A mutation in switch I alters the load-dependent kinetics of myosin Va

Fig. 1. Mini-ensemble laser trap assay and representative displacement records. a A schematic of the mini-ensemble laser trap assay depicting the geometry of the three-bead laser trap assay. Displacement of the bead-actin-bead assembly was detected using a quadrant photodiode (detector). The myosin Va S1 constructs were adhered to the coverslip surface using c-Myc monoclonal antibody (Thermo-Fisher Scientific Inc., catalog # 13-2500). b, c Representative displacement vs. time records for both constructs (WT and S217A) in the absence and presence of 30 mM Pi, and at two of the three trap stiffnesses used in the study. Actomyosin binding events were detected using a custom algorithm within the R project for statistical computing (see Methods). The detected events are indicated as the change in color from black to the corresponding color for the laser trap stiffness and [Pi]. The duration of the binding event was determined using the same custom algorithm, and the peak force was determined from the peak displacement that was sustained for 5 msec (ton), multiplied by the appropriate laser trap stiffness (Force=ktrap x displacement from trap center). The scale for the displacement and time…

Fig. 2. Actomyosin binding event attachment times. a Main graphs, cumulative distribution of binding event lifetimes for each construct in the presence and absence of Pi as indicated. The three trap stiffness values used (0.04, 0.06 and 0.10 pN.nm−1) are represented by increasing darker shades of either blue (WT) or maroon (S217A), darkest shade is highest stiffness. Insets show the same data as a bar graph with the corresponding conditions as indicated. b Mean ± SEM of binding event lifetimes for both constructs and each stiffness in the presence and absence of 30 mM Pi as indicated. Significant differences were detected using a non-parametric Kruskal–Wallis ANOVA followed by tests for independent samples using SPSS®. * indicates significantly (p < 0.05) different from the control solution (0 mM Pi) for each myosin Va construct. # indicates significantly different from the attached lifetime from WT myosin at the corresponding [Pi] and laser trap stiffness. ‡ indicates 0.10 pN.nm−1 in the absence of Pi is significantly different from the lifetime at 0.04 pN.nm−1 in the absence of Pi for each myosin construct. The number of binding events was 1017, 578 and 1295 for WT in the absence of…

Fig. 3. Binding event displacements and forces. a A schematic of the laser trap assay. Force was equal to trap stiffness (ktrap) multiplied by the displacement from trap center (δ) b Raw displacement records at increasing trap stiffness values from 0.04 (black) to 0.06 (pink) to 0.10 pN.nm−1 (gray). c The power density function (PDF) at each trap stiffness from 0.04 to 0.10 pN.nm−1, determined from the raw displacement records of bead-actin-bead assembly alone (Fig. 3b). Significant differences were detected using a non-parametric Kruskal-Wallis ANOVA followed by two-tailed tests for independent samples using SPSS®. No adjustment was made for multiple comparisons d Mean ± SEM for peak displacements at each trap stiffness, in the absence and presence of 30 mM Pi. * indicates significantly (p < 0.05) different from the control, 0 mM Pi. # indicates significantly different from the attached lifetime for WT myosin at the corresponding [Pi] and laser trap stiffness. ‡ indicates that 0.10 pN.nm−1 in the absence of Pi is significantly different from the lifetime at 0.04 pN.nm−1 in the absence of Pi for each myosin construct. e Mean ± SEM of the peak force generated at each stiffness and [Pi].…

Fig. 4. Load-dependence of the Pi-induced detachment rate and the hypothesized effect on the energy landscape. a A raw displacement record depicting the method used to determine the time spent at peak force (TPF), identified by sliding a 5 ms window over the data. Once the peak was determined the time spent at that peak force was determined until the run was terminated (shaded area), see Methods for more detail. b The durations at peak force were converted to rates (kdet) and plotted against the peak force. Values represent the mean ± SEM. Data were fit with the Bell-bond equation: kdet = ko x exp(Fd/kT). The fit parameters for WT were ko = 16.8 ± 1.8 s−1 (mean ± SEM) and the d value was −0.9 ± 0.3 nm (mean ± SEM) in the absence of Pi and 13.7 ± 0.2 s−1 for ko and 0.6 nm for d in the presence of 30 mM Pi. For the S217A construct ko was 11.1 ± 0.3.0 s−1 and a d-value of −0.2 ± 0.7 nm in the absence of Pi, and ko was 2.5 ± 1.1 s−1and the d-value 4.1 ± 0.9 nm in the presence of Pi. c Bar graph of means ± SEM of the fit parameters to the Bell-bond equation. Differences between the fit parameters to the Bell-bond equation were determined by constructing 95% confidence intervals around the…