Endocytic myosin-1 is a force-insensitive, power-generating motor

Figures

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  Figure 1:. Models for the functions of actin assembly and myosin activity during membrane deformation for clathrin-mediated endocytosis Cartoon diagram illustrating the organization of actin filaments and Myo5 molecules at endocytic sites. Actin filaments are bound by coat proteins at the tip of the growing membrane invagination and oriented with their growing ends toward the plasma membrane, powering membrane invagination. The type I myosin Myo5 could either anchor the actin network in a favorable orientation (left) or provide an assisting force (right).

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  Figure 2:. In-solution, population biochemical characterization of Myo5 (A) Coomassie-stained SDS-polyacrylamide gels showing example preparations of the purified Myo5 motor/lever construct and calmodulin (Cmd1, light chain) used in all experiments. (B) The actin concentration dependence of the steady-state ATPase activity of 100 nM unphosphorylated (grey circles) and phosphorylated Myo5 (black circles). Each data point represents the average of 6–7 time courses, which were 100 s each. The orange line is a best fit of the phosphorylated Myo5 data to a rectangular hyperbola. (C) Schematic pathway for the Myo5 ATPase cycle. Blue motors are in tightly bound conformations, green motors are weakly bound/unbound. (D) Example light scattering transients reporting on ATP-induced dissociation of phosphorylated (left, kobs=17 s−1) and unphosphorylated (right, kobs=64.1 s−1) actoMyo5, obtained by mixing 100 nM actoMyo5 (AM) with 94 µm and 72 µM ATP, respectively, as shown in the inset schematic. The black line is the fit of a single exponential function to the data. (E) ATP concentration dependence of dissociation of 100 nM unphosphorylated (grey circles) and phosphorylated actoMyo5 (black circles).…

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  Figure 3:. Single molecule, optical trap analysis of Myo5 step size and kinetics (A) Cartoon schematic of the 3-bead optical trapping setup. A biotinylated actin filament is tethered between two neutravidin-coated beads that are trapped in a dual beam optical trap. This bead-actin-bead “dumbbell” is lowered onto pedestal beads that have been sparsely coated with His6 antibody to attach Myo5-motor/lever-Avi-Tev-His9. (B-D) Single Myo5 displacements of a single bead position and covariance traces, calculated using both beads, showing single molecule interactions acquired in the presence of 1 µM (B) 10 µM (C) and 1000 µM ATP. (D). Blue bars indicate attachment events as identified by covariance (gray) decreases. The threshold of event detection by the covariance traces are indicated by dashed gray lines. (E) Schematic of displacement traces depicting the 2-step nature of actomyosin displacements in the optical trap. (F-H) Binding events were synchronized at their beginnings (left) or ends (right) and averaged forward or backward in time, respectively. Measured total displacement of Myo5 was 5.0 nm at 10 µM ATP, with the 1st substep contributing a 4.8 nm displacement (arrow 1. in G) and the 2nd…

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  Figure 4:. Myo5 attachment lifetimes are substantially less force-dependent than other known type I myosins An isometric optical force clamp was utilized to determine the force-sensitivity of the detachment of Myo5 from actin. (A) Durations of individual actomyosin attachments as a function of force, plotted on a semi-log scale (B) The solid black line shows the force dependence of the detachment rates determined by MLE fitting of unaveraged points in A. For illustration purposes, attachment durations from (A) were binned by force at every 10 points, averaged, and converted to rates. Best- fit parameters were determined by MLE fitting and 95% confidence intervals were calculated via bootstrapping. The solid black line is calculated from best fit parameters (k=67.6 s−1, d=1.14 nm), while the gray shaded region is the 95% confidence interval (k=67.6−72.9 s−1, d=1.03−1.26 nm). All MLE fitting was performed on unaveraged data and was corrected for instrument deadtime. (C) The force dependent detachment rate of Myo5 (from panel B) plotted alongside the force dependent detachment rates for Myo1b, Myo1c, and β-cardiac muscle myosin, Myh7. (D) Power output for the same four myosins calculated over…