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.
Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.
Marion
M
Jasnin
Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.
Mary
M
Ecke
Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.
Wolfgang
W
Baumeister
Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany. Electronic address: gerisch@biochem.mpg.de.
Günther
G
Gerisch
D008841:Actin Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure*; D000199:Actins / Q000737:chemistry* / Q000378:metabolism; D004023:Dictyostelium / Q000378:metabolism / Q000648:ultrastructure; D055032:Electron Microscope Tomography / Q000379:methods; D015800:Protozoan Proteins / Q000737:chemistry* / Q000378:metabolism
D000199:Actins; D015800:Protozoan Proteins