Using Single Myosin and Small Myosin Ensembles to Examine Thin Filament Activation in a Laser Trap Assay

Abstract

Thin filament activation in muscle requires both Ca++ binding to troponin and myosin strongly binding to actin. Despite extensive study, the relative importance of each of these processes to overall thin filament activation remains unclear. To determine the role of Ca++ we examined the effect of increasing Ca++ concentrations on the frequency of single molecule binding events in a single molecule laser trap assay using reconstituted thin filaments. The frequency of actomyosin binding events increased from 0.03 at low Ca++ (pCa7), to 0.30 at pCa6 to 0.44 events/sec at pCa5. Increasing the myosin concentration so that the regulated filament interacted with a mini-ensemble of myosin (∼6 heads) in the laser trap assay was performed to gain insight into the cooperative nature of actomyosin binding. Binding events were broadly distributed at the highest Ca++ (pCa5), generating forces from 0.2 to 6.75pN, but averaged 1.38±0.05pN. As Ca++ was decreased the high force events were lost, reducing the average force to 0.76pN at pCa6, 0.61 at pCa7 and 0.2pN at pCa9. In addition the total number of actomyosin interactions decreased from 3.7/sec at pCa5 to just 0.05/sec at pCa9. Finally, we measured filament velocity in a motility assay as a function of Ca++ and ATP to distinguish rigor activation vs. Ca++ activation in a large ensemble of myosin. At pCa9 filaments were activated at ATP levels <200 μM while at intermediate Ca++ levels (pCa6.5) filaments achieved only half of the maximum velocity observed at saturating Ca++ (pCa5). Preliminary modeling efforts suggest that these observations are consistent with a model where Ca++ regulates myosin's weak to strong binding transition but once bound strongly an actomyosin interaction activates the filament locally, facilitating neighboring molecules to bind.