Actin Network Organization by the Monomeric Myosin IXa

Abstract

Class IX myosins are thought to act as motorized signaling molecules, as they contain a Rho-GTPase-activating domain (RhoGAP) in the tail, which can alter the dynamics of the actin cytoskeleton. Myosin-IXa is localized in acto-myosin networks at cell-cell adhesions, where it was identified as a key requirement for collective cell migration. Many processes depend on the movement of groups of cells; during tumor progression cancer cells invade the tissue surrounding a primary tumor, while in wound healing epithelial cells have to move close to the wound. We have expressed a variety of human myosin-IXa constructs comprising the motor domain and the calmodulin-binding lever arm. Using quantitative analysis of TIRF microscopy data we found that myosin-IXa assembles actin into bundles of up to seven or even more filaments. Electron microscopy combined with single particle image processing revealed that the bundles consisted of highly ordered 2D 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. Intriguingly, the polarity of the actin filaments in the bundles changed in the presence of calcium. By comparing real EM data with simulated EM of modelled F-actin, we find that approx. 75% of filaments were anti-parallel (in-phase and out-of-phase) at pCa4. This polarity switching is currently investigated in further detail by TIRF microscopy. In addition, we are characterizing the single molecule mechanics of myosin-IXa using an optical-tweezer transducer. Our results indicate that actin lattices induced by the molecular motor myosin-IXa provide orientated actin tracks and a network of regularly spaced platforms for localized Rho-GAP activity in cell polarization and collective cell migration.