A Combination of Diffusion and Active Translocation Localizes Myosin 10 to the Filopodial Tip

Myosin 10 is an actin-based molecular motor that localizes to the tips of filopodia in mammalian cells. To understand how it is targeted to this distinct region of the cell we have used total internal reflection fluorescence microscopy (TIRFM) to study the movement of individual full length and truncated GFP-tagged molecules. Truncation mutants lacking the motor region failed to localize to filopodial tips but still bound transiently at the plasma membrane. Deletion of the single alpha helical (SAH) and anti-parallel coiled-coil forming regions, which lie between the motor and PH domains, reduced the instantaneous velocity of intrafilopodial movement but did not affect the number of substrate adherent filopodia. Deletion of the anti-parallel coiled-coil forming region, but not the EKR rich region of the SAH domain restored intrafilopodial trafficking, suggesting this region is important in determining myosin 10 motility. We propose a model by which myosin 10 rapidly targets to the filopodial tip via a sequential reduction in dimensionality, first undergoing rapid diffusion within the 3-dimensional volume of the cell body combined with periods of slower, 2-dimensional diffusion in the plane of the plasma membrane then making a 1-dimensional motorized movement along the polarized actin filament bundle within the filopodium until reaching the tip becoming confined at a single point. Here we have observed directly each phase of the trafficking process using single molecule fluorescence imaging of live cells and have quantified our observations using single particle tracking, autocorrelation analysis and kymographs.