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...