A function for filamentous alpha-smooth muscle actin: retardation of motility in fibroblasts.
Actins are known to comprise six mammalian isoforms of which beta- and gamma-nonmuscle actins are present in all cells, whereas alpha-smooth muscle (alpha-sm) actin is normally restricted to cells of the smooth muscle lineages. alpha-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 alpha-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 alpha-sm actin is a signal for retardation of migratory behavior in fibroblasts. Comparison in a migration assay of fibroblast cell strains with and without alpha-sm actin revealed migratory restraint in alpha-sm actin-positive fibroblasts. Electroporation of monoclonal antibody (mAb) 1A4, which recognizes specifically the NH2-terminal Ac-EEED sequence of alpha-sm actin, significantly increased the frequency of migrating cells over that obtained with an unrelated antibody or a mAb against beta-actin. Time-lapse video microscopy revealed migratory rates of 4.8 and 3.0 microns/h, respectively. To knock out the alpha-sm actin protein, several antisense phosphorothioate oligodeoxynucleotide (ODNs) were tested. One of these, 3'UTI, which is complementary to a highly evolutionary conserved 3' untranslated (3'UT) sequence of alpha-sm actin mRNA, was found to block alpha-sm actin synthesis completely without affecting the synthesis of any other proteins as analyzed by two-dimensional gel electrophoresis. Targeting by antisense 3'UTI significantly increased motility compared with the corresponding sense ODN. alpha-Sm actin inhibition also led to the formation of less prominent focal adhesions as revealed by immunofluorescence staining against vinculin, talin, and beta1-integrin. We propose that an important function of filamentous alpha-sm actin is to immobilize the cells.
Rønnov-Jessen
L
L
Structural Cell Biology Unit, Institute of Medical Anatomy, the Panum Institute, Copenhagen, Denmark.
Petersen
O W
OW
D008841:Actin Cytoskeleton / Q000648:ultrastructure; D000199:Actins / Q000502:physiology*; D001483:Base Sequence; D001940:Breast / Q000166:cytology; D002465:Cell Movement; D016373:DNA, Antisense / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D005109:Extracellular Matrix / Q000502:physiology; D005260:Female; D005347:Fibroblasts / Q000502:physiology*; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000737:chemistry; D014741:Video Recording
D000199:Actins; D016373:DNA, Antisense