Myosin VI Dimerizes And Walks Processively Along Actin

Abstract

In order to assess how lever arm length affects the three-dimensional motions of myosin V during processive motility, two constructs were studied using single molecule polarized total internal reflection fluorescence (polTIRF) microscopy. MyoV6IQ and MyoV4IQ contain 6 and 4 calmodulin (CaM) binding IQ motifs, and otherwise consist of the native myosin V excluding the tail domain. Bifunctional rhodamine labeled CaM replaced a native CaM, giving probe angles b P relative to the actin axis and a P , the azimuth around actin. As with other processive myosins, a P and b P exhibited tilting of the probe with each step. With MyoV6IQ, a P often returned to its initial value after two steps, as expected for nearly straight walking. This behavior enabled us to determine the orientation of the lever arm, a L and b L , as well as q L and f L , the probe angles relative to CaM. b L was 100 and 40 in the leading and trailing heads, respectively. In MyoV4IQ, b P was similar to 6IQ, but a P seldom returned to its earlier value after two steps. This indicates considerable net azimuthal rotation, as expected for smaller step sizes. Thus, lever arm length determines the azimuthal angular path, whereas the axial orientation is likely determined by structural constraints in the motor domain. Modified gliding filament assays were performed using polTIRF to detect twirling of actin about its axis during motility. MyoV6IQ twirled almost exclusively left-handed with a pitch of 1.4 mm. My-oV4IQ twirled with both right-and left-handed pitches of 1.0 and 1.2 mm, respectively. Bidirectional twirling of MyoV4IQ contrasts with every isoform of myosin previously tested (II, native V, VI and X) all of which twirled with a single handedness.