Comparisons of endothelial cell G- and F-actin distribution in situ and in vitro.
Numerous studies have described the F-actin cytoskeleton; however, little information relevant to G-actin is available. The actin pools of bovine aortic endothelial cells were examined using in situ and in vitro conditions and fluorescent probes for G-(deoxyribonuclease I, 0.3 microM) or F-actin (phalloidin, 0.2 microM). Cells in situ displayed a diffuse G-actin distribution, while F-actin was concentrated in the cell periphery and in fine stress fibers that traversed some cells. Cells of subconfluent or just confluent cultures demonstrated intense fluorescence, with many F-actin stress fibers. Postconfluent cultures resembled the condition in situ; peripheral F-actin was prominent, traversing actin stress fibers were greatly reduced and fluorescent intensity was diminished. Postconfluency had little influence on G-actin, with only an enhancement in the intensity of G-actin punctate fluorescence. When post-confluent cultures were incubated with cytochalasin D (15 min; 10(-4) M), F-actin networks were disrupted and actin punctate and diffuse fluorescence increased. G-actin fluorescence was not altered by the incubation. Although its unstructured nature may account for the minor changes observed, the stability of the G-actin pool in the presence of notable F-actin modulations suggested that filamentous actin was the key constituent involved in these actin cytoskeletal alterations. A separate finding illustrated that the concomitant use of actin probes with image enhancement and fluorescent microscopy could reveal simultaneously the G- and F-actin pools within the same cell.
DuBose
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DA
U.S. Army Research Institute of Environmental Medicine, Natick, Massachusetts 01581.
Haugland
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R
D000199:Actins / Q000032:analysis*; D000818:Animals; D001011:Aorta / Q000737:chemistry; D002417:Cattle; D015638:Cytochalasin D / Q000494:pharmacology; D003850:Deoxyribonuclease I; D004730:Endothelium, Vascular / Q000737:chemistry*; D005453:Fluorescence; D005456:Fluorescent Dyes; D066298:In Vitro Techniques
D000199:Actins; D005456:Fluorescent Dyes; D015638:Cytochalasin D; D003850:Deoxyribonuclease I