The specific NH2-terminal sequence Ac-EEED of alpha-smooth muscle actin plays a role in polymerization in vitro and in vivo.
The blocking effect of the NH2-terminal decapeptide of alpha-smooth muscle (SM) actin AcEEED-STALVC on the binding of the specific monoclonal antibody anti-alpha SM-1 (Skalli, O., P. Ropraz, A. Trzeviak, G. Benzonana, D. Gillessen, and G. Gabbiani. 1986. J. Cell Biol. 103:2787-2796) was compared with that of synthetic peptides modified by changing the acetyl group or by substituting an amino acid in positions 1 to 5. Using immunofluorescence and immunoblotting techniques, anti-alpha SM-1 binding was abolished by the native peptide and by peptides with a substitution in position 5, indicating that AcEEED is the epitope for anti-alpha SM-1. Incubation of anti-alpha SM-1 (or of its Fab fragment) with arterial SM actin increased polymerization in physiological salt conditions; the antibody binding did not hinder the incorporation of the actin antibody complex into the filaments. This action was not exerted on skeletal muscle actin. After microinjection of the alpha-SM actin NH2-terminal decapeptide or of the epitopic peptide into cultured aortic smooth muscle cells, double immunofluorescence for alpha-SM actin and total actin showed a selective disappearance of alpha-SM actin staining, detectable at approximately 30 min. When a control peptide (e.g. alpha-skeletal [SK] actin NH2-terminal peptide) was microinjected, this was not seen. This effect is compatible with the possibility that the epitopic peptide traps a protein involved in alpha-SM actin polymerization during the dynamic filament turnover in stress fibers. Whatever the mechanism, this is the first evidence that the NH2 terminus of an actin isoform plays a role in the regulation of polymerization in vitro and in vivo.
Chaponnier
C
C
Department of Pathology, University of Geneva, Switzerland.
Goethals
M
M
Janmey
P A
PA
Gabbiani
F
F
Gabbiani
G
G
Vandekerckhove
J
J
D000199:Actins / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure; D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D001011:Aorta / Q000166:cytology; D001667:Binding, Competitive; D002498:Centrifugation; D000939:Epitopes; D015151:Immunoblotting; D008845:Microinjections; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D009842:Oligopeptides / Q000276:immunology / Q000378:metabolism*; D010446:Peptide Fragments / Q000276:immunology; D011108:Polymers / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D017208:Rats, Wistar
D000199:Actins; D000939:Epitopes; D009842:Oligopeptides; D010446:Peptide Fragments; D011108:Polymers