Fluorescent phallotoxin, a tool for the visualization of cellular actin.

Abstract

A fluorescent derivative of phalloidin has been synthesized possessing high affinity to filamentous actin. This compound was used for visualization of actin-containing structures in eukaryotic nonmuscle cells. Due to its low molecular weight (1250), fixation by formaldehyde was sufficient to render the membrane permeable for the labeled peptide. Bundles of microfilaments are the predominant pattern in the flat rat kangaroo PtK1 cells, whereas a net of concentric fibers characterizes the more spherical bovine kidney MDBK cells. Specificity of staining was confirmed by competition experiments with unlabeled phalloidin.The phallotoxins, a family of poisonous bicyclic heptapeptides from the mushroom Amanita phalloides (for review see ref.1), whose main representative is phalloidin (Ta), form tight complexes with F actin from liver cells and from muscle. As Staining of Cells. Cells grown on glass coverslips were rinsed briefly in phosphate-buffered saline (Pi/NaCI) (pH 7.4), fixed for 5 min in 3.7% formaldehyde in Pi/NaCl at room temperature and washed extensively in P1/NaCl. After fixation, cells were either used directly for fluorescent staining or dehydrated in absolute acetone for 4 min at -20°C and air dried or rendered permeable by a 2-min exposure to 0.1% Triton X-100 in Abbreviations: FL, fluorescein; Pi/NaCl, phosphate-buffered saline.t To whom reprint requests should be addressed.

Localization and dynamics of nonfilamentous actin in cultured cells [published erratum appears in J Cell Biol 1993 Nov;123(3):following 767]

“…In this study, we have defined the state of cellular actin molecules based on their reactivities toward different probes. Filamentous actin was detected as those reacting positively with fluorescent phalloidin (Wulf et al, 1979;Barak et al, 1980). Actin antibodies and fluorescent analogs of actin were used to examine the overall distribution of both filamentous and nonfilamentous actin (Lessard, 1988).…”

“…These inelude anti-actin antibodies that bind to G-and F-aetin (Lin, 1981;Lessard, 1988), fluorescent phalloidin that binds specifically to F-actin 0Vieland, 1977;Wulf et al, 1979) and vitamin D-binding protein (DBP) 1 that binds specifically to G-actin (Van Baelen et al, 1980;Goldsehmidt-Clermont et al, 1985;Lee and Galbraith, 1992). In addition, using multiple probes and ratio imaging, we have been able to map the relative distribution of nonfilamentous actin in motile cells.…”

Morphology and DNA degeneration during autoschizic cell death in bladder carcinoma T24 cells induced by ascorbate and menadione treatment

“…Coverslips were seeded with T24 cells (1 ϫ 10 5 ) and incubated for 24 hr. Then the cells were overlaid for 1, 2, and 4 hr with media containing vitamins or with only media and treated with the phalloidin conjugate using the technique of Wulf et al (1979). Briefly, a 0.2 mg/mL stock solution Phalloidin-FITC (P5282; Sigma) was prepared in methanol.…”

Interaction partners of Dlk/ZIP kinase: co-expression of Dlk/ZIP kinase and Par-4 results in cytoplasmic retention and apoptosis

“…The cells were incubated with a rabbit anti-Par-4 antibody (1 : 10 000) for 1 h and, after washing, with goat anti-rabbit Cy TM 3-conjugated secondary antibody (Dianova, Hamburg, Germany) (1 : 500) for 30 min. Actin staining was performed either with TRITC-labeled or cumarin-labeled phalloidin according to Wulf et al (1979) or with anti-actin antibodies (Dianova) and FITC-conjugated secondary antibodies. DAPI staining of nuclei was performed after ®xation and permeabilization of cells with 1 mg/ml of DAPI (4,6-Diamidino-2-phenylindole) for 15 min and subsequent washing as described above.…”