title: Role of actin filaments in endothelial cell-cell adhesion and membrane stability under fluid shear stress.
abstract: Clostridium botulinum C2 toxin (C2 toxin) and purified ADP-ribosylated-alpha-actin (ADP-r-alpha-actin) cause specific actin depolymerisation in living cells. This effect was used to investigate the actin microfilament system with particular emphasis on cell-cell adhesion and plasma membrane integrity in endothelial cells. C2 toxin caused time- and dose-dependent (15-100 ng/ml) changes in endothelial surface morphology (investigated by atomic force microscopy), intercellular gap formation and cell detachment under shear stress. Low concentrations of C2 toxin (1.5 ng/ml), however, did not induce cell detachment but inhibited shear stress-dependent cell alignment. Gap formation as well as cell loss under shear stress was also observed in cells microinjected with purified ADP-r-alpha-actin. Intercellular gap formation was mediated by increased alpha-catenin solubility (40%) due to actin filament depolymerisation. Disintegration of plasma membranes (measured by LDH release) and cell fragmentation during simultaneous exposure to shear stress and C2 toxin were due to a loss of more than 50% of membrane-associated actin. These data show that small disturbances in actin dynamics inhibit shear stress-dependent cell alignment; that depolymerisation of actin filaments increases the solubility of alpha-catenin, thus resulting in cell dissociation and that actin filaments of the membrane cytoskeleton are required to protect the cells from haemodynamic injury such as shear stress. Together, the study shows a heterogeneous regulation of actin filament dynamics at subcellular locations. Junction-associated actin filaments displayed the highest sensitivity whereas stress fibres were far more stable.
authors.affiliation: Institut für Physiologie, TU-Dresden, Fiedlerstrasse 42, 01307 Dresden, Germany. Hans.Schnittler@mailbox.TU-Dresden.de
authors.forename: H J
authors.initials: HJ
authors.lastname: Schnittler
authors.forename: S W
authors.initials: SW
authors.lastname: Schneider
authors.forename: H
authors.initials: H
authors.lastname: Raifer
authors.forename: F
authors.initials: F
authors.lastname: Luo
authors.forename: P
authors.initials: P
authors.lastname: Dieterich
authors.forename: I
authors.initials: I
authors.lastname: Just
authors.forename: K
authors.initials: K
authors.lastname: Aktories
mesh_terms: D008841:Actin Cytoskeleton / Q000502:physiology*; D000199:Actins / Q000378:metabolism*; D000818:Animals; D001905:Botulinum Toxins / Q000494:pharmacology; D015820:Cadherins / Q000378:metabolism; D002448:Cell Adhesion / Q000502:physiology*; D002458:Cell Fractionation; D002462:Cell Membrane / Q000378:metabolism; D022081:Cell Surface Extensions / Q000378:metabolism; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D015151:Immunoblotting; D007365:Intercellular Junctions / Q000378:metabolism*; D008845:Microinjections; D018625:Microscopy, Atomic Force; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011108:Polymers / Q000378:metabolism; D013314:Stress, Mechanical; D013552:Swine; D051178:alpha Catenin
chemical_list: D000199:Actins; D015820:Cadherins; D003598:Cytoskeletal Proteins; D011108:Polymers; D051178:alpha Catenin; D011065:Poly(ADP-ribose) Polymerases; D001905:Botulinum Toxins; C096324:botulinum toxin type C