Swinholide A Is a Microfilament Disrupting Marine Toxin That Stabilizes Actin Dimers and Severs Actin Filaments

doi:10.1074/jbc.270.8.3463

Swinholide A Is a Microfilament Disrupting Marine Toxin That Stabilizes Actin Dimers and Severs Actin Filaments (*)

From the (1)Laboratory of Cell Biology, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, the
Department of Physiology and Biophysics, Health Science Center, State University of New York, Stony Brook, New York 11794, and the
(2)Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel

^¶ To whom correspondence should be addressed:
Bldg. 3, Rm. B1-22, NIH, Bethesda, MD 20892.
Tel.: 301-496-1616; Fax: 301-402-1519.

↵^§ Present address: University of Florida College of Medicine, J. Hillis Miller Health Center, Gainesville, FL 32610-0277.

Abstract

Swinholide A, isolated from the marine sponge Theonella swinhoei, is a 44-carbon ring dimeric dilactone macrolide with a 2-fold axis of symmetry. Recent studies have elucidated its unusual structure and shown that it has potent cytotoxic activity. We now report that swinholide A disrupts the actin cytoskeleton of cells grown in culture, sequesters actin dimers in vitro in both polymerizing and non-polymerizing buffers with a binding stoichiometry of one swinholide A molecule per actin dimer, and rapidly severs F-actin in vitro with high cooperativity. These unique properties are sufficient to explain the cytotoxicity of swinholide A. They also suggest that swinholide A might be a model for studies of the mechanism of action of F-actin severing proteins and be therapeutically useful in conditions where filamentous actin contributes to pathologically high viscosities.

Footnotes

↵* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

TRITC-phalloidin

tetramethylrhodaminyl-phalloidin

C.I.

confidence interval

PDM

N,N′-1,4phenylenedimaleimide

pyrenyl-actin

N-pyrenylcarboxymethylamidoethyl-actin.
↵²The value of K for highly cooperative binding of two actin subunits to swinholide A was determined by the best fit of the data to the equation, K = [A₂S]/c²•[S], where c is the actin critical concentration, [S] is the concentration of free swinholide A (total minus [A₂S]), and [A₂S] is the concentration of swinholide with two bound actin subunits. This equation implies infinite cooperativity since the concentration of swinholide A complexed to a single actin subunit is assumed to be zero. K = [A₂S]/[A₂]•[S], where [A₂] = c/2, assuming that at steady state all unpolymerized actin not complexed to swinholide A is dimer. The experiments described in Fig. 2 do not distinguish between these two models because the state of the unpolymerized actin is indeterminant, but sedimentation velocity analysis under the same conditions established that the unpolymerized actin was predominantly, if not exclusively, monomer (data not shown). Therefore, K does not apply. The values of c and [A₂S] were determined by linear regression analysis of all of the steady-state fluorescence data in Fig. 2B.
↵³The rate of depolymerization was about 15% higher in the absence of swinholide A when measured by stopped-flow fluorescense than when measured in a steady-state fluorimeter either because the initial rate was determined more accurately or because of fragmentation during injection.
↵⁴The two ends of the polarized filament are designated “barbed” and “pointed” from the arrowhead-like appearance of electron microscopic images of filaments decorated with myosin.
↵⁵While the authors (24) mention that tolytoxin may have severing activity, their data are consistent with formation of a 1:1 complex with actin monomer.
- Received November 17, 1994.
- Revision received December 20, 1994.