Localization of isoactins in isolated smooth muscle thin filaments by double gold immunolabeling

Abstract

Smooth muscle cells contain both smooth muscle-specific and cytoplasmic actin isoforms. We tested the hypothesis that these two classes of isoactins are segregated into separate "contractile" vs. "cytoskeletal" populations of thin filaments. Isolated thin filaments from adult swine stomach were double-labeled with isoactin-specific peptide antibodies with the use of two different size protein A-gold markers to localize the isoactins on individual filaments. The large and small gold beads bound to individual filaments were counted on electron micrographs, and the results were analyzed statistically. Both classes of actin isoforms were present in every filament, and the relative proportions of smooth muscle vs. cytoplasmic actin isoforms in each filament showed no significant deviation from a single random population. Furthermore, the distribution of the isoactins along each filament was also random; no significant clustering of isoforms was observed. Controls containing added skeletal muscle F-actin showed that the techniques used could have detected nonrandom isoform distributions if they had been present.