Fluorescence staining of the actin cytoskeleton in living cells with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin.

Abstract

An active fluorescent derivative of the actin-binding mushroom [Amanita phalloides] toxin phallacidin was synthesized. Convenient methods were developed to stain actin cytoskeletal structures in living and fixed cultured animal cells [mouse fibroblast 3T3 cells and chicken embryonic cells] and actively streaming algal [Chara australis] cells. Actin binding specificity was demonstrated by competitive binding experiments and comparative staining of well-known structures. Large populations of living animal cells in culture were readily stained by using a relatively mild lysolecithin permeabilization procedure facilitated by the small molecular size of the label. Actin in animal cells was stained in stress fibers, ruffles, the cellular geodome and in diffuse appearing distributions apparently associated with the plasma membrane. Staining of actin cables in algae with nitrobenzoxadiazole (NBD)-phallacidin did not inhibit cytoplasmic streaming. NBD-phallacidin provides a convenient actin-specific fluorescent label for cellular cytoskeletal structures with promise for use in studies of actin dynamics in living systems.