In Vitro Motility Analysis of Actin-Tropomyosin Regulation by Troponin and Calcium

Abstract

In striated muscles, contractility is controlled by Ca[IMG] binding to the regulatory protein complex troponin, which is a component of the thin filaments. Troponin is an allosteric inhibitor acting on tropomyosin to switch the thin filament between ``on'' and ``off'' states. We have used an in vitro motility assay to examine troponin regulation of individual actin-tropomyosin filaments moving over immobilized skeletal muscle heavy meromyosin. The most striking observation is that the actin-tropomyosin filament appears to be regulated as a single unit. At pCa 9.0, addition of up to 4 nM troponin causes the proportion of filaments motile to decrease from >85% to 20% with no dissociation of the filaments from the heavy meromyosin surface or change in velocity. Increasing Ca[IMG] concentration causes the filaments to be switched back on with half-maximal increase in the proportion of filaments motile at pCa 5.8-6.0 and a modest increase in filament velocity. This is an ``all or none'' process in which an entire filament, up to 15 {micro}m long, switches rapidly as a single cooperative unit. Thus, the effect of Ca[IMG] upon the thin filament is to recruit motile filaments.