Myosin-x selects fascin-actin bundles for processive motility using a novel structural adaptation

The essential role of cytoskeletal motor proteins in organizing cellular compartments and cargoes is widely accepted. However, many of the molecular details of the overall transport and organization process are poorly understood. These essential features include: how motors engage cargoes at their source; how motors are activated once they engage cargo; how they choose the correct set of cytoskeletal tracks; how they disengage from cargo at their destination and how they return to sources of cargo. Errors at any of these stages may lead to faults in cellular organization and misplaced cargoes.;To begin to address these questions we have focused on a particular motor protein, myosin X, due to its ability to navigate to precise locations within the cell as Myosin X is mainly found in interphase cells at the tips of filopodia. These long, slender projections at the leading edge of migrating cells are involved in cell motility and environmental sensing.;The work presented in this thesis shows that myosin X selects the fascin-actin bundle at the filopodial core for motility. In single molecule assays, processive runs of individual myosin X dimers on single actin filaments are short and rare. However, single myosin X motors move robustly and processively along fascin-actin bundles, taking ∼18 nm steps along the bundle as determined by optical trapping experiments. This selection requires parallel, closely spaced filaments, as myosin X is also processive on artificial actin bundles formed by molecular crowding. To determine the structural features that drive bundle selection, we employed a domain swapping approach with the nonselective myosin V to identify the 'selectivity module' of myosin X. We found a surprising role of the myosin X tail region (post IQ) in supporting long runs on bundles. We find that the tail is structured and biases the orientation of the myosin X heads, as a targeted insertion that introduces flexibility in the tail abolishes selectivity. Moreover, the myosin X head is kinetically adapted for initiating processive runs on bundles. Together, these results demonstrate how a cell can manipulate molecular trafficking through a modification of the actin cytoskeleton architecture. By establishing parallel unidirectional filaments, through various cross-linkers and actin capping and polymerizing machinery, the cell can recruit and localize myosin X function in space and time.