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Abstract

Myosin-10 an unconventional myosin motor that localizes to tips of filopodia, long finger- like projections from cells, to help relocate its cargo proteins to the tips and bodies of filopodia. While it is known that myosin-10 moves along filopodial actin, how myosin-10 specifically selects for filopodial actin tracks is currently unknown. Three research groups attempted to determine myosin-10’s track selection ability using single molecule techniques to measure myosin-10 actin bundle selection behavior. Due to the low dimerization affinity for myosin-10 without a high local motor concentration encouraged by cargo binding, additional nucleation domains were needed to be attached to shortened constructs with the regulatory cargo-binding domains removed. These studies disagreed about myosin-10 track sensitivity because one construct demonstrated clear bundle selection not seen the other two constructs. After these studies were performed, the native myosin-10 coiled-coil structure was solved and showed that all three research groups had inadvertently fused an antiparallel coiled- coil to a parallel coiled-coil. We studied the coiled-coils of the three previously designed constructs to determine the source of different behaviors between constructs and link myosin- 10 bundle selection back to full-length wild-type myosin-10. The bundle selective construct, which attempted to create a continuous coiled-coil, forms an antiparallel oriented coiled-coil. Additionally this project tests the flexibility of a single-alpha helix domain connecting the motor domain that binds actin to the coiled-coil to demonstrate how rigidity and orientation of the coiled-coil in myosin-10 leads to actin track selection.

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