The reorganization of microfilaments, centrosomes, and microtubules during in vitro small wound reendothelialization

Abstract

The repair of small endothelial wounds is an important process by which endothelial cells maintain endothelial integrity. An in vitro wound model system was used in which precise wounds were made in a confluent endothelial monolayer. The repair process was observed by time-lapse cinemicrophotography. Using fluorescence and immunofluorescence microscopy, the cellular morphological events were correlated with the localization and distribution of actin microfilament bundles and vinculin plaques, and centrosomes and their associated microtubules. Single to four-cell wounds underwent closure by cell spreading while wounds seven to nine cells in size closed by initially spreading which was then followed at ~1 h after wounding by cell migration. These two processes showed different cytoskeletal patterns. Cell spreading occurred independent of centrosome location. However, centrosome redistribution to the front of the cell occurred as the cells began to elongate and migrate. While the peripheral actin microfilament bundles (i.e., the dense peripheral band) remained intact during cell spreading, they broke down during migration and were associated with a reduction in peripheral vinculin plaque staining. Thus, the major events characterizing the closure of endothelial wounds were precise in nature, followed a specific sequence, and were associated with specific cytoskeletal patterns which most likely were important in maintaining directionality of migration and reducing the adhesion of the cells to their neighbors within the monolayer.