(Received for publication, November 17, 1994; and in revised form, December 20, 1994)
From the
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.
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.
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.
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 () and with 10 µM swinholide A (
). 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 (
),
0.3 (
), 0.9 (
), 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
= 9.2
10
M
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
. 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 (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) .
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
containing 0 (-), 152
(+), 280 (
), 480(- - -),
and 1000 (
) 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 (
) or 0.1 (-) mM MgCl
with either 0 (
) or 158 nM (
) 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
in the absence (
) or presence (
) 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
.
If swinholide A,
like the protein gelsolin, not only severs actin filaments but also
caps the barbed ends ()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.
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. ()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.