrelated small interfering RNA (siNR) or siRNA against cof (siCof) for 48 h as described under MATERIALS METHODS. The efficiency of siCof was evaluated by Western blots, using tubulin as a loading control (n = 9). B: cells grown in 6-well plates were transfected with siNR or siCof, and 48 h later they were serum-deprived, preincubated in isotonic medium for 10 min, and exposed to iso- (I) or hypertonicity (H, 300 mM sucrose) for an additional 5 min. Subsequently, cells were fixed, permeabilized, incubated with 0.33 μM rhodmine-labeled phalloidin for 1 h, and extensively washed. Bound phalloidin (as a measure of total F-actin) was extracted with methanol and quantified fluorimetrically as described under MATERIALS AND METHODS (n = 9). Both visual inspection and parallel protein determinations verified that the confluent layers remained intact during the extraction procedure. C: wild-type (WT) LLC-PK1 cells were incubated with vehicle or 10 μM Y-27632 for 10 min and then challenged with iso- or hypertonic medium (300 mM sucrose) for 5 min. F-actin content was measured as above (n = 5). D: LLC-PK1 cells, stably expressing AA-MLC, a nonphosphorylatable, dominant negative form of the myosin light chain, were treated as in C (n = 6). Bottom: hyperosmolarity induced cof phosphorylation in AA-MLC cells as well is shown. E: expression of AA-MLC inhibits the osmotically triggered phosphorylation of MLC. Wild-type and AA-MLC cells were treated iso- or hypertonically for 5 min. Lysates obtained from these cells were probed with antibodies against Myc (to detect Myc-tagged AA-MLC), phospho-MLC and MLC, as indicated. F: effect of hyperosmolarity and Y-27632 on F-actin content was investigated in ELA cells, under iso- and hypertonic conditions (n = 4).