Self-organization of actin networks by a monomeric myosin.

The organization of actomyosin networks lies at the center of many types of cellular motility, including cell polarization and collective cell migration during development and morphogenesis. Myosin-IXa is critically involved in these processes. Using total internal reflection fluorescence microscopy, we resolved actin bundles assembled by myosin-IXa. Electron microscopic data revealed that the bundles consisted of highly ordered lattices with parallel actin polarity. The myosin-IXa motor domains aligned across the network, forming cross-links at a repeat distance of precisely 36 nm, matching the helical repeat of actin. Single-particle image processing resolved three distinct conformations of myosin-IXa in the absence of nucleotide. Using cross-correlation of a modeled actomyosin crystal structure, we identified sites of additional mass, which can only be accounted for by the large insert in loop 2 exclusively found in the motor domain of class IX myosins. We show that the large insert in loop 2 binds calmodulin and creates two coordinated actin-binding sites that constrain the actomyosin interactions generating the actin lattices. The actin lattices introduce orientated tracks at specific sites in the cell, which might install platforms allowing Rho-GTPase-activating protein (RhoGAP) activity to be focused at a definite locus. In addition, the lattices might introduce a myosin-related, force-sensing mechanism into the cytoskeleton in cell polarization and collective cell migration.

Motor domain phosphorylation increases nucleotide exchange and turns MYO6 into a faster and stronger motor.

Myosin motors perform many fundamental functions in eukaryotic cells by providing force generation, transport or tethering capacity. Motor activity control within the cell involves on/off switches, however, few examples are known of how myosins regulate speed or processivity and fine-tune their activity to a specific cellular task. Here, we describe a phosphorylation event for myosins of class VI (MYO6) in the motor domain, which accelerates its ATPase activity leading to a 4-fold increase in motor speed determined by actin-gliding assays, single molecule mechanics and stopped flow kinetics. We demonstrate that the serine/threonine kinase DYRK2 phosphorylates MYO6 at S267 in vitro. Single-molecule optical-tweezers studies at low load reveal that S267-phosphorylation results in faster nucleotide-exchange kinetics without change in the working stroke of the motor. The selective increase in stiffness of the acto-MYO6 complex when proceeding load-dependently into the nucleotide-free rigor state demonstrates that S267-phosphorylation turns MYO6 into a stronger motor. Finally, molecular dynamic simulations of the nucleotide-free motor reveal an alternative interaction network within insert-1 upon phosphorylation, suggesting a molecular mechanism, which regulates insert-1 positioning, turning the S267-phosphorylated MYO6 into a faster motor.

The highly efficient holding function of the mollusc 'catch' muscle is not based on decelerated myosin head cross-bridge cycles.

Certain smooth muscles are able to reduce energy consumption greatly when holding without shortening. For instance, this is the case with muscles surrounding blood vessels used for regulating blood flow and pressure. The phenomenon is most conspicuous in 'catch' muscles of molluscs, which have been used as models for investigating this important physiological property of smooth muscle. When the shells of mussels are held closed, the responsible muscles enter the highly energy-efficient state of catch. According to the traditional view, the state of catch is caused by the slowing down of the force-generating cycles of the molecular motors, the myosin heads. Here, we show that catch can still be induced and maintained when the myosin heads are prevented from generating force. This new evidence proves that the long-held explanation of the state of catch being due to the slowing down of force producing myosin head cycles is not valid and that the highly economic holding state is caused by the formation of a rigid network of inter-myofilament connections based on passive molecular structures.