Reassembly of contractile actin cortex in cell blebs.
Contractile actin cortex is involved in cell morphogenesis, movement, and cytokinesis, but its organization and assembly are poorly understood. During blebbing, the membrane detaches from the cortex and inflates. As expansion ceases, contractile cortex re-assembles under the membrane and drives bleb retraction. This cycle enabled us to measure the temporal sequence of protein recruitment to the membrane during cortex reassembly and to explore dependency relationships. Expanding blebs were devoid of actin, but proteins of the erythrocytic submembranous cytoskeleton were present. When expansion ceased, ezrin was recruited to the membrane first, followed by actin, actin-bundling proteins, and, finally, contractile proteins. Complete assembly of the contractile cortex, which was organized into a cagelike mesh of filaments, took approximately 30 s. Cytochalasin D blocked recruitment of actin and alpha-actinin, but had no effect on membrane association of ankyrin B and ezrin. Ezrin played no role in actin nucleation, but was essential for tethering the membrane to the cortex. The Rho pathway was important for cortex assembly in blebs.
Charras
Guillaume T
GT
Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA. gcharras@hms.harvard.edu
Hu
Chi-Kuo
CK
Coughlin
Margaret
M
Mitchison
Timothy J
TJ
D000199:Actins / Q000235:genetics / Q000378:metabolism*; D003285:Contractile Proteins / Q000235:genetics / Q000378:metabolism*; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism* / Q000648:ultrastructure; D004910:Erythrocyte Membrane / Q000378:metabolism* / Q000648:ultrastructure; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D008845:Microinjections; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning; D046529:Microscopy, Electron, Transmission; D018715:Microscopy, Video; D009154:Mutation; D024462:Myosin Type II / Q000378:metabolism; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014162:Transfection; D020742:rhoA GTP-Binding Protein / Q000378:metabolism; D000097573:Red Fluorescent Protein; D000099149:Ezrin
D000199:Actins; D003285:Contractile Proteins; D003598:Cytoskeletal Proteins; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D008840:Microfilament Proteins; D024462:Myosin Type II; D011993:Recombinant Fusion Proteins; D020742:rhoA GTP-Binding Protein; D000097573:Red Fluorescent Protein; D000099149:Ezrin; C066737:RHOA protein, human