The Function of Myosin-10 In Vitro and Inside Filopodia

Abstract

Myosin-10 is an actin based motor that localizes to actin rich areas within the cell and carries cargos including netrin receptors and β integrins. Using transient kinetic and single molecule assays, a number of groups have shown that myosin-10 can act as a processivemotor, capable of translocating along actin as a single molecule. However, both the mechanical and kinetic parameters are quite diverse amongst different groups (Kerber & Cheney 2011, J. Cell Sci. 124:3733-3741 and references therein). Negative stain electron microscopy and single particle image processing performed using a truncated myosin-10 construct (1 - 940 a.a.) containing a leucine-zipper at the C-terminal end confirmed dimerization of the molecule with a distance between motor domains of ∼50 nm. When bound to F-actin, the myosin-10 lever arm makes a shallower angle with respect to the filament axis than seen for acto-myosin-5 or acto-myosin-2. In vitro actin gliding assays showed myosin-10-HMM moved F-actin at ∼410 nm.s−1 similar to the speed of GFP-tagged, intact myosin-10 we observed moving within filopodia of live mammalian cells (∼600 nm.s−1) using TIRF microscopy. Optical trapping experiments revealed the average work-stroke size was ∼17 nm with a single actomyosin-10 stiffness of ∼0.4 pN.nm−1 and an ADP-release limited detachment rate of ∼13 s−1. In most raw data traces, we observed unitary displacements, however at low [ATP] (<500 nM) and low optical trap stiffness (<0.005 pN.nm−1) we observed staircase-like interactions with an average movement of ∼35 nm between steps; a behavior characteristic of a processive molecular motor. We will discuss these measurements in the context of structure, mechano-chemical coupling and the functional significance of this motor in the living cell.