©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Swinholide A Is a Microfilament Disrupting Marine Toxin That Stabilizes Actin Dimers and Severs Actin Filaments (*)

(Received for publication, November 17, 1994; and in revised form, December 20, 1994)

Michael R. Bubb (§) Ilan Spector (1) Alexander D. Bershadsky (2) Edward D. Korn (¶)

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

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

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.


INTRODUCTION

Swinholide A, which was discovered in a search for marine natural products with biological activity(1) , is an unusual 44-carbon dimeric dilactone ring macrolide with a 2-fold axis of symmetry(2, 3, 4) . It has become an important target for chemical studies because of its striking structural features and biomedical potential(5) . The total synthesis of swinholide A was recently reported(6) . Swinholide A has antifungal activity (1) and is highly cytotoxic to a variety of cancer cell lines (3, 5) , but the mechanism by which it exerts its cytotoxicity is unknown. In this paper, we describe the effects of swinholide A on actin filaments in vivo and its interactions with F-actin and G-actin in vitro.


EXPERIMENTAL PROCEDURES

Cytology

Balb/c 3T3 and Swiss 3T3 cells (American Type Culture Collection) were plated in 35-mm tissue culture dishes in Dulbecco's modified Eagle's medium supplemented with 10% calf serum (Life Technologies, Inc.) at 37 °C in a humidified atmosphere of 7.5% CO(2) in air. Stock solutions of swinholide A (5 and 50 µM) in dimethyl sulfoxide were stored at 4 °C. Swinholide A was added to the culture dishes at final concentrations of 5-100 nM, and cells were examined over a 1-24-h period. For fluorescence microscopy, treated and untreated cells grown on coverslips were fixed with 3% paraformaldehyde in phosphatebuffered saline and then permeabilized with 0.2% Triton X-100 in phosphate-buffered saline and labeled with TRITC-phalloidin (^1)to visualize F-actin (7) and with 4`,6-diamido-2-phenylindole to visualize the nucleus(8) . The stained cells were examined with a Zeiss Axiophot microscope equipped with epifluorescence illumination.

Actin Dimer Formation

Pyrenyl-actin was prepared (9) from gel-filtered rabbit skeletal muscle actin(10) . The ability of swinholide A to sequester unpolymerized actin subunits was quantified by the increase in the apparent critical concentration of pyrenyl-actin at steady state in buffer containing 2.0 mM MgCl(2), 0.1 mM CaCl(2), 0.2 mM dithiothreitol, 0.2 mM ATP, 0.01% sodium azide, and 5.0 mM Tris, pH 7.8 (buffer F). Pyrenyl-actin fluorescence intensity was measured at 21 °C at an excitation wavelength of 366 nm and emission wavelength of 386 nm. Sedimentation velocity data were obtained at 53,000 rpm at 18 °C in a Beckman XLA analytical ultracentrifuge. Optical absorbance scans at 290 nm, with and without swinholide A, were obtained at 7-min intervals in buffer G (buffer F without MgCl(2)). To cross-link actin oligomers, actin (20 µM) was incubated with an equal volume of PDM in 20 mM sodium borate for 5 min at room temperature; the products were analyzed by SDS-polyacrylamide gel electrophoresis(11) . Purified recombinant gelsolin (12) was provided by Dr. Alan Weeds, MRC, Cambridge, UK.

Actin-severing Assays

Ca-F-actin (10 µM) was prepared by polymerizing Ca-pyrenyl Ca-G-actin in buffer G by the addition of MgCl(2) to a final concentration of 2.0 mM. To prepare Mg-F-actin (10 µM), the stock Ca-pyrenyl G-actin was first converted to Mg-pyrenyl G-actin by addition of 125 µM EGTA and 50 µM MgCl(2) (final concentrations) and then polymerized by addition of 2.0 mM MgCl(2) (final concentration); the divalent cation does not readily exchange unless actin is preincubated in EGTA and MgCl(2) prior to polymerization(13) . Fluorescence intensity was measured at 18 °C in either a 27-µl cuvette, for stopped-flow measurements, or after 10 s of mixing by inversion in a conventional 1.8-ml cuvette, with the first measurement occurring 25 s after dilution.


RESULTS

Effects of Swinholide A on Cell Morphology and the Actin Cytoskeleton

At concentrations as low as 80 nM, swinholide A caused rounding of cultured mouse embryo 3T3 fibroblast cells within 1 h and massive destruction of the actin cytoskeleton as monitored by TRITC-phalloidin staining (data not shown). At concentrations of 10-50 nM swinholide A, partial cell retraction or arborization and diminution of microfilament bundles (stress fibers) began after 2-4 h, with complete loss of stress fibers by 5-7 h. The effects of 50 nM swinholide A on cells grown to subconfluence are shown in Fig. 1, A and B, and the effects of 10 nM swinholide A on exponentially growing cells in Fig. 1, D and E. The intensely fluorescent, well developed arrays of stress fibers (Fig. 1A) completely disappeared in cells exposed to 50 nM swinholide A for 24 h with the appearance of weakly fluorescent patches randomly scattered throughout the cytoplasm (Fig. 1B). Exponentially growing cells (Fig. 1C) exposed to 10 nM swinholide A for 24 h became arborized with diffuse cytoplasmic staining and fluorescent punctate structures (Fig. 1D). Almost all of these cells became binuclear (Fig. 1E), indicating that swinholide A did not interfere with the progression of cells through mitosis but inhibited cytokinesis, presumably by inhibiting formation and function of the contractile ring. Swinholide A did not affect the integrity and organization of the microtubule system (data not shown).


Figure 1: Effects of swinholide A on mouse fibroblasts. Fluorescence micrographs of Balb/c 3T3 (A and B) and Swiss 3T3 (C and D) cells labeled with TRITC-phalloidin. The decrease in fluorescence intensity in B and D was much greater than it appears to be because the exposure times were varied to optimize visualization. A, control cells grown to subconfluence; B, subconfluent cells treated for 24 h with 50 nM swinholide A. C, sparse cultures of exponentially growing control cells. D and E, exponentially growing cells treated with 10 nM swinholide A for 24 h and labeled with TRITC-phalloidin (D) and 4`,6-diamido-2-phenylindole (E); both cells are binuclear. Bar = 20 µm.



Sequestration of Actin Dimers by Swinholide A

To elucidate the mechanism by which swinholide A disrupts the cytoskeleton, its interactions with G-actin and F-actin were studied in vitro. Sedimentation velocity data (Fig. 2A) showed that swinholide A converted monomeric G-actin, s(w) = 3.4 S(14) , into a single species with a sedimentation coefficient s(w) = 5.1 S which, assuming a partial specific volume of 0.73 ml/g(14) , is exactly the predicted value (15) for a dimer consisting of two contiguous spheres. In experiments with limiting amounts of swinholide A, the molar concentration of actin dimer formed from G-actin was equal to the total concentration of swinholide A (data not shown). The same results were obtained when swinholide A was added to actin in buffer F (data not shown), demonstrating that swinholide A also can induce the formation of an actin dimer under polymerizing conditions. As quantified by the increase in the apparent critical concentration of pyrenyl-actin in buffer F (Fig. 2B), the amount of actin sequestered by swinholide A was consistent with highly cooperative binding of two actin monomers to one molecule of swinholide A (^2)with an equilibrium association constant, K(a) = 9.2bullet10M (95% C.I., 7.8bullet10 to 1.06bullet10). For comparison, this is equivalent to the binding of one actin dimer to one swinholide A^2 with K(a) = 4bullet10^7M (C.I., 3.6bullet10^7 to 4.6bullet10^7). The ability of swinholide A to bind actin dimers is reasonable given its 2-fold axis of symmetry(2) .


Figure 2: Sequestration of dimeric actin by swinholide A. A, sedimentation velocity analysis of the swinholide A-induced actin dimer. Monomeric actin (15 µM) was added to buffer G alone (up triangle) and with 10 µM swinholide A (box). B, effect of swinholide A on the apparent actin critical concentration. Pyrenyl-F-actin was diluted in buffer F to the concentrations shown in the presence of 0 (box), 0.3 (up triangle), 0.9 (circle), or 1.8 () µM swinholide A, and the fluorescence was determined at steady state. The solidlines are the theoretical predictions of the total expected fluorescence if swinholide A binds two actin subunits with K = 9.2bullet10M and infinite cooperativity, the critical concentration is 0.22 µM (95% C.I., 0.20-0.24), and filamentous actin is 33 times more fluorescent than unpolymerized actin, as independently determined (data not shown). If swinholide A binds two actin subunits with finite cooperativity, the higher concentrations of swinholide A would be less likely to be saturated and, thus, more likely to reveal any deviation from infinite cooperativity. Therefore, the consistent deviation of the data for 0.9 µM swinholide A to the left of the theoretical line could imply the existence of a small amount of swinholide A (less than 10%) with a single bound actin subunit and finite cooperativity, i.e. the apparently non-random residuals observed when the data are compared to the theoretical curve imply that the model may be imperfect. C, SDS-polyacrylamide gel electrophoresis analysis of PDM-cross-linked actin. Lanes1-3, actin cross-linked in buffer G (actin:PDM = 2:1) in the presence of 0, 5, and 15 µM swinholide A. Lane4, actin cross-linked in the absence of swinholide A by addition of PDM (actin:PDM = 2:1) simultaneously with 2 mM MgCl(2). The dimer observed in lanes2-4 had the same electrophoretic mobility (apparent mass, 86 kDa) as the dimer obtained by cross-linking the gelsolin-actin dimer complex (data not shown) in which the two actin subunits are anti-parallel (16) . Lane 5, filamentous actin cross-linked in buffer F (actin:PDM = 1:2) in the absence of swinholide A produced a ladder of actin oligomers including a dimer (apparent molecular mass, 116 kDa) resulting from cross-linking two adjacent parallel subunits across the genetic helix(19) .



In the presence of swinholide A, actin was cross-linked by PDM in a non-polymerizing buffer to a species (Fig. 2C, lanes2 and 3) with identical electrophoretic mobility to that of the actin dimer formed when the cross-linking reagent was added immediately after addition of MgCl(2) (Fig. 2C, lane4, and (16) ). This is the same electrophoretic mobility as the dimer obtained by cross-linking the two actin subunits that bind to the F-actin severing protein, gelsolin(12) , and faster than the electrophoretic mobility of the dimer formed when adjacent subunits along the genetic helix of F-actin are cross-linked (Fig. 2C, lane5 and (17) ). Maximum cross-linking efficiency in the presence of swinholide A was obtained at a molar ratio of 1.0 PDM to 2.0 actin subunits (data not shown), as expected if the two subunits in the actin dimer were oriented in anti-parallel fashion so that the Cys-374 residues of the subunits could be cross-linked, as previously suggested(12, 16) .

Severing and Depolymerization of F-actin by Swinholide A

In addition to sequestering non-polymerized actin subunits, stoichiometric concentrations of swinholide A severed F-actin. As determined by stopped-flow fluorescence measurements, (^3)the rate of depolymerization induced by swinholide A increased during the first several seconds after dilution (Fig. 3A), which suggested that swinholide A increased the number of filament ends by severing F-actin. Swinholide A was more effective at higher Mg concentrations (Fig. 3B), as is gelsolin (18) , but Ca-F-actin was also severed (Fig. 3, C and D). Depolymerization was highly cooperative with respect to swinholide A concentration (Fig. 3, C and D), implying that swinholide A must bind to several neighboring subunits before the filament breaks. This is in contrast to severing by the protein, gelsolin, for which severing is non-cooperative, but is qualitatively similar to the severing activity of actophorin, a protein isolated from the soil amoeba, Acanthamoeba castellanii(19) .


Figure 3: Severing of F-actin by swinholide A as detected by the effect on the time course of depolymerization. A, initial depolymerization rates of Mg-pyrenyl F-actin. F-actin was diluted in a 3-syringe stopped-flow apparatus to a final concentration of 400 nM in 2.0 mM MgCl(2) containing 0 (-), 152 (+), 280 (bulletbulletbulletbullet), 480(- - -), and 1000 (circle) nM swinholide A, and the decrease in fluorescence was measured over time. The injection was complete at t = 0.5 s. B, dependence of the severing activity of swinholide A on the Mg concentration. Mg-pyrenyl F-actin was diluted at time t = 0 to a final concentration of 190 nM actin and either 2.0 (bulletbulletbulletbullet) or 0.1 (-) mM MgCl(2) with either 0 (box) or 158 nM (circle) swinholide A. Depolymerization was followed by the decrease in fluorescence. C, depolymerization rates of Ca-pyrenyl F-actin. F-actin was diluted, as in B, in varying concentrations of swinholide A in 0.1 mM MgCl(2) in the absence (circle) or presence (box) of gelsolin-actin dimer added to the stock F-actin at a concentration of 1 complex:20 F-actin subunits immediately prior to dilution. D, depolymerization rates of Ca-pyrenyl F-actin as in C but in 2.0 mM MgCl(2).



If swinholide A, like the protein gelsolin, not only severs actin filaments but also caps the barbed ends (^4)of severed filaments, the rates of depolymerization of uncapped and gelsolin-capped filaments should converge at high concentrations of swinholide A(20) . In fact, the opposite behavior was observed (Fig. 3, C and D) indicating that the barbed ends of filaments severed in the absence of gelsolin remained uncapped. If swinholide A caps the pointed ends of actin filaments, the rate of depolymerization of gelsolin-capped filaments should have decreased in the presence of swinholide A, contrary to what was observed (Fig. 3, C and D), because both ends would then have been blocked. Therefore, it is highly unlikely that swinholide A caps either end of actin filaments.

The increase in the rate of F-actin depolymerization was unlikely to have been due simply to sequestration of actin subunits by swinholide A because: (i) under the conditions of the experiments in Fig. 3, the initial concentration of actin monomers was very low; (ii) the effect of swinholide A was greater at higher Mg concentration (Fig. 3, B, C, and D) where the monomer concentration is lower; and (iii) successive increments in swinholide A concentration had a progressively greater effect on both gelsolin-capped and uncapped filaments (Fig. 3, C and D), the opposite of what would have been expected from simple mass action.

Swinholide A could actively destabilize F-actin by either complexing to and increasing the off rate of terminal subunits or by severing actin filaments, thus creating more filament ends. The first possibility is inconsistent with the observation that the depolymerization rate was not directly proportional to the concentration of swinholide A (Fig. 3, C and D). Also, the observed increase in depolymerization rate with time (Fig. 3A) is most consistent with an increase in the number of filament ends as a result of severing (21) . The highly cooperative dependence of the polymerization on the swinholide A concentration (Fig. 3, C and D) implies that swinholide A must bind to several neighboring subunits before the filament breaks. While the data are inconsistent with the possibility that swinholide A increases actin depolymerization rates by binding directly to the filament ends, the available data do not rule out the possibility that swinholide A binds cooperatively to the sides of actin filaments, and in doing so, increases the off rates of terminal subunits. Video microscopic observation of filament depolymerization (22) will be required to rule out this intriguing, but never previously observed, possibility.


DISCUSSION

Latrunculin and tolytoxin, two other macrolides that decrease the concentration of F-actin, appear to sequester actin monomers (7, 23, 24) and not to sever F-actin. (^5)Neither of these macrolides has the 2-fold axis of symmetry that presumably accounts for the interaction of swinholide A with an actin dimer. Although cytochalasins have been reported to induce the transient formation of an actin dimer and may have some severing activity, their principal activity is to cap the barbed end of actin filaments and serve as nuclei for filament elongation(25) . Cytochalasins do not usually cause a decrease in the concentration of F-actin (26, 27, 28) and do not form a stable complex with dimeric actin as determined by sedimentation velocity experiments(29) . The effects of swinholide A on the actin cytoskeleton and cell morphology are more like those of latrunculins (7) than of cytochalasins. It is not surprising that interactions of actin with swinholide A and cytochalasins differ as swinholide A lacks the carbonate or acetate carbonyl groups that are common to all cytochalasin derivatives that affect actin filament length(30) .

The interactions of swinholide A with actin are similar to those of gelsolin in that gelsolin's ability to sever F-actin also increases with increasing Mg concentration (18) and the actin dimers formed by gelsolin have the same electrophoretic mobility as those formed by swinholide A when both are covalently cross-linked. In contrast to swinholide A, gelsolin caps actin filaments with high affinity and its severing activity is non-cooperative. Fragments of gelsolin expressed by recombinant protein technology, however, do show cooperative effects in terms of their actin binding properties(31) . This implies that swinholide A may be a valuable tool for dissecting the mechanism by which gelsolin interacts with actin filaments. Actophorin, an actin-severing protein from the soil amoeba A. castellanii, demonstrates cooperativity that is qualitatively similar to swinholide A but appears to sever much less effectively and apparently binds to actin monomers(19) . Further studies of swinholide A may help elucidate the mechanisms of action of this and other actin-severing proteins in vitro and in vivo.

Therapeutic agents that target the cytoskeleton are mostly compounds that affect microtubules. Recently, however, several drugs that directly or indirectly affect the organization of the actin cytoskeleton have been investigated as potential therapies for neoplastic, immunologic, and cardiovascular disease(32, 33, 34, 35, 36) . Furthermore, it has been shown that filamentous actin contributes to the viscosity of sputum from patients with cystic fibrosis and that the actin filament-severing protein, gelsolin, reduces sputum viscosity in vitro(37) . The unique properties of swinholide A described in this paper not only probably explain its cytotoxicity but also make it (or a derivative less permeable to cell membranes) a candidate for therapeutic drug intervention in clinical situations where high viscosity due to filamentous actin is pathologic.


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.

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

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

(^1)
The abbreviations used are: TRITC-phalloidin, tetramethylrhodaminyl-phalloidin; C.I., confidence interval; PDM, N,N`-1,4phenylenedimaleimide; pyrenyl-actin, N-pyrenylcarboxymethylamidoethyl-actin.

(^2)
The 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 = [A(2)S]/c^2bullet[S], where c is the actin critical concentration, [S] is the concentration of free swinholide A (total minus [A(2)S]), and [A(2)S] 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 = [A(2)S]/[A(2)]bullet[S], where [A(2)] = c/2, assuming that at steady state all unpolymerized actin not complexed to swinholide A is dimer. The experiments described in Fig. 2do 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 [A(2)S] were determined by linear regression analysis of all of the steady-state fluorescence data in Fig. 2B.

(^3)
The 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.

(^4)
The 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.

(^5)
While the authors (24) mention that tolytoxin may have severing activity, their data are consistent with formation of a 1:1 complex with actin monomer.


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