jasplakinolide and dolastatin-11

To study cellular actin dynamics, a cell-free assay based on fluorescence anisotropy was developed. Using G-actin-Alexa as a probe, we found that anisotropy enhancement reflects F-actin elongation. Anisotropy enhancement varies with the concentration of magnesium and calcium cations and with ethylenediaminetetraacetate or well-known effectors of the polymerization. This assay gives the overall status of actin dynamics in cell extracts which are the closest conditions to in vivo, implying most of the regulating proteins that are missing in purified actin measurements. It can be used in a large-scale screening for chemical compounds which modulate actin polymerization.

Topics: Actins; Animals; Cations, Divalent; Cell Transformation, Neoplastic; Cell-Free System; Chelating Agents; Cytosol; Depsipeptides; Fluorescence Polarization; Fluorescent Dyes; In Vitro Techniques; Mice; NIH 3T3 Cells; Rabbits; Succinimides; Thermodynamics

Knowledge of the phalloidin binding position in F-actin and the relevant understanding of the mechanism of F-actin stabilization would help to define the structural characteristics of the F-actin filament. To determine the position of bound phalloidin experimentally, x-ray fiber diffraction data were obtained from well-oriented sols of F-actin and the phalloidin-F-actin complex. The differences in the layer-line intensity distributions, which were clearly observed even at low resolution (8 A), produced well-resolved peaks corresponding to interphalloidin vectors in the cylindrically averaged difference-Patterson map, from which the radial binding position was determined to be approximately 10 A from the filament axis. Then, the azimuthal and axial positions were determined by single isomorphous replacement phasing and a cross-Patterson map in radial projection to be approximately 84 degrees and 0.5 A relative to the actin mass center. The refined position was close to the position found by prior researchers. The position of rhodamine attached to phalloidin in the rhodamine-phalloidin-F-actin complex was also determined, in which the conjugated Leu(OH)(7) residue was found to face the outside of the filament. The position and orientation of the bound phalloidin so determined explain the increase in the interactions between long-pitch strands of F-actin and would also account for the inhibition of phosphate release, which might also contribute to the F-actin stabilization. The method of analysis developed in this study is applicable for the determination of binding positions of other drugs, such as jasplakinolide and dolastatin 11.

Topics: Actin Cytoskeleton; Actins; Animals; Antineoplastic Agents; Biophysics; Depsipeptides; Electrons; Macromolecular Substances; Models, Molecular; Models, Statistical; Phalloidine; Protein Binding; Protein Conformation; Rhodamines; X-Ray Diffraction

The successful synthesis of dolastatin 11, a depsipeptide originally isolated from the mollusk Dolabella auricularia, permitted us to study its effects on cells. The compound arrested cells at cytokinesis by causing a rapid and massive rearrangement of the cellular actin filament network. In a dose-and time-dependent manner, F-actin was rearranged into aggregates, and subsequently the cells displayed dramatic cytoplasmic retraction. The effects of dolastatin 11 were most similar to those of the sponge-derived depsipeptide jasplakinolide, but dolastatin 11 was about 3-fold more cytotoxic than jasplakinolide in the cells studied. Like jasplakinolide, dolastatin 11 induced the hyperassembly of purified actin into filaments of apparently normal morphology. Dolastatin 11 was qualitatively more active than jasplakinolide and, in a quantitative assay we developed, dolastatin 11 was twice as active as jasplakinolide and 4-fold more active than phalloidin. However, in contrast to jasplakinolide and phalloidin, dolastatin 11 did not inhibit the binding of a fluorescent phalloidin derivative to actin polymer nor was it able to displace the phalloidin derivative from polymer. Thus, despite its structural similarity to other agents that induce actin assembly (all are peptides or depsipeptides), dolastatin 11 may interact with actin polymers at a distinct drug binding site.

Topics: Actin Cytoskeleton; Actins; Animals; Antineoplastic Agents; Bacterial Proteins; Cell Division; Cells, Cultured; Depsipeptides; Dipodomys; Fluorescent Dyes; Isothiocyanates; Oligopeptides; Peptides; Peptides, Cyclic; Phalloidine