Tropomyosin and Caldesmon Enhance the Binding Force of Unphosphorylated Myosin to Thin Filaments

Abstract

Smooth muscle is unique in its ability to maintain force for long periods of time at low energy consumption. This property, called the latch-state, has been postulated to be a consequence of the dephosphorylation of myosin molecules when attached to the actin thin filament. Alternatively, unphosphorylated (unPHOS) myosin could potentially attach to the thin filaments, contributing to force maintenance. Thus, in this study we verified if unPHOS myosin can bind to actin in presence of tropomyosin and caldesmon and we measured its binding force using the laser trap. Briefly, a microsphere captured in a laser trap was attached to an actin filament. The filament was brought in contact with a pedestal coated with unPHOS myosin. The pedestal was then moved away from the trap at constant velocity. The microsphere followed the pedestal until the force exerted by the trap on the microsphere was sufficient to overcome the binding force of myosin to the actin filament. At this point, the microsphere sprang back into the trap center. The force of unbinding (Funb) was calculated as the product of the trap stiffness and the maximal distance between the trapped microsphere and the trap center. Funb was normalized by the number of myosin molecules estimated per actin filament length. Funb of unregulated actin filaments (0.118±0.007 pN; mean±SE) was enhanced in presence of tropomyosin (0.16±0.008 pN; p<0.05), caldesmon (0.169±0.017 pN; p<0.05) and a mix of both regulatory proteins (0.174±0.022 pN; p<0.05). Our data demonstrate that unPHOS myosin binds to regulated actin filaments and that each regulatory protein increases this binding force but that their effect is not cumulative. Thus, the actin regulatory proteins might play a major role in the latch-state, by enhancing the binding of unPHOS myosin.