Organization of actin cytoskeleton during early endothelial regeneration in vitro

ABSTRACT The pattern of early cell movement after an experimental ‘wound’ and the organization of actin in stationary and moving cultured endothelial cells have been studied by means of: (1) time-lapse photography; (2) indirect immunofluorescence using anti-actin antibodies with and without pretreatment with the actin destabilizing factor present in human plasma; and (3) differential centrifugation and densitometric analysis of stained sodium dodecylsulphate/polyacrylamide gels in order to evaluate the total and relative amounts of G and F-actin. Up to 5 h after a single scratch, movement consists of a coordinate spreading and translocation of a band of about 10 cells from the wound edge. Compared to stationary cells, moving endothelial cells show: (1) no significant changes in the intensity and distribution of immunofluorescent staining with anti-actin antibodies, but an increased sensitivity of cytoplasmic actin, including stress fibres, to the actin-destabilizing factor purified from human plasma; and (2) no significant change in the total amount of actin, but a decreased relative amount of F-actin and a corresponding increased relative amount of G-actin. We conclude that endothelial cell movement in vitro is accompanied by a rapid change in the state of actin organization characterized by an overall decrease in cytoplasmic F-actin.