Characterizing the concentration and load dependence of phosphate binding to rabbit fast skeletal actomyosin

Figures

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  Fig. 1.. Miniensemble laser trap assay and representative displacement records. (A) A schematic of the miniensemble laser trap assay. Displacements of the actin filament were detected with a quadrant photodiode (detector) using backfocal plane interferometry. (B and C) Representative displacement records obtained in the absence and presence of increasing concentrations of Pi at two of the three trap stiffness values used. Laser trap stiffnesses are shown in picoNewtons per nanometer (pN·nm−1). A custom algorithm within R was used to detect binding events (Materials and Methods), with events colored based on the [Pi] and laser trap stiffness. The algorithm determined the duration of each binding event (red double arrow, ton) based on the time spent >8 nm above average baseline. Peak force was determined from the peak displacement, for a 5 ms window, multiplied by the corresponding laser trap stiffness.

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  Fig. 2.. Actomyosin binding event lifetimes. (A) Cumulative distributions of binding event lifetimes (main graphs). Increasing laser trap stiffnesses (0.04, 0.10, and 0.20 pN·nm−1) indicated by progressively darker shades of green (0 mM-Pi), blue (5 mM-Pi), purple (10 mM-Pi), and orange (30 mM-Pi), with the darkest shades indicating the highest stiffness values. Insets are dot-plot distributions of the individual event lifetimes. (B) Bar graph of mean ± SEM for binding event lifetimes at each laser trap stiffness and [Pi] (colored the same as in A). Significant differences were detected using a nonparametric Kruskal–Wallis ANOVA followed by two-tailed tests for independent samples using SPSS®. A Bonferroni adjustment was made for multiple comparisons. * indicates significantly (P < 0.05) different from the control condition (0 mM-Pi) at each laser trap stiffness. # indicates significantly different from the attached lifetime at 0.04 pN·nm−1 stiffness and the same [Pi]. ‡ indicates significantly different from all [Pi] at the same laser trap stiffness. The sample sizes for each condition are listed in the legend of Table 1.

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  Fig. 3.. Binding event lifetimes at low [ATP]. (A and B) Representative displacement vs. time records from the miniensemble assay at 5 µM ATP, in the absence (green) and the presence (orange) of 30 mM Pi. Actomyosin binding events were detected using the custom algorithm within the R software program described in the Materials and Methods. (C) Cumulative distribution of binding event lifetimes at 5 µM ATP in the absence (n = 658 events) and presence (n = 668 events) of 30 mM-Pi. (D) Mean ± SEM of the binding event lifetimes generated at each [Pi] at laser trap stiffness of 0.04 pN·nm−1. * indicates significantly (P < 0.05) different from the control (0 mM-Pi) at the corresponding trap stiffness. # indicates significantly (P < 0.05) different from the binding event lifetime at 100 µM-ATP at the corresponding [Pi].

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  Fig. 4.. Force generated by myosin ensembles. (A) Schematic of miniensemble laser trap assay. Force is the product of trap stiffness (ktrap) and the displacement from trap center (δ) (Bottom) raw displacement records at laser trap stiffnesses of 0.04 (black), 0.10 (yellow), and 0.20 pN·nm−1 (gray). (B) Distributions of peak forces attained at each [Pi]. (C) Mean ± SEM of the peak force generated at each stiffness and [Pi]. Significant differences were detected using a nonparametric Kruskal–Wallis ANOVA followed by two-tailed tests for independent samples within SPSS®. A Bonferroni adjustment was made for multiple comparisons. * indicates significantly (P < 0.05) different from control (0 mM added Pi) at the corresponding trap stiffness. # indicates significantly different from the attached lifetime at 0.04 pN·nm−1 stiffness at the same [Pi]. ‡ indicates significantly different from all [Pi] at the same laser trap stiffness. (D) Relative decrease in force as a function [Pi] for each stiffness level as indicated. Data fit to a dose–response curve with EC50 values being 24 ± 4 mM, 20 ± 3 mM and 2 ± 0.5 mM for 0.04, 0.10, and 0.20 pN·nm−1 trap stiffness, respectively. Differences between the…

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  Fig. 5.. Binding event displacements. (A) Representative records of miniensemble runs at each [Pi] at 0.20 pN nm−1 laser trap stiffness. Records are 250 ms in duration. (B) Mean ± SEM of the peak displacements generated at each stiffness and [Pi] (colored as in Fig. 2B). Significant differences determined using a nonparametric Kruskal–Wallis ANOVA followed by two-tailed tests for independent samples in SPSS®. A Bonferroni adjustment was made for multiple comparisons. * indicates significantly (P < 0.05) different from the control condition (0 mM added Pi) at the corresponding trap stiffness. # indicates significantly different from 0.04 pN nm−1 stiffness at the same [Pi]. ‡ indicates significantly different from all [Pi] at the same stiffness. (C) Cumulative distribution of time to peak displacements. After the analysis identified the start of an event (Materials and Methods), the attachment period from the beginning of a binding event to the peak displacement (Tpeak) was determined. (D) Mean ± SEM for rate of force development (rate of force) at each stiffness and [Pi]. *, # and ‡ same as in Fig. 3A.

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  Fig. 6.. Load dependence of the Pi-induced detachment rate. (A) A raw displacement record depicting the method used for determining the time spent at peak force (TPF) within a binding event, which was identified using a 5 ms moving window (shaded area); see Materials and Methods for more detail. (B) kdet (1/lifetime) plotted against the peak force (mean ± SEM). Data fit to the Bell-bond equation, kdet = k0 × exp(Fd/kT); where kdet is the observed detachment rate (1/lifetime), k0 the predicted detachment rate in the absence of load, F the peak force, d the fit parameter defining the curvature of the relationship, k the Boltzmann constant and T the temperature in Kelvin. The solid line represents 0 mM-Pi (green) and dashed lines in the presence of 5 mM (blue), 10 mM (purple), and 30 mM-Pi (red). The fit parameters (mean ± SEM) were k0 = 124.39 ± 9.47 s−1 and d-value was −0.89 ± 0.12 nm, R2 = 0.99 (0 mM added Pi); k0 = 119.91 ± 10.20 s−1 and d = −0.21 ± 0.11 nm, R2 = 0.80 (5 mM Pi); k0 = 122.93 ± 6.84 s−1 and d = −0.03 ± 0.08 nm, R2 = 0.14 (10 mM Pi); and 112.25 ± 7.67 s−1 for k0 and d = 0.34 ± 0.09 nm, R2 = 0.94 (30 mM Pi). (C) Bar graphs of the means ± SEM of the fit parameters to the…

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  Fig. 7.. Model fits to data. (A) Fig. 7 Branched Pathway and Powerstroke Reversal models reproduce our measurements. (A) The chemomechanical schemes used for simulations. The left pathway depicts the Branched Pathway model (green) and the right the scheme used for the Powerstroke Reversal model (red). In these schemes, D stands for ADP, P inorganic phosphate, and T ATP. States with myosin bound to actin are on the top, unbound states on the bottom, and the powerstroke is indicated by a rotation of the myosin head in the cartoon. Each reaction is labeled with a rate constant, and force-dependent rate constants are indicated by (F). (B, Top) Raw experimental displacement records of the actin filament in the laser trap assay in the absence (Left) and presence (Right) of 30 mM Pi at low and high stiffness, respectively. Detected binding events are colored in turquoise. Middle traces are from simulations from the Branched Pathway model (green) and the lowest trace are records from the Powerstroke Reversal model (red). (C) Violin plots of the distribution of the RFD obtained from simulations of each model superimposed on the experimental distributions. Points represent means and lines connect…