Myosin VI–coated nanosphere movement on extracted keratocyte actin networks does not result in or require actin filament reorganization. (a, left) Platinum replica micrograph of an extracted keratocyte prepared after movement of myosin VI artificial dimer-coated nanospheres on its surface. (a, middle) A higher resolution image showing the size of nanospheres relative to the F-actin network size. (a, right) A schematic diagram depicting the relative sizes of a nanosphere (highlighted by the green circle in the center and right panels), the myosin VI dimers, and the keratocyte actin network. The right panel shows two dimers bound to actin filaments, coupled through the nanosphere (green line), and bound to the nanosphere at the yellow circles. (b) Trajectories of movement of myosin VI dimer-coated nanospheres on a keratocyte extracted and fixed with 2% formaldehyde for 10 min. (c) Sample trajectories from panel b without exception show movement toward the cell center. Bars, 1 µm.

Movement of myosin VI monomer-coated nanospheres on extracted keratocyte actin networks assessed from the experiment and simulation. (a) A schematic of the simplified model used. (a, left) Four monomers are coupled through the nanospheres, all bound to the actin filaments (the myosin VI flexibility is depicted as a spring and the centroid of the nanosphere is shown as a yellow circle). (a, middle) The monomer on the right releases from actin upon binding to ATP and searches for the next binding site. (a, right) The monomer binds at random to an actin filament, and then strokes (curved arrow). The stroke causes a distension of all monomers (depicted as distension of connecting springs) along with movement of the centroid (depicted by movement of the yellow circle). The extent of movement is determined by the balance of force between the four connected monomers. (b) Simulated end–end speed for limited (1 nm), moderate (3 nm), and very large (50 nm) distensions permitted. (c) Simulated trajectories of monomer-coated nanosphere motion for moderate distensions. (d) Experimental trajectories of myosin VI monomer-coated nanospheres moving along an extracted keratocyte. (e) Sample trajectories from panel d aligned such that the cell periphery is at the top and the cell center is at the bottom. All movement, without exception, is toward the cell center. (f) Comparison of end–end speeds from the experiment (blue) and simulation (red). (g) Comparison of frame–frame speeds between the experiment (blue) and simulation (red). Bars, 1 µm.

Interaction of myosin VI artificial dimer-coated nanospheres with extracted keratocyte lamellipodial F-actin networks. (a) Fluorescence micrograph of a fish epidermal keratocyte after extraction. (b) Platinum replica electron micrograph of an extracted fish epidermal keratocyte showing the dense, interlaced actin network in the keratocyte lamellipodium. (c) Trajectories of movement of 200-nm-diameter nanospheres coated with a single myosin VI dimer on the extracted keratocyte. (d) Sample trajectories from panel c aligned such that the cell periphery is at the top and the cell center is at the bottom. (e and f) Trajectories of movement of nanospheres coated with ∼10 myosin VI dimers, such that approximately two dimers interact with the keratocyte lamellipodium. All nanospheres, without exception, move toward the cell center. A nanosphere landing near the cell edge can traverse the entire length of the keratocyte lamellipodium. (g) Sample trajectories from panel f aligned such that the cell periphery is at the top and cell center is at the bottom. Myosin VI is a pointed end–directed motor; hence, the polarity of F-actin, inferred from the observed movement, is indicated by barbed end (+) and pointed end (−) in panels d and g. Bars, 1 µm.

Movement of myosin VI artificial dimer-coated nanospheres on extracted keratocyte actin network assessed from experimentation and simulation. (a) A digitized interlaced actin network used in the simulation (blue) overlaid on the platinum replica micrograph from which it was obtained. (b) A schematic drawing of the simplified model used for the simulation. (b, left) Two dimers bound to actin filaments, coupled through the nanosphere (green line), and bound to the nanosphere at the yellow circle. (b, center) The dimer on the left steps first to its next binding site. (b, right) The dimer on the right steps to the next binding site. Binding sites are selected to maintain the distance between the nanosphere attachment points (length of the green line). (c) Sample trajectories of simulated movement of nanospheres on the extracted keratocyte lamellipodium. (d) Comparison of end–end speeds from the experiment (blue) and simulation (red). (e) Comparison of frame–frame speeds between the experiment (blue) and simulation (red). Bars, 1 µm.