Over‐expression of smooth muscle caldesmon in mouse fibroblasts

Caldesmon is an actin, calmodulin, tropomyosin, and myosin binding protein implicated in the regulation of actomyosin interactions. We have investigated the effect of overexpression of the higher molecular weight smooth muscle isoform of caldesmon on mouse L cell physiology. Mouse L(TK-) cells were transfected stably with plasmids carrying the TK+ gene and a full length human smooth muscle caldesmon cDNA under control of the adenovirus major late promoter. Two clones displaying four and eight times the level of the endogenous mouse high molecular weight caldesmon were isolated. These cells acquire a distinct phenotype characterized by an altered morphology, including an increased number of processes and larger area due to enhanced cell spreading, and a significantly slower growth rate than that of untransfected control cells, or cells transfected with the TK+ gene alone. The majority of the overexpressed caldesmon appears to be active and localized on cytoskeleton structures as determined by detergent lysis. Immunofluorescence analysis of the clones revealed that the caldesmon is localized as punctate staining on stress-fibers and in membrane ruffles. The immunofluorescence images suggest that caldesmon overexpressing cells have more total filaments than control cells. The effects of excess caldesmon on cell mobility are ambiguous: one clone displayed increased motility compared to the control, while the motility of the second clone was decreased relative to the control.