Simultaneous visualization of G- and F-actin in endothelial cells.

Haugland RP, You W, Paragas VB, Wells KS, DuBose DA
J Histochem Cytochem. 1994 42 (3): 345-50

PMID: 8308251 · DOI:10.1177/42.3.8308251

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.

MeSH Terms (12)

Actins Animals Antibodies, Monoclonal Blotting, Western Cattle Cells, Cultured Deoxyribonuclease I Electrophoresis, Polyacrylamide Gel Endothelium, Vascular Fluorescent Dyes Microscopy, Fluorescence Phalloidine

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