Transient external force induces phenotypic reversion of malignant epithelial structures via nitric oxide signaling.

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.

To pull or be pulled: parsing the multiple modes of mechanotransduction.

A cell embedded in a multicellular organism will experience a wide range of mechanical stimuli over the course of its life. Fluid flows and 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 on the basis of 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.

The stepping pattern of myosin X is adapted for processive motility on bundled actin.

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 myosin motor that selects bundled actin for motility.

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.

Optimization and generality of a small deoxyribozyme that ligates RNA.

In vitro evolution was previously used to identify a small deoxyribozyme, 7Q10, that ligates RNA with formation of a 2'-5' phosphodiester linkage from a 2',3'-cyclic phosphate and a 5'-hydroxyl group. Ligation occurs in a convenient "binding arms" format analogous to that of the well-known 10-23 and 8-17 RNA-cleaving deoxyribozymes. Here, we report the optimization and generality of 7Q10 as a 2'-5' RNA ligase. By comprehensive mutagenesis of its 16-nucleotide enzyme region, the parent 7Q10 sequence is shown to be optimal for RNA ligation yield, although several mutations are capable of increasing the ligation rate approximately fivefold at the expense of yield. The 7Q10 deoxyribozyme ligates any RNA substrates that form the sequence motif UA GR (arrowhead=ligation site and R=purine), providing at least 30% yield of ligated RNA in approximately 1-2 hours at 37 degrees C and pH 9.0. Comparable yields are obtained in approximately 12-24 hours at pH 7.5, which may be more suitable for larger RNAs that are more sensitive to non-specific degradation. For RNA substrates that form the related ligation junction UA GY (Y=pyrimidine), somewhat lower yields are obtained, but significant ligation activity is still observed. These data establish that 7Q10 is a generally applicable RNA ligase. A plot of log(k(obs)) versus pH from pH 6.9 to 9.0 has a slope of just under 1, suggesting that a single deprotonation occurs during the rate-determining reaction step. The compact 7Q10 deoxyribozyme has both practical utility and the potential for increasing our structural and mechanistic understanding of how nucleic acids can mediate chemical reactions.