Stepwise Movement Of Myosin-10 Within The Filopodium Of Live Mammalian Cells

Myosin-10 moves along fascin-bundled actin filaments inside filopodia and accumulates at the filopodial tip. Initial reports suggest that clusters of myosin-10 moved towards the plus-end of actin filaments at a velocity of 0.2µm.s−1 at 23°C (Berg & Cheney, 2002). Subsequently, single-molecule imaging revealed that individual myosin-10 molecules move at higher velocities of around 0.6µm.s−1 (at 23°C) (Kerber et al. 2009). Here, we have used high-speed (100fps), single molecule imaging to measure the movement of individual eGFP-tagged myosin-10 molecules in intact live mammalian cells at 37°C. We find that single molecules of myosin-10 move by a combination of 3-D and 2-D diffusion within the cell body and at the plasma membrane to navigate towards the cell periphery. After arriving at the filopodial base, myosin-10 switches to highly-directed and persistent 1-D motion towards the plus-end of the actin bundle comprising the filopodial core. Individual molecules moved from the base to the tip (a distance of 5-10µm) with an average velocity of 1.4µm.s−1 at 37°C (Baboolal et al. 2016). Some molecules exhibited brief intervals of random, back-and-forth motion but rapidly reverted to the smooth forward motion. The path of individual molecules was tracked with a precision of ∼20nm in the x,y-plane and ∼40nm along the z-axis. By plotting the path taken by many individual molecules we mapped the, 100nm diameter, fascin-bundled actin core. In addition, high-speed imaging allowed us, for the first time, to measure the individual ATP-driven steps taken by individual myosin-10 molecules as they step along the actin filament lattice in intact, live cells. The fast velocity of myosin-10 can be explained by the large step size (36nm per head) and fast ATP turnover (35s−1 at 37°C).