Single-molecule analysis reveals that regulatory light chains fine-tune skeletal myosin II function

Abstract

Myosin II is the main force-generating motor during muscle contraction. Myosin II exists as different isoforms that are involved in diverse physiological functions. One outstanding question is whether the myosin heavy chain (MHC) isoforms alone account for these distinct physiological properties. Unique sets of essential and regulatory light chains (RLCs) are known to assemble with specific MHCs, raising the intriguing possibility that light chains contribute to specialized myosin functions. Here, we asked whether different RLCs contribute to this functional diversification. To this end, we generated chimeric motors by reconstituting the MHC fast isoform (MyHC-IId) and slow isoform (MHC-I) with different light-chain variants. As a result of the RLC swapping, actin filament sliding velocity increased by ∼10-fold for the slow myosin and decreased by >3-fold for the fast myosin. Results from ensemble molecule solution kinetics and single-molecule optical trapping measurements provided in-depth insights into altered chemo-mechanical properties of the myosin motors that affect the sliding speed. Notably, we found that the mechanical output of both slow and fast myosins is sensitive to the RLC isoform. We therefore propose that RLCs are crucial for fine-tuning the myosin function.

Keywords: actin; actin filament gliding assay; molecular imaging; molecular motor; muscle contraction; myosin; myosin II; optical trapping; optical tweezers; regulatory light chain; single-molecule analysis; single-molecule biophysics; solution kinetics.

Figure 1.

Myosin S1 structure and ATPase cycle. A, crystal structure of scallop myosin II-subfragment-1 (Protein Data Bank code 1SR6) (62). The myosin heavy and light chains are shown as a ribbon diagram; S1 is indicated with the key parts of the motor domain. B, kinetic scheme for actomyosin ATPase. Myosin II with different nucleotide states is illustrated: A, actin; M, myosin; T, ATP; D, ADP; Pi, P_i. Strong and weak interaction states of myosin with actin are labeled in black or green, respectively. Rate constants for forward and reverse reactions are indicated as k_+n and k_−n, respectively.

Figure 2.

Reconstitution of chimeric motors. A, scheme illustrates in vitro reconstituted motors with different combinations of RLCs with fast (dark blue) and slow myosin II (sky blue) heavy chains. Papain digestion of full-length myosin II generated single-headed myosin S1. Regulatory light chains are color-coded to indicate the exchange: MLC2B (pink), MLC2v (green), and cgmRLC (orange). B, in vitro motility assay using native myosin S1 (WT-S1f and WT-S1s) or with chimeric motors. Motors were immobilized on a nitrocellulose-coated surface. Speed of movement was measured at saturating ATP concentration of 2 mm at room temperature (22 °C). Bar diagrams show the reduction in mean velocity from 1.2 ± 0.19 for WT-S1f to 0.44 ± 0.13 μm/s for S1f-MLC2v (N = 72 and 100 actin filaments, respectively) and the increase in mean velocity for WT-S1s from 0.047 ± 0.05 to 0.40 ± 0.05 μm/s for S1s-cgmRLC (N = 82 and 166 filaments, respectively). The motility experiments were performed with at least three different preparations of myosin motors and chimeras and were highly reproducible. For S1f-cgmRLC, N = 70; for S1f-MLC2B, N = 110; for S1s-MLC2v, N = 56; and for S1s-MLC2B, N = 55. Error bars, S.E. Statistical significance was calculated using unpaired t test for the following pairs of motors: WT-S1f and S1f-cgmRLC, p < 0.0001; WT-S1f and S1f-MLC2v, p < 0.0001; WT-S1s and S1s-cgmRLC, p < 0.0001; WT-S1s and S1s-MLC2B, p < 0.0001.

Figure 3.

Ensemble kinetic experiments. Plots of ATP turnover rates as a function of F-actin concentration for fast (A) and slow (B) myosins. Data were fitted to hyperbolas using the Michaelis–Menten formula. The catalytic efficiency, k_cat/K_app, was obtained from the initial slope of the hyperbola. C and D, ADP-release kinetics from actomyosin. The rate of ADP dissociation from the actomyosin complex was measured by rapidly mixing actomyosin-mantADP complex with excess ADP. The decrease in mant fluorescence followed single exponential kinetics in the case of the fast myosins (C). For the slow myosin constructs, the fluorescence transients were best described by two exponentials with a fast rate constant (k_−AD^fast) and a slow rate constant (k_−AD^slow) indicative of two-step ADP-release kinetics (D). ATP turnover experiments of actomyosin constructs used coumarin-labeled phosphate-binding protein (MDCC-PBP; Thermo Fisher Scientific) as a sensor for the detection of liberated P_i from fast (E) and slow (F) myosins. The transients follow single exponentials with a corresponding rate constant for P_i release (k_−Pi). All kinetic parameters are summarized in Table 1.

Figure 4.

Single-molecule optical trapping: lifetime of AM interactions. A, the experimental setup for three-bead optical trapping measurements. Note that the indicated components, bead size, and protein dimensions are not to scale. B, original displacement over time data records; actin-myosin interaction can be observed as a reduction in the large thermal fluctuations from a bead at 10 μm ATP. The top and bottom panels show records collected from fast and slow WT myosin S1, respectively. Some of the interaction events are shown as green (fast WT) and red (slow WT) lines to indicate the duration of lifetimes, t_on. Measurements were done by applying positive feedback and triangular wave (∼600 Hz). C–J, for WT-S1f, WT-S1s, and chimeras, event lifetimes (t_on) are plotted in histograms and fitted with a single-exponential decay function to determine the average time constant. K, the bar diagram shows the average t_on for WT and chimeric motors at 10 μm ATP concentration and room temperature of ∼22 °C. Error bars, S.E. (from the fits). For WT-S1f, n = 5000 events, N = 21; for WT-S1s; n = 1675 events, N = 19, for S1f-cgmRLC; n = 1517 events, N = 10, for S1f-MLC2B; n = 1953 events, N = 16, for S1f-MLC2v; n = 5714 events, N = 22, for S1s-cgmRLC; n = 1468 events, N = 12, for S1s-MLC2v; n = 400 events, N = 4, for S1s-MLC2B; n = 900 events, N = 8 (where N = number of individual motor molecules and n = number of AM interaction events). Statistical significance was calculated for the following pairs of motors using nonparametric Mann–Whitney U test: WT-S1f and WT-S1s, p < 0.00001; WT-S1s and S1s-cgmRLC, p < 0.0001; WT-S1s and S1s-MLC2B, p < 0.0001; WT-S1f and S1f-MLC2B, p = 0.0691; WT-S1f and S1f-MLC2v, p < 0.0001, WT-S1f and S1f-cgmRLC, p = 0.114; WT-S1s and S1s-MLC2v, p = 0.0941. p < 0.05 was considered statistically significant; p > 0.05 was considered not statistically significantly different.

Figure 5.

Powerstroke size of chimeric motors. A–H, the average stroke size/mean displacement was determined by histogram shift (δ) from mean free dumbbell noise. The histograms fitted with Gaussian function to determine the average stroke size for the indicated myosin motors. I, the average stroke size determined from the Gaussian fits for WT and chimeric motors were compared in a bar diagram. Error bars, S.E. from the fits. The statistically significant difference in the powerstroke size was calculated for the following pairs of motors: WT-S1f and WT-S1s, p < 0.0001; WT-S1f and S1f-cgmRLC, p = 0.8841; S1f and S1f-MLC2B, p = 0.0053; S1f and S1f-MLC2v, p = 0.0025; WT-S1s and S1s-cgmRLC, p = 0.1002; WT-S1s and S1s-MLC2B, p < 0.0001; WT-S1s and S1s-MLC2v, p < 0.0001. Statistical significance was calculated using unpaired t test.

Figure 6.

Stiffness. A and B, original data records acquired in optical trapping experiments. Bead position is plotted over time for both the left and right bead of the dumbbell. Positive position feedback was used to increase the amplitude of thermal fluctuations, which effectively increased the variance ratio between binding events and free dumbbell noise for both traps in the direction of the actin filament axis. C and D, variance versus time plotted for the records shown in A and B. Variance was calculated for a rolling window of 20 ms, at our sampling rate of 10 kHz. For the example shown here, the variance-Hidden-Markov method yielded a combined trap stiffness of 0.078 pN/nm and a myosin head stiffness of 0.85 pN/nm. E, box plot with overlapped data points, the stiffness measured for WT-S1f, WT-S1s, and chimeric motors. Each rhombus represents the measured stiffness from individual myosins. Average stiffness with S.D. was as follows: for WT-S1f, −1.12 ± 0.03; for S1f-MLC2B, −1.24 ± 0.09; for S1f-MLC2v, −1 ± 0.07; for WT-S1s, −0.437 ± 0.08; for S1s-cgmRLC, −0.585 ± 0.024; for S1s-MLC2B, −1.62 ± 0.27; for S1s-MLC2v, −0.533 ± 0.11. Unpaired t test was used to calculate the statistical significance. WT-S1f (n = 20) and WT-S1s (n = 13) displayed statistically significant difference with p < 0.0001. No statistically significant difference between WT-S1f and S1f-MLC2B (N = 12, p = 0.72) or WT-S1f and S1f-MLC2v (N = 15, p = 0.128) was found. For WT-S1s and S1s-cgmRLC (N = 10), marginal but statistically significant difference was found (p = 0.0493). For WT-S1s and S1s-MLC2B, N = 6 and p < 0.0001. For WT-S1s and S1s-MLC2v, n = 11 and p = 0.4735. N = number of individual motor molecules.