Temperature change does not affect force between regulated actin filaments and heavy meromyosin in single‐molecule experiments

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Abstract
The temperature dependence of sliding velocity, force and the number of cross‐bridges was studied on regulated actin filaments (reconstituted thin filaments) when they were placed on heavy meromyosin (HMM) attached to a glass surface. The regulated actin filaments were used because our previous study on muscle fibres demonstrated that the temperature effect was much reduced in the absence of regulatory proteins. A fluorescently labelled thin filament was attached to the gelsolin‐coated surface of a polystyrene bead. The bead was trapped by optical tweezers, and HMM–thin filament interaction was performed at 20–35°C to study the temperature dependence of force at the single‐molecule level. Our experiments showed that there was a small increase in force with temperature (Q10= 1.43) and sliding velocity (Q10= 1.46). The small increase in force was correlated with the small increase in the number of cross‐bridges (Q10= 1.49), and when force was divided by the number of cross‐bridges, the result did not depend on the temperature (Q10= 1.03). These results demonstrate that the force each cross‐bridge generates is fixed and independent of temperature. Our additional experiments demonstrate that tropomyosin (Tm) in the presence of troponin (Tn) and Ca2+ enhances both force and velocity, and a truncated mutant, Δ23Tm, diminishes force and velocity. These results are consistent with the hypothesis that Tm in the presence of Tn and Ca2+ exerts a positive allosteric effect on actin to make actomyosin linkage more secure so that larger forces can be generated.