title: Temperature change does not affect force between regulated actin filaments and heavy meromyosin in single-molecule experiments.
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 degrees 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 (Q(10) = 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, Delta23Tm, 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.
authors.affiliation: Department of Anatomy and Cell Biology, University of Iowa, Iowa City, IA 52242, USA. masataka-kawai@uiowa.edu
authors.forename: Masataka
authors.initials: M
authors.lastname: Kawai
authors.forename: Takanori
authors.initials: T
authors.lastname: Kido
authors.forename: Martin
authors.initials: M
authors.lastname: Vogel
authors.forename: Rainer H A
authors.initials: RH
authors.lastname: Fink
authors.forename: Shin'ichi
authors.initials: S
authors.lastname: Ishiwata
mesh_terms: D008841:Actin Cytoskeleton / Q000737:chemistry*; D008967:Molecular Biology / Q000379:methods; D020409:Molecular Motor Proteins / Q000737:chemistry*; D018482:Muscle, Skeletal / Q000737:chemistry*; D015879:Myosin Subfragments / Q000737:chemistry*; D011485:Protein Binding; D012518:Sarcomeres / Q000737:chemistry*; D013314:Stress, Mechanical; D013696:Temperature
chemical_list: D020409:Molecular Motor Proteins; D015879:Myosin Subfragments