Structured Post-IQ Domain Governs Selectivity of Myosin X for Fascin-Actin Bundles

Abstract

Without guidance cues, cytoskeletal motors would traffic components to the wrong destination with disastrous consequences for the cell. Recently, we identified a motor protein, myosin X, that identifies bundled actin filaments for transport. These bundles direct myosin X to a unique destination, the tips of cellular filopodia. Because the structural and kinetic features that drive bundle selection are unknown, 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. Moreover, the myosin X head is adapted for initiating processive runs on bundles. We found that the tail is structured and biases the orientation of the two myosin X heads because a targeted insertion that introduces flexibility in the tail abolishes selectivity. Together, these results suggest how myosin motors may manage to read cellular addresses.The essential role of cytoskeletal motor proteins in organizing cellular compartments and cargoes is widely accepted (1). 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. Clearly, errors at any of these stages would 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 a precise location within the cell (2). Myosin X is found at the mitotic and meiotic spindle, where it sets the orientation of the spindle relative to the substrate and influences spindle length (3, 4). However, myosin X is more commonly found in interphase cells at the tips of filopodia (5). These long, slender projections at the leading edge of migrating cells are involved in cell motility and environmental sensing. Myosin X transports components such as Ena/VASP and integrins that are found in the filopodial tip complex (2, 6). The early work by Berg et al. (5) demonstrated that myosin X reaches filopodial tips under its own power. Recently, we found that myosin X identifies filopodia by recognizing the fascin-bundled actin filaments found in the filopodial core (7). Myosin X takes long processive runs along these tightly bundled actin filaments while largely ignoring isolated actin filaments found throughout the cell. In contrast, myosin V does not distinguish between single actin filaments and bundled actin filaments when taking processive runs.Here, we seek to identify the "selectivity module" that allows myosin X to recognize bundled actin filaments. Our first approach is to swap similar domains between the selective myosin X and the nonselective myosin V. In this approach, we are aided by the fact that both m...

Antiparallel coiled-coil–mediated dimerization of myosin X

“…Several recent papers by Rock and colleagues have suggested that Myo10 displays strong selectivity for bundled actin (17,18). This finding contradicts the findings of a recent study by Sun et al (16).…”

“…Thus, we believe that the bundled actin selectivity of the Myo10 chimera shown in the Rock et al studies is likely due to the conformational change of the anti-CC dimer induced by the GCN4 fusion. Nonetheless, the detailed single-molecule stepping assays performed in the Rock et al studies have provided additional evidence, albeit from a different angle, supporting the critical role that the anti-CC plays in Myo10's processive movement along actin filaments (17,18).…”

“…Thus, the Myo5 coiled coil should enhance the dimerization of Myo10 without altering the dimer's anti-CC conformation. In the Nagy et al studies (17,18), the parallel GCN4 coiled coil was fused to Leu920 of Myo10. Leu920 is in the middle of αB of the Myo10 anti-CC dimer, and the truncation of Myo10 and fusion of GCN4 at this residue is likely to Values are means ± SD from three independent experiments with 10 cells per experiment, using unpaired t test, *P < 0.05, *P < 0.01, ***P < 0.001.…”

“…Both in vitro single-molecule-based experiments and in vivo near single-molecule imaging assays have shown that Myo10 is capable of processive movements along actin filaments; thus it is expected that Myo10 can dimerize (15,16). Additionally, the role that Myo10 plays in filopodial induction suggests that it may function as a bundled actin filament-selective motor (17,18). However, neither Myo10's supposed coiled-coil dimerization nor its selectivity for bundled actin is well substantiated.…”

Myosin-10 produces its power-stroke in two phases and moves processively along a single actin filament under low load

“…Each ATP-driven step taken by such truncated chimeras of myosin-10 has been reported to be in the range of 18-34 nm long (16)(17)(18)(19). Furthermore, live cell fluorescence imaging studies (20, 21) using a full-length GFP-tagged myosin-10 construct showed that single molecules move in a highly directed manner within the filopodia.…”