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 crosslinks 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 crosscorrelation 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.electron microscopy | unconventional myosin | actin network T he actin-based cytoskeleton, composed of actin filaments and actin-binding partners, including a large variety of myosin motor proteins, is responsible for highly diverse forms of cellular motility. The actomyosin cytoskeleton continuously undergoes major structural reorganizations in the lamellipodium of migrating cells and during polarization in epithelium cells in morphogenesis (1-4). The molecular mechanical mechanisms of these local actomyosin networks, however, are still largely unknown.Myosin class IX, a monomeric myosin with two mammalian isoforms, has been shown to play an important role in these processes. Whereas myosin-IXb is found in migrating cells of the immune system, myosin-IXa is abundantly expressed in the brain and testis and at lower levels in the kidney, adrenal gland, lung, and spleen and has been shown to play a critical role in epithelial differentiation and morphology (5-8). The N-terminal catalytic domain of myosin-IX binds actin and hydrolyses ATP, whereas the following light-chain binding-neck region acts as a lever arm to amplify the small conformational changes in the catalytic motor domain into nanometer displacements at the end of the lever. The C-terminal tail domain comprises one or two C1 zinc-binding domains and a Rho-GTPase-activating protein (RhoGAP) domain that inactivates the small GTPase Rho (5). Interestingly, the motor head of class IX myosins features a unique domain not found in any other class of myosin, namely a very large inser...

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.

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