Journal
PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY
Volume 442, Issue 5, Pages 675-687Publisher
SPRINGER-VERLAG
DOI: 10.1007/s004240100589
Keywords
C botulinum C2 toxin; membrane cytoskeleton; stress fibres; dense peripheral band; adherens junctions; cell adhesion; catenin
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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.
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