Simultaneous visualization of G- and F-actin in endothelial cells.
We developed site-specific fluorescent probes that permit simultaneous microscopic observation of G- and F-actin in bovine endothelial cells. G-actin distribution was visualized with fluorescein-deoxyribonuclease I (DNAse I). F-actin was labeled with phalloidin conjugated to the new long-wavelength fluorophore BODIPY 581/591 (581-nm excitation, 591-nm emission), which is spectrally similar to Texas Red. The G-actin appeared as pervasive green fluorescence that was more intense in the nuclear region, where cell thickness is greater and stress fibers are less frequent. In addition, we observed a punctate fluorescein pattern around the nuclei and in other parts of the cells, suggesting that some G-actin is localized to small discrete sites. F-actin was observed as red fluorescent filaments. Unlabeled DNAse I effectively prevented staining of G-actin by the fluorescent DNAse I conjugates. The specificity of DNAse I for G-actin was confirmed by the presence of a single labeled band with molecular weight corresponding to actin in a Western blot of total cytoplasmic endothelial proteins reacted with biotin-DNAse I-streptavidin-alkaline phosphatase. Anti-actin antibody, which associates with both G- and F-actin, in conjunction with fluorescent secondary antibody produced a pattern similar to that obtained by simultaneous visualization with fluorescein-DNAse I and BODIPY 581/591- or rhodamine-phalloidin.
Haugland
R P
RP
Molecular Probes, Inc., Eugene Oregon 97402.
You
W
W
Paragas
V B
VB
Wells
K S
KS
DuBose
D A
DA
D000199:Actins / Q000032:analysis*; D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D003850:Deoxyribonuclease I; D004591:Electrophoresis, Polyacrylamide Gel; D004730:Endothelium, Vascular / Q000737:chemistry* / Q000166:cytology; D005456:Fluorescent Dyes; D008856:Microscopy, Fluorescence; D010590:Phalloidine
D000199:Actins; D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D010590:Phalloidine; D003850:Deoxyribonuclease I