Nonmuscle myosin-2: mix and match

Members of the nonmuscle myosin-2 (NM-2) family of actin-based molecular motors catalyze the conversion of chemical energy into directed movement and force thereby acting as central regulatory components of the eukaryotic cytoskeleton. By cyclically interacting with adenosine triphosphate and F-actin, NM-2 isoforms promote cytoskeletal force generation in established cellular processes like cell migration, shape changes, adhesion dynamics, endo- and exo-cytosis, and cytokinesis. Novel functions of the NM-2 family members in autophagy and viral infection are emerging, making NM-2 isoforms regulators of nearly all cellular processes that require the spatiotemporal organization of cytoskeletal scaffolding. Here, we assess current views about the role of NM-2 isoforms in these activities including the tight regulation of NM-2 assembly and activation through phosphorylation and how NM-2-mediated changes in cytoskeletal dynamics and mechanics affect cell physiological functions in health and disease

Drosophila non-muscle myosin II motor activity determines the rate of tissue folding

Non-muscle cell contractility is critical for tissues to adopt shape changes. Although, the non-muscle myosin II holoenzyme (myosin) is a molecular motor that powers contraction of actin cytoskeleton networks, recent studies have questioned the importance of myosin motor activity cell and tissue shape changes. Here, combining the biochemical analysis of enzymatic and motile properties for purified myosin mutants with in vivo measurements of apical constriction for the same mutants, we show that in vivo constriction rate scales with myosin motor activity. We show that so-called phosphomimetic mutants of the Drosophila regulatory light chain (RLC) do not mimic the phosphorylated RLC state in vitro. The defect in the myosin motor activity in these mutants is evident in developing Drosophila embryos where tissue recoil following laser ablation is decreased compared to wild-type tissue. Overall, our data highlights that myosin activity is required for rapid cell contraction and tissue folding in developing Drosophila embryos

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

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

Functional characterization of the human myosin-7a motor domain

Myosin-7a participates in auditory and visual processes. Defects in MYO7A, the gene encoding the myosin-7a heavy chain, are causative for Usher syndrome 1B, the most frequent cause of deaf-blindness in humans. In the present study, we performed a detailed kinetic and functional characterization of the isolated human myosin-7a motor domain to elucidate the details of chemomechanical coupling and the regulation of motor function. A rate-limiting, slow ADP release step causes long lifetimes of strong actin-binding intermediates and results in a high duty ratio. Moreover, our results reveal a Mg2+-sensitive regulatory mechanism tuning the kinetic and mechanical properties of the myosin-7a motor domain. We obtained direct evidence that changes in the concentration of free Mg2+ ions affect the motor properties of human myosin-7a using an in vitro motility assay system. Our results suggest that in a cellular environment, compartment-specific fluctuations in free Mg2+ ions can mediate the conditional switching of myosin-7a between cargo moving and tension bearing modes

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

Competition between myosin II and βH-spectrin regulates cytoskeletal tension

Spectrins are membrane cytoskeletal proteins generally thought to function as heterotetramers comprising two α-spectrins and two β-spectrins. They influence cell shape and Hippo signaling, but the mechanism by which they influence Hippo signaling has remained unclear. We have investigated the role and regulation of the Drosophila β-heavy spectrin (βH-spectrin, encoded by the karst gene) in wing imaginal discs. Our results establish that βH-spectrin regulates Hippo signaling through the Jub biomechanical pathway due to its influence on cytoskeletal tension. While we find that α-spectrin also regulates Hippo signaling through Jub, unexpectedly, we find that βH-spectrin localizes and functions independently of α-spectrin. Instead, βH-spectrin co-localizes with and reciprocally regulates and is regulated by myosin. In vivo and in vitro experiments support a model in which βH-spectrin and myosin directly compete for binding to apical F-actin. This competition can explain the influence of βH-spectrin on cytoskeletal tension and myosin accumulation. It also provides new insight into how βH-spectrin participates in ratcheting mechanisms associated with cell shape change