β actin and its mRNA are localized at the plasma membrane and the regions of moving cytoplasm during the cellular response to injury

Abstract

Previous work in our laboratory has shown that microvascular pericytes sort muscle and nonmuscle actin isoforms into discrete cytoplasmic domains (Herman, I. M., and P. A. D'Amore. 1985. J. Cell Biol. 101:43-52; DeNofrio, D., T. C. Hoock, and I. M. Herman. J. Cell. Biol. 109:191-202). Specifically, muscle (α-smooth) actin is present on the stress fibers while nonmuscle actins (β and γ) are located on stress fibers and in regions of moving cytoplasm (e.g., ruffles, lamellae). To determine the form and function of β actin in microvascular pericytes and endothelial cells recovering from injury, we prepared isoformspecific antibodies and cDNA probes for immunolocalization, Western and Northern blotting, as well as in situ hybridization. Anti-β actin IgG was prepared by adsorption and release of β actin-specific IgG from electrophoretically purified pericyte β actin bound to nitrocellulose paper. Anti-β actin IgGs prepared by this affinity selection procedure showed exclusive binding to β actin present in crude cell lysates containing all three actin isoforms. For controls, we localized β actin as a bright rim of staining beneath the erythrocyte plasma membrane. Anti-β actin IgG, absorbed with β actin bound to nitrocellulose, failed to stain erythrocytes. Simultaneous localization of β actin with the entire F-actin pool was performed on microvascular pericytes or endothelial cells and 3T3 fibroblasts recovering from injury using anti-β actin IgG in combination with fluorescent phalloidin. Results of these experiments revealed that pericyte β actin is localized beneath the plasma membrane in association with filopods, pseudopods, and fan lamellae. Additionally, we observed bright focal fluorescence within fan lamellae and in association with the ends of stress fibers that are preferentially associated with the ventral plasmalemma. Whereas fluorescent phalloidin staining along the stress fibers is continuous, anti-β actin IgG localization is discontinuous. When injured endothelial and 3T3 cells were stained through wound closure, we localized β actin only in motile cytoplasm at the wound edge. Staining disappeared as cells became quiescent upon monolayer restoration. Appearance of β actin at the wound edge correlated with a two- to threefold increase in steady-state levels of β actin mRNA, which rose within 15-60 min after injury and returned to noninjury levels during monolayer restoration. In situ hybridization revealed that transcripts encoding β actin were localized at the wound edge in association with the repositioned protein. Results of these experiments indicate that β actin and its encoded mRNA are polarized at the membrane-cytoskeletal interface within regions of moving cytoplasm.