Beta actin and its mRNA are localized at the plasma membrane and the regions of moving cytoplasm during the cellular response to injury.
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 (alpha-smooth) actin is present on the stress fibers while nonmuscle actins (beta and gamma) are located on stress fibers and in regions of moving cytoplasm (e.g., ruffles, lamellae). To determine the form and function of beta actin in microvascular pericytes and endothelial cells recovering from injury, we prepared isoform-specific antibodies and cDNA probes for immunolocalization, Western and Northern blotting, as well as in situ hybridization. Anti-beta actin IgG was prepared by adsorption and release of beta actin-specific IgG from electrophoretically purified pericyte beta actin bound to nitrocellulose paper. Anti-beta actin IgGs prepared by this affinity selection procedure showed exclusive binding to beta actin present in crude cell lysates containing all three actin isoforms. For controls, we localized beta actin as a bright rim of staining beneath the erythrocyte plasma membrane. Anti-beta actin IgG, absorbed with beta actin bound to nitrocellulose, failed to stain erythrocytes. Simultaneous localization of beta actin with the entire F-actin pool was performed on microvascular pericytes or endothelial cells and 3T3 fibroblasts recovering from injury using anti-beta actin IgG in combination with fluorescent phalloidin. Results of these experiments revealed that pericyte beta 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-beta actin IgG localization is discontinuous. When injured endothelial and 3T3 cells were stained through wound closure, we localized beta actin only in motile cytoplasm at the wound edge. Staining disappeared as cells became quiescent upon monolayer restoration. Appearance of beta actin at the wound edge correlated with a two- to threefold increase in steady-state levels of beta 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 beta actin were localized at the wound edge in association with the repositioned protein. Results of these experiments indicate that beta actin and its encoded mRNA are polarized at the membrane-cytoskeletal interface within regions of moving cytoplasm.
Hoock
T C
TC
Program in Cell, Molecular and Developmental Biology, Tufts University Health Science Schools, Boston, Massachusetts 02111.
Newcomb
P M
PM
Herman
I M
IM
D000199:Actins / Q000032:analysis* / Q000235:genetics; D000818:Animals; D002417:Cattle; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000737:chemistry*; D002465:Cell Movement / Q000502:physiology; D002478:Cells, Cultured; D003593:Cytoplasm / Q000737:chemistry*; D004730:Endothelium, Vascular / Q000737:chemistry* / Q000293:injuries; D004912:Erythrocytes / Q000737:chemistry; D005347:Fibroblasts / Q000737:chemistry; D006801:Humans; D007074:Immunoglobulin G / Q000302:isolation & purification; D007150:Immunohistochemistry; D009131:Muscle, Smooth, Vascular / Q000737:chemistry*; D009693:Nucleic Acid Hybridization; D012333:RNA, Messenger / Q000032:analysis; D014945:Wound Healing / Q000502:physiology
D000199:Actins; D007074:Immunoglobulin G; D012333:RNA, Messenger