Dynamics of Myosin XI: The Family Speed Demon

Abstract

Myosin XI is the fastest known processive motor, and is implicated in organelle transport and propulsion of cytoplasmic streaming in plant cells. We studied the motility and dynamics of a recombinant myosin XI motor domain hybridized with the myosin Va lever arm with 6 calmodulin-bearing IQ motifs and the dimerization motif. Single molecule fluorescence tracking (FIONA) and polarization (polTIRF) measurements support the hand-over-hand stepping model for myosin XI. Its step size (∼34.5 nm) is slightly shorter than the helical pitch of actin filaments, suggesting an overall left-handed helical walking path. The path was confirmed by polTIRF azimuthal angles as well as bead motility on suspended actin filaments. The hybrid myosin XI showed a maximal velocity of at least 4 μm/s, which is ∼7-fold faster than its structurally similar cousin, myosin V, but they have similar run lengths of ∼1 μm. FIONA measurements showed faster myosin XI stepping rates on actin filaments as compared to bundled actin. The gliding filament assay gave 5-10-fold slower velocities than single molecule processive runs. PolTIRF experiments identified several classes of molecules, with different leading and trailing probe angles. These classes may represent placement of the bifunctional rhodmaine probe on different calmodulins. Compared with myosin V, myosin XI showed more variable localization between steps and more off-axis motion, which may relate to myosin XI's role in cytoplasmic streaming on bundled actin beams. Supported by NIH grant GM086352.