A function for filamentous α-smooth muscle actin: Retardation of motility in fibroblasts

Abstract

Actins are known to comprise six mammalian isoforms of which β- and γ- nonmuscle actins are present in all cells, whereas α-smooth muscle (α-sm) actin is normally restricted to cells of the smooth muscle lineages. α-Sm actin has been found also to be expressed transiently in certain nonmuscle cells, in particular fibroblasts, which are referred to as myofibroblasts. The functional significance of α-sm actin in fibroblasts is unknown. However, myofibroblasts appear to play a prominent role in stromal reaction in breast cancer, at the site of wound repair, and in fibrotic reactions. Here, we show that the presence of α-sm actin is a signal for retardation of migratory behavior in fibroblasts. Comparison in a migration assay of fibroblast cell strains with and without α-sm actin revealed migratory restraint in α-sm actin-positive fibroblasts. Electroporation of monoclonal antibody (mAb) 1A4, which recognizes specifically the NH2-terminal Ac-EEED sequence of α-sm actin, significantly increased the frequency of migrating cells over that obtained with an unrelated antibody or a mAb against β- actin. Time-lapse video microscopy revealed migratory rates of 4.8 and 3.0 μm/h, respectively. To knock out the α-sm actin protein, several antisense phosphorothioate oligodeoxynucleotide (ODNs) were tested. One of these, 3'UT1, which is complementary to a highly evolutionary conserved 3' untranslated (3'UT) sequence of α-sm actin mRNA, was found to block α-sm actin synthesis completely without affecting the synthesis of any other proteins as analyzed by two-dimensional gel electrophoresis. Targeting by antisense 3'UT1 significantly increased motility compared with the corresponding sense ODN. α-Sm actin inhibition also led to the formation of less prominent focal adhesions as revealed by immunofluorescence staining against vinculin, talin, and β1-integrin. We propose that an important function of filamentous α-sm actin is to immobilize the cells.