Actin Organization in Cells Responding to a Perforated Surface, Revealed by Live Imaging and Cryo-Electron Tomography

In a 3D environment, motile cells accommodate their protruding and retracting activities to geometrical cues. Dictyostelium cells migrating on a perforated film explored its holes by forming actin rings around their border and extending protrusions through the free space. The response was initiated when an actin wave passed a hole, and the rings persisted only in the PIP3-rich territories surrounded by a wave. To reconstruct actin structures from cryo-electron tomograms, actin rings were identified by cryo-correlative light and electron microscopy, and thin wedges of relevant regions were obtained by cryo-focused ion-beam milling. Retracting stages were distinguished from protruding ones by the accumulation of myosin-II. Early actin rings consisted of filaments pointing upright from the membrane, entangled with a meshwork of filaments close to the membrane. Branches identified at later stages suggested that formin-based nucleation of filaments was followed by Arp2/3-mediated network stabilization, which prevented buckling of the force-generating filaments. [Display omitted] •Migrating cells sense substrate curvature to assemble force-generating actin networks•Live imaging and cryo-electron tomography reveal network dynamics and architecture•Close-to-life network structures are visualized in the context of whole vitrified cells•Ion-beam milling and automated filament segmentation are key to structural analysis Cells migrating on structured surfaces respond to geometrical cues by the assembly of force-generating actin networks. Jasnin et al. reveal their architecture by cryo-electron tomography, correlative light and electron microscopy, and focused ion-beam milling.