Gravin, a 300 kDa intracellular protein, is downregulated in confluent endothelial cell cultures, but is upregulated at the wound edge in wounded endothelial cell monplayers. Inhibition of gravin expression using antisense oligonucleotides inhibits endothelial wound healing suggesting that this protein may play a role in cell spreading and migration. To better understand how gravin may play a role in cell adhesion and migration, the intracellular distribution of gravin in endothelial cells and its relationship to the cytoskeleton were examined. Cultured human umbilical vein endothelial cells were double-labelled with anti-gravin antibodies and probes for cytoskeletal proteins and examined by fluorescence and confocal microscopy. Fluorescence microscopy showed that gravin had a punctate distribution which did not colocalize with actin filaments, microtubules, or intermediate filaments. Treatment of cells with either cytochalasin D or colchicine disrupted all three cytoskeletal components, but did not significantly alter gravin distribution. Three dimensional reconstruction of images collected by confocal microscopy clearly revealed that gravin was concentrated in the cortical cytoplasm adjacent to both the apical and basal plasma membranes. Immunoelectron microscopy confirmed this with gold labelling being associated with the apical and basal plasma membranes. These data indicate that gravin is not a component of stress fibers, microtubules, or intermediate filaments. However, it is hypothesized that gravin is a membrane skeleton protein that plays a role in endothelial wound healing through interactions with components of either the plasma membrane or the membrane skeleton. Studies are underway to test this hypothesis.
|Original language||English (US)|
|State||Published - 1997|
ASJC Scopus subject areas
- Molecular Biology