Figure 1.

Single-molecule method for observing mDia1-mediated actin polymerization. (A) Shown here is a schematic of the actin dumbbell. The gray ball represents the truncated mDia1-bound bead. Because G-actin is present in solution (small spheres), an actin filament elongates toward the bead (gray), to which an actin filament barbed end is attached via mDia1. (B) Shown here is a schematic of single- and double-trap experiments. In the double-trap experiment (left), the distance between the two trap centers is extended to generate higher tensile force (up to ∼5.5 pN), whereas in the single-trap experiment (right) the avidin bead (orange) is trapped whereas the mDia1 bead is free. (C) Shown here is kymography of the phase-contrast images of two beads. The time course of the two beads on the straight line across their centers (left image, yellow line) is shown. Double-trap experiments were performed until 12 s (blue line), when the mDia1 bead was released and stage movement was begun. Data were collected for 0.2 μM ATP-G-actin. (D) Shown here is the time course of upper bead displacement from the trap center and the bead-to-bead distance (from (C)). Tensile force was estimated by converting the upper (avidin) bead displacement from the trap center (right axis). The instantaneous change of bead-to-bead distance (arrowheads) arose from the rotation, which occurred just after repositioning the trap center, as shown in Fig. S3. To see this figure in color, go online.