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The Specific NH2-Terminal Sequence Ac-EEED of α-Smooth Muscle Actin Plays a Role in Polymerization in vitro and in vivo

Christine Chaponnier, Marc Goethals, Paul. A. Janmey, Françoise Gabbiani, Giulio Gabbiani and Joël Vandekerckhove
The Journal of Cell Biology
Vol. 130, No. 4 (Aug., 1995), pp. 887-895
Stable URL: http://www.jstor.org/stable/1617074
Page Count: 9
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The Specific  NH2-Terminal Sequence Ac-EEED of α-Smooth Muscle Actin Plays a Role in Polymerization in vitro and in vivo
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Abstract

The blocking effect of the NH2-terminal decapeptide of α-smooth muscle (SM) actin AcEEED-STALVC on the binding of the specific monoclonal antibody anti-αSM-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-αSM-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-αSM-1. Incubation of anti-αSM-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 α-SM actin NH2-terminal decapeptide or of the epitopic peptide into cultured aortic smooth muscle cells, double immunofluorescence for α-SM actin and total actin showed a selective disappearance of α-SM actin staining, detectable at ∼30 min. When a control peptide (e.g. α-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 α-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.

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