Eukaryotic cells organize their contents through trafficking along cytoskeletal filaments. The leading edge of a typical metazoan cytoskeleton consists of a dense and complex arrangement of cortical actin. A dendritic mesh is found across the broad lamellopodium, with long parallel bundles at microspikes and filopodia. It is currently unclear whether and how myosin motors identify the few actin filaments that lead to the correct destination, when presented with many similar alternatives within the cortex. Here we show that myosin X, an actin-based motor that concentrates at the distal tips of filopodia, selects the fascin-actin bundle at the filopodial core for motility. Myosin X moves individual actin filaments poorly in vitro, often supercoiling actin into plectonemes. However, single myosin X motors move robustly and processively along fascin-actin bundles. This selection requires only parallel, closely spaced filaments, as myosin X is also processive on artificial actin bundles formed by molecular crowding. Myosin X filopodial localization is perturbed in fascin-depleted HeLa cells, demonstrating that fascin bundles also direct motility in vivo. Our results demonstrate that myosin X recognizes the local structural arrangement of filaments in long bundles, providing a mechanism for sorting cargo to distant target sites.fascin ͉ motor navigation ͉ myosin X ͉ filopodia ͉ single-molecule fluorescence I n a dense mesh of cellular actin, if all filaments are functionally equivalent, then myosins will move in a random walk as they translate, detach, and reattach to new filaments. Alternatively, myosins may identify and walk along certain subpopulations of actin that lead to target locations, based on a set of common structural features. One possibility is that different myosin classes may partition among filaments based on actin isoforms. However, no significant difference between ␤␥-actin versus ␣-actin has been detected for myosin V, the only motor to be tested on both tracks (1). A second possibility is that actinassociated proteins, in particular tropomyosins, modulate the binding of myosin to actin. Indeed, class I myosins are excluded from tropomyosin-decorated actin in stress-fibers, whereas class II myosins are not (2, 3). In addition to this direct regulatory mechanism, tropomyosins may also direct myosin traffic by stabilizing particular actin tracks (4). However, tropomyosins are not typically found in regions of actively polymerizing, dynamic actin, such as the leading edge of the cell. Therefore, additional mechanisms likely exist for directing myosin traffic.To identify factors that could direct myosins to specific locations, we searched for myosin classes that are localized to limited populations of actin even in the presence of nearby dynamic actin. The class X myosin meets these criteria for a selective motor. Myosin X travels to and is highly concentrated at the distal tips of filopodia: long, slender projections often found at the leading edge of migrating cells (5-7). Filopodia are used for envi...

Myosin X is a molecular motor that is adapted to select bundled actin filaments over single actin filaments for processive motility. Its unique form of motility suggests that myosin X's stepping mechanism takes advantage of the arrangement of actin filaments and the additional target binding sites found within a bundle. Here we use fluorescence imaging with one-nanometer accuracy to show that myosin X takes steps of ∼18 nm along a fascin-actin bundle. This step-size is well short of the 36-nm step-size observed in myosin V and myosin VI that corresponds to the actin pseudohelical repeat distance. Myosin X is able to walk along bundles with this step-size if it straddles two actin filaments, but would be quickly forced to spiral into the constrained interior of the bundle if it were to use only a single actin filament. We also demonstrate that myosin X takes many sideways steps as it walks along a bundle, suggesting that it can switch actin filament pairs within the bundle as it walks. Sideways steps to the left or the right occur on bundles with equal frequency, suggesting a degree of lateral flexibility such that the motor's working stroke does not bias it to the left or to the right. On single actin filaments, we find a broad mixture of 10-20-nm steps, which again falls short of the 36-nm actin repeat. Moreover, the motor leans to the right as it walks along single filaments, which may require myosin X to adopt strained configurations. As a control, we also tracked myosin V stepping along actin filaments and fascin-actin bundles. We find that myosin V follows a narrower path on both structures, walking primarily along one surface of an actin filament and following a single filament within a bundle while occasionally switching to neighboring filaments. Together, these results delineate some of the structural features of the motor and the track that allow myosin X to recognize actin filament bundles.

A cell embedded in a multicellular organism will experience a wide range of mechanical stimuli over the course of its life. Fluid flows or neighboring cells actively exert stresses on the cell, while the cell’s environment presents a set of passive mechanical properties that constrain its physical behavior. Cells respond to these varied mechanical cues through biological responses that regulate activities such as differentiation, morphogenesis, and proliferation, as well as material responses involving compression, stretching, and relaxation. Here, we break down recent studies of mechanotransduction into categories based on the input mechanical stimuli acting upon the cell and the output response of the cell. This framework provides a useful starting point for identifying overlaps in molecular players and sensing modalities, and it highlights how different timescales involved in biological and material responses to mechanical inputs could serve as a means for filtering important mechanical signals from noise.

Non-malignant breast epithelial cells cultured in three-dimensional laminin-rich extracellular matrix (lrECM) form well organized, growth-arrested acini, whereas malignant cells form continuously growing disorganized structures. While the mechanical properties of the microenvironment have been shown to contribute to formation of tissue-specific architecture, how transient external force influences this behavior remains largely unexplored. Here, we show that brief transient compression applied to single malignant breast cells in lrECM stimulated them to form acinar-like structures, a phenomenon we term ‘mechanical reversion.’ This is analogous to previously described phenotypic ‘reversion’ using biochemical inhibitors of oncogenic pathways. Compression stimulated nitric oxide production by malignant cells. Inhibition of nitric oxide production blocked mechanical reversion. Compression also restored coherent rotation in malignant cells, a behavior that is essential for acinus formation. We propose that external forces applied to single malignant cells restore cell-lrECM engagement and signaling lost in malignancy, allowing them to reestablish normal-like tissue architecture.

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