Single‐Molecule Investigation of Load‐Dependent Actomyosin Dissociation Kinetics for Cardiac and Slow Skeletal Myosin

Figures

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  Figure 1. A) Stationary dual beam set up. The actin filament is held between the two beads (blue) B1 and B2 trapped with focused laser beams L1 and L2. The laser beam position remains fixed. Myosin is adsorbed onto the pedestal bead (white). Illustrated elements are not to scale. B) Actomyosin interaction when the dumbbell exerts external force (F) in the same or opposite direction to that of the powerstroke direction. “+” and ‘−’ signs indicate the actin polarity and represent the plus and minus end of an actin filament. C) Typical bead position time record elucidating the dumbbell undergoing a Brownian motion shown as large amplitude fluctuations. The intermittent interaction between actin and myosin impedes the free dumbbell movement, thus reducing the fluctuation amplitude as indicated in the record with red horizontal lines (longer‐duration events) or blue points (shorter‐duration events). Displacement (nm) and trap stiffness (pN nm−1) are used to estimate the force shown on the right Y‐axis. D) Scatter plot showing individual actomyosin interaction lifetime events at different forces measured at 500 µm ATP. The plot was fitted with a single exponential Arrhenius equation (red line,…

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  Figure 2. Square wave scanning method. A) The laser beams (L1 and L2) were synchronized to switch between rapid mobile and long stationary phases. The dumbbell thus alternates the direction along the actin filament axis (X‐direction), i.e., sweeps back and forth parallel to an actin filament past the myosin on the platform bead. B) The blue square‐shaped form shows the displacement over time trace when the trapped beads alternate between the mobile and stationary phases as they follow the laser motion. The mobile phase lasts for 0.5 ms while the stationary phase was set between 0.5 and 5 sec as per the experimental requirement. Myosin can bind to actin during the mobile phase and stationary phase. The binding events during the mobile phase are analyzed for load dependence. The actomyosin binding events are indicated during the upward (green) or downward (red) movement. ‘D’ is the distance between mean bead and the laser position, which is used to estimate the external force. Fmin and Fmax are minimum and maximum force experienced by the actomyosin crossbridge depending on the binding position during the 180 nm excursion. C) Original data trace showing the bead motion (blue) and the…

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  Figure 3. Actomyosin interaction lifetime. A) Duration of actomyosin interaction over external force measured for SolM‐II at 500 µm ATP using a square wave scanning approach. Red dots indicate individual data points. Event lifetimes under assisting and resisting load are shown with a light green and red background, respectively. B) Events shorter than 1 s (dashed box in A) are further expanded to highlight the events with the increased lifetimes under high loads. Total events n = 5112 from 54 individual myosin molecules. C) Cumulative frequency distribution of the binding events are plotted over lifetimes at different resisting loads as indicated. D) The cumulative distribution for binding events pooled from 4‐pN binwidth (i.e., 4 −7.99 pN) fitted with a double exponential function indicated as red fit, n = 1257. Double exponential fit to the distribution yields two populations of events with amplitudes Af and As with corresponding time constants τf and τs, respectively. E,F) Fast and slow detachment rates, kf and ks derived at 10 and 500 µm ATP are compared for their response to load. kf and k s were estimated from a reciprocal of time constants τf and τs, for different force range as…

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  Figure 4. Actomyosin detachment kinetics of βM‐II. A) A representative record from stationary optical trap set up with bead displacement over time trace displaying intermittent βM‐II interaction with actin at 80 µm ATP. Discernible interaction events marked with stars observed on positive displacement side in the data trace. Force values are displayed on the right Y‐axis scale. B) Scatter plot showing individual actomyosin interaction lifetime events measured at load range of −2–+2 pN using stationary trap setup. Longer lifetime events have been observed with increasing resisting load. Data was fitted with the single exponential (fit shown as a purple line) as described in Figure 1D to estimate “k 0” and “d”, N = 5, n = 1482. C) Histogram of lifetimes of the binding events. Events shown in dotted rectangle in Figure B are binned, n = 541. Maximum likelihood estimation (MLE) was employed to calculate the average lifetime (τ) and thereby detachment rate (1/τ) of 33.5 ± 2.78 s−1, n = 422. D) Detachment rates over force plot. Binwidth of 0.5 pN was applied to pool the events within corresponding force range. Events in 1–2 pN force range were pooled for the last data points. Plot fitted with…

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  Figure 5. A) For βM‐II, actomyosin binding likelihood during actin filament excursion in alternating directions using square wave scanning at 1 µm ATP; n = 733 and 713 on the resisting and assisting sides, respectively, yielding a ratio of 1.02. B,C) Actomyosin detachment rates are compared between βM‐II at 1 µm ATP and SolM‐II at 10 µm ATP. For βM‐II, events were pooled for 4‐pN binwidth, and the average lifetime (τ) was determined by fitting the events with two exponential fits as explained in Figure 3D. Reciprocal of time constants, i.e., detachment rates (kf and ks ) estimated and plotted over external force. The detachment rates were plotted at increasing resisting and assisting load. D) The fraction of events (Af) corresponding to first population (i.e., kf) at various loads are compared for βM‐II and SolM‐II. At 1 µm ATP; resisting load n4 = 101, n4−8 = 206, n8−12 = 344, n12−16 = 101, Assisting load; n4 = 91, n4−8 = 205, n8−12 = 329, n12−16 = 109, total events = 1486, N = 30 n = number of events, N = number of myosin molecules.

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  Figure 6. Actomyosin crossbridge cycle. Key intermediate states are shown. As the myosin interacts with actin, strongly bound transition states are indicated (blue myosin heads). Our proposed post‐powerstroke AM.ADP.Pi state is indicated with a grey motor head. The three load‐responsive states identified in our studies as are shown. Only single myosin heads are shown. A – Actin, M – myosin. A‐M indicates weak interaction between actin and myosin, AM – strong interaction.