class="enumerated" style="list-style-type:decimal">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.
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