Cytoskeletal targeting of calponin in differentiated, contractile smooth muscle cells of the ferret

摘要

Biochemical and quantitative image analysis methods were used to investigate the anatomical basis for the previously described agonist-induced redistribution of calponin.At 140 nm resolution, the quantitative distribution of calponin in resting cells was statistically indistinguishable from that of filament bundles containing α-smooth muscle actin and myosin, but was significantly different from that of filaments containing β-non-muscle actin. Conversely, in stimulated cells, the distribution of calponin was not significantly different from that of β-actin filaments in the subplasmalemmal cell cortex but was significantly different from the distribution of α-actin- and myosin-containing filamentous bundles.The distribution of calponin significantly differed from that of the intermediate filament proteins vimentin and desmin as well as that of the dense body protein α-actinin either by ratio analysis of the subcellular distribution or by colocalization analysis.The imaging results, although limited to 140 nm spatial resolution, suggested the hypothesis that the agonist-induced redistribution involves the binding of calponin to isoform-specific actin filaments. This hypothesis was tested by quantifying the relative affinity of calponin for purified α- and β-actin. Light scattering measurements showed that calponin induces bundle formation with β-actin more readily than α-actin, indicating that calponin may be preferentially sequestered by β-actin under appropriate conditions.These results are consistent with a model whereby agonist activation decreases calponin's binding to filaments, but the tighter binding to β-actin filaments results in a spatial redistribution of calponin to the submembranous cortex.