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.

Etherification of alpha-amanitin with tritiated methyl iodide yielded a radioactively labeled amatoxin of high specific activity (similar to or approximately 4 Ci/mmol) which, in its inhibition capacity for RNA polymerase II, was very similar to alpha-amanitin. The labeled toxin was used successfully in binding assays with RNA polymerases II and in radioimmunological determinations of amatoxins. If long-chained alkyl bromides were reacted with alpha-amanitin, lipophilic ether derivatives were obtained with a facilitated penetration capacity into cells. As a consequence of the improved permeability, two derivatives, O-hexyl- and O-decyl-alpha-amanitin, were more toxic in vivo than alpha-amanitin, although their affinity to RNA polymerases II was much reduce. By reaction of N-tert-butyloxy-carbonyl-N'-(6-bromocaproyl)ethylenediamine with alpha-amanitin, a ten-atom spacer with a terminal amino group could be introduced into the toxin, which allowed the attachment of alpha-amanitin to proteins, solid-phase supports, or reporter groups. For example, by reaction with fluoresceinyl isothiocyanate, a fluorescent amatoxin was prepared for visualizing amatoxin-binding structures in cells. After succinylation of the spacer moiety, alpha-amanitin could be attached to proteins, e.g., fetuin, yielding a derivative with good antigenic properties. When an alpha-amanitin derivative was coupled to Sepharose 6B, an adsorbent for affinity chromatography was obtained suitable for a one-step purification of amatoxin-binding immunoglobulins from the sera of immunized rabbits.

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