A Myosin II-Based Nanomachine Devised for the Study of Ca2+-Dependent Mechanisms of Muscle Regulation

Figure 1. (a) Cartoon illustrating sarcomere shortening due to the reciprocal sliding between the myosin filament (black) originating from the center of the sarcomere (M line) and the actin filament (yellow) originating from the Z line at the end of the sarcomere (green), powered by the interdomain structural change in the S1 fragment of the myosin motor (orange) from the pre- (upper panel) to the post-working stroke conformation (lower panel). (b) Schematic representation of the half-sarcomere protein assembly at rest. Shown are actin (yellow), tropomyosin (Tm, red) and troponin complex (Tn, light and dark gray and brown) on the thin filament. On the thick filament (black), most of the S1 fragments of myosin dimers (orange) lie tilted back (OFF state) and the S1 fragments of two dimers move away with the tilting of their S2 rod-like domain (ON state); the MyBP-C (blue) lies on the thick filament with the C-terminus and extends to the thin filament with the N-terminus. Titin (pink) in the I-band connects the Z line to the tip of the thick filament and in the A-band runs on the surface of the thick filament up to the M line at the center of the sarcomere.

Figure 2. Performance of the rabbit HMM-based nanomachine. (a) Schematic representation of two snapshots during the interaction between the actin filament and the motor ensemble. Upper panel: in position clamp at F0; lower panel: in force clamp at 0.4 F0. (b) Recording of the actin filament sliding (L, upper trace, red) and force (F, lower trace, blue) during an interaction. Numbers bounded by dashed lines identify the different time intervals (it) as detailed in the text. (c) Frequency distribution of F0. Data are plotted in classes of 3 pN; dark gray bars, measurements in 0.1 mM CaCl2 (= 77 µM free [Ca2+]); light gray dashed bars, in the absence of Ca2+. (d) F-V relation in 0.1 mM CaCl2 (dark gray open circles) and in Ca2+-free solution (light gray open circles). Points are mean ± SD from individual experiments, grouped in classes of force 0.15 F0 wide. Dark and light gray filled symbols on the ordinate are the Vf in the in vitro motility assay (IVMA) on rabbit HMM with and without Ca2+, respectively. The dashed lines are Hill’s hyperbolic equation fits to the data with the same color code as symbols. Data in c and d are from 28 experiments in 0.1 mM CaCl2 and 23 experiments in Ca2+-free…

Figure 3. Model simulation. (a) Kinetic scheme from [10], with three states of the myosin motor (blue): D, detached; A1 and A2, attached to an actin monomer (brown). During shortening, the motor attached in the A2 state can slip to the next actin monomer farther from the center of the sarcomere (red) within the same ATPase cycle. The probability of a second slipping to the pink monomer is limited to 1/10 of that of the first slip. (b) Simulation of the F-V relation of fast mammalian muscle (adapted from supplementary figure 6f in [10]). The experimental F-V relation (filled circles) is calculated for the half-sarcomere at full overlap (294 motors available) from the data of fast mammalian muscle at room temperature [31,32]. The dashed line is Hill’s hyperbolic equation fitted to the results of the model simulation with a series compliance similar to that in situ (0.01 nm pN-1). (c) P-F relation calculated from Hill’s fit in b (adapted from supplementary figure 6g in [10]). (d) Experimental F-V relation obtained by pooling data in the absence and the presence of Ca2+ from Figure 2 (open circles) and fitted by Hill’s hyperbolic equation (dashed line), and its simulation calculated for a…