Structural and functional dissection of allosteric pathways in myosin motor domains

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So far, yet so close: α-Catenin dimers help migrating cells get together

Epithelial cells in tissues use their actin cytoskeletons to stick together, whereas unattached cells make active plasma membrane protrusions to migrate. In this issue, Wood et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201612006) show that the junction component α-catenin is critical in freely moving cells to promote adhesion and migration

Measurement of junctional tension in epithelial cells at the onset of primitive streak formation in the chick embryo via non-destructive optical manipulation

Directional cell intercalations of epithelial cells during gastrulation has in several organisms been shown to be associated with a planar cell polarity in the organisation of the actin-myosin cytoskeleton and is postulated to reflect directional tension that drives oriented cell intercalations. We have characterised and applied a recently introduced non-destructive optical manipulation technique to measure the tension in individual epithelial cell junctions of cells in various locations and orientations in the epiblast of chick embryos in the early stages of primitive streak formation. Junctional tension of mesendoderm precursors in the epiblast is higher in junctions oriented in the direction of intercalation than in junctions oriented perpendicular to the direction of intercalation and higher than in junctions of other cells in the epiblast. The kinetic data are fitted best with a simple visco-elastic Maxwell model and we find that junctional tension and to a lesser extent viscoelastic relaxation time are dependent on myosin activity

Mechanism and specificity of pentachloropseudilin-mediated inhibition of myosin motor activity.

Here, we report that the natural compound pentachloropseudilin (PClP) acts as a reversible and allosteric inhibitor of myosin ATPase and motor activity. IC(50) values are in the range from 1 to 5 μm for mammalian class-1 myosins and greater than 90 μm for class-2 and class-5 myosins, and no inhibition was observed with class-6 and class-7 myosins. We show that in mammalian cells, PClP selectively inhibits myosin-1c function. To elucidate the structural basis for PClP-induced allosteric coupling and isoform-specific differences in the inhibitory potency of the compound, we used a multifaceted approach combining direct functional, crystallographic, and in silico modeling studies. Our results indicate that allosteric inhibition by PClP is mediated by the combined effects of global changes in protein dynamics and direct communication between the catalytic and allosteric sites via a cascade of small conformational changes along a conserved communication pathway

Kinetic properties and small-molecule inhibition of human myosin-6.

Myosin-6 is an actin-based motor protein that moves its cargo towards the minus-end of actin filaments. Mutations in the gene encoding the myosin-6 heavy chain and changes in the cellular abundance of the protein have been linked to hypertrophic cardiomyopathy, neurodegenerative diseases, and cancer. Here, we present a detailed kinetic characterization of the human myosin-6 motor domain, describe the effect of 2,4,6-triiodophenol on the interaction of myosin-6 with F-actin and nucleotides, and show how addition of the drug reduces the number of myosin-6-dependent vesicle fusion events at the plasma membrane during constitutive secretion

The role of myosin 1c and myosin 1b in surfactant exocytosis

Actin and actin-associated proteins have a pivotal effect on regulated exocytosis in secretory cells and influence pre-fusion as well as post-fusion stages of exocytosis. Actin polymerization on secretory granules during the post-fusion phase (formation of an actin coat) is especially important in cells with large secretory vesicles or poorly soluble secretions. Alveolar type II (ATII) cells secrete hydrophobic lipo-protein surfactant, which does not easily diffuse from fused vesicles. Previous work showed that compression of actin coat is necessary for surfactant extrusion. Here, we investigate the role of class 1 myosins as possible linkers between actin and membranes during exocytosis. Live-cell microscopy showed translocation of fluorescently labeled myosin 1b and myosin 1c to the secretory vesicle membrane after fusion. Myosin 1c translocation was dependent on its pleckstrin homology domain. Expression of myosin 1b and myosin 1c constructs influenced vesicle compression rate, whereas only the inhibition of myosin 1c reduced exocytosis. These findings suggest that class 1 myosins participate in several stages of ATII cell exocytosis and link actin coats to the secretory vesicle membrane to influence vesicle compression

Vinculin controls talin engagement with the actomyosin machinery

The link between extracellular-matrix-bound integrins and intracellular F-actin is essential for cell spreading and migration. Here, we demonstrate how the actin-binding proteins talin and vinculin cooperate to provide this link. By expressing structure-based talin mutants in talin null cells, we show that while the C-terminal actin-binding site (ABS3) in talin is required for adhesion complex assembly, the central ABS2 is essential for focal adhesion (FA) maturation. Thus, although ABS2 mutants support cell spreading, the cells lack FAs, fail to polarize and exert reduced force on the surrounding matrix. ABS2 is inhibited by the preceding mechanosensitive vinculin-binding R3 domain, and deletion of R2R3 or expression of constitutively active vinculin generates stable force-independent FAs, although cell polarity is compromised. Our data suggest a model whereby force acting on integrin-talin complexes via ABS3 promotes R3 unfolding and vinculin binding, activating ABS2 and locking talin into an actin-binding configuration that stabilizes FAs