Role of actin filaments in endothelial cell-cell adhesion and membrane stability under fluid shear stress

Clostridium botulinum C2 toxin (C2 toxin) and purified ADP-ribosylated-alph a -actin (ADP-r-alpha -actin) cause specific actin depolymerisation in livi ng cells. This effect was used to investigate the actin microfilament syste m with particular emphasis on cell-cell adhesion and plasma membrane integr ity in endothelial cells. C2 toxin caused time- and dose-dependent (15-100 ng/ml) changes in endothelial surface morphology (investigated by atomic fo rce microscopy), intercellular gap formation and cell detachment under shea r stress. Low concentrations of C2 toxin (1.5 ng/ml), however, did not indu ce cell detachment but inhibited shear stress-dependent cell alignment. Gap formation as well as cell loss under shear stress was also observed in cel ls microinjected with purified ADP-r-alpha -actin. Intercellular gap format ion 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 s tress and C2 toxin were due to a loss of more than 50% of membrane-associat ed actin. These data show that small disturbances in actin dynamics inhibit shear stress-dependent cell alignment; that depolymerisation of actin fila ments increases the solubility of alpha -catenin, thus resulting in cell di ssociation and that actin filaments of the membrane cytoskeleton are requir ed to protect the cells from haemodynamic injury such as shear stress. Toge ther, the study shows a heterogeneous regulation of actin filament dynamics at subcellular locations. Junction-associated actin filaments displayed th e highest sensitivity whereas stress fibres were far more stable.