MINIREVIEW
Marine toxins and the cytoskeleton: a new view of
palytoxin toxicity
M. Carmen Louzao, Isabel R. Ares and Eva Cagide
Departamento de Farmacologia, Facultad de Veterinaria, Universidad de Santiago de Compostela, Lugo, Spain
Introduction
Palytoxin is a potent marine toxin that was first
isolated from a coelenterate tentatively identified as
Palythoa sp. This toxin was shown to be extremely
toxic to mammals, with a reported LD
50
value of just
0.45 lgÆkg
)1
after intraperitoneal injection into mice
[1]. In addition to being highly toxic, palytoxin has
large and complex structure (Fig. 1), which was deter-
mined in the 1980s [2]. This water-soluble molecule
consists of a long, partially unsaturated, aliphatic
backbone with spaced cyclic ethers and 64 chiral
centers. Examination of the structure shows that there
is, in fact, a group of different palytoxins, whose
molecular weights vary according to the species from
which they are obtained. Several biogenic origins of
palytoxins have been proposed, as these toxins have
been found not only in zooanthids but in sea anemo-
nes, polychaete worms, crabs, and herbivorous fishes,
probably due to an accumulation of the toxin by the
food chain in the organisms living close to the zoan-
thid colonies [3].
It has been reported that dinoflagellates of the genus

+
-ATPase has been proposed as receptor for palytoxin. The marine
toxin is known to act on the Na
+
pump and elicit an increase in Na
+
per-
meability, which leads to depolarization and a secondary Ca
2+
influx,
interfering with some functions of cells. Studies on the cellular cytoskeleton
have revealed that the signaling cascade triggered by palytoxin leads to
actin filament system distortion. The activity of palytoxin on the actin cyto-
skeleton is only partially associated with the cytosolic Ca
2+
changes; there-
fore, this ion represents an important factor in altering this structure, but it
is not the only cause. The goal of the present minireview is to compile the
findings reported to date about: (a) how palytoxin and analogs are able to
modify the actin cytoskeleton within different cellular models; and (b) what
signaling mechanisms could be involved in the modulation of cytoskeletal
dynamics by palytoxin.
Abbreviations
F-actin, filamentous actin; G-actin, globular actin.
FEBS Journal 275 (2008) 6067–6074 ª 2008 The Authors Journal compilation ª 2008 FEBS 6067
the producing species [6]. Among the nine different
Ostreopsis species existing, five of them have been
reported as producers of toxic substances [6], but just
four of them have been named: Ostreopsis siamensis
was reported to produce ostreocin-D (Fig. 1), a potent

serious respiratory distress, mild dyspnea, wheezes,
and in some cases conjunctivitis [7,8]. Italian coasts
are not the only seawaters where Ostreopsis species
have appear; they have been found in the waters
around Spain and Greece as well, indicating that the
expansion in the Mediterranean Sea of these species
in recent years is becoming a potential risk for
human health in Europe.
Fig. 1. Palytoxin and ostreocin-D structures.
Palytoxin activity against the cytoskeleton M. C. Louzao et al.
6068 FEBS Journal 275 (2008) 6067–6074 ª 2008 The Authors Journal compilation ª 2008 FEBS
Distortion of the Na
+
pump
At the cellular level, a broad range of studies have
indicated that the Na
+
pump or Na
+
⁄ K
+
-ATPase is
the higher-affinity cellular receptor for palytoxin [9,10].
This protein transports three Na
+
out the cell in
exchange for two K
+
that are driven in by hydrolyzing
one molecule of ATP. This generated electrochemical

2+
influx [11,12] that may
lead to multiple events regulated by Ca
2+
-dependent
pathways. The mechanisms by which this rise in intra-
cellular Ca
2+
([Ca
2+
]
i
) is produced have not been
completely elucidated. Nevertheless, in excitable cells,
at least three mechanisms have been reported to be
involved in the intracellular Ca
2+
increase: (a) voltage-
dependent Ca
2+
channels activated by the initial depo-
larization of the membrane; (b) Na
+
–Ca
2+
exchanger
in reverse mode, which drives Ca
2+
into the cells
because of the intracellular Na

functions. In eukaryotic cells, the actin cytoskeleton is
required for cell motility and surface remodeling; it is
essential for several contractile activities, such as mus-
cle contraction and the separation of daughter cells by
the contractile ring during cytokinesis; it controls cell–
cell and cell–substrate interactions, together with adhe-
sion molecules; and it actively participates in signal
transduction, cell volume regulation, secretion, and
surface receptor modulation [18–23].
Although palytoxin has been investigated as a
compound that is able to interfere with some of these
cellular functions [24–27], its effects on the actin cyto-
skeleton were not studied until few years ago. The
same is true of other palytoxin-like compounds, such
as ostreocin-D and ovatoxin-a, although in these cases
almost no information is available concerning their
biological activity. The first investigations assaying
palytoxin and ostreocin-D toxicities were performed in
freshly isolated intestinal cells (rabbit enterocytes from
the duodenum–jejunum), using fluorescent phalloidin
as an F-actin marker and laser-scanning cytometry
and confocal microscopy as techniques for analysis
[28]. There were two reasons for starting this type of
study with an intestinal model: (a) the great complexity
of the actin cytoskeleton in these cells; and (b) the
severe gastrointestinal toxicity exerted by palytoxin
in vivo [29,30]. Nanomolar concentrations (75 nm)of
palytoxin or ostreocin-D, and 4 h of incubation, were
enough to induce sharp F-actin disassembly and to
almost halve the quantity of F-actin on intestinal cells.

maintenance of cells in suspension was not responsible
for the absence of shape changes when palytoxins were
probed. Another interesting feature observed in the
case of latrunculin-A was some brush border disorga-
nization in the apical region of intestinal cells that was
not found after palytoxin or ostreocin-D treatments.
New findings have demonstrated again the palytoxin
activity on the cytoskeleton of intestinal cells. This
toxin induced dose- and time-dependent F-actin dis-
ruption in cultured Caco-2 cells [36], a human carcino-
genic line that undergoes spontaneous in vitro
enterocytic differentiation [37]. Interestingly, a correla-
tion among partial F-actin breakdown after 1 h of
palytoxin treatment (100 nm), morphological altera-
tions and cell detachment from substratum was also
found in that study. In this respect, the outcome of
ostreocin-D treatment of CaCo-2 cells remains
unknown.
The impact of palytoxin action on actin filament sys-
tem is not restricted to epithelial cells. In a recent
report, Louzao et al. [12] demonstrated that palytoxins
were able also to interfere with the cytoskeleton of
neuronal cells. These assays were carried out on the
human neuroblastoma cell line BE(2)-M17, an excit-
able model previously utilized for exploring anticyto-
skeletal effects and changes in ion fluxes in response to
marine toxins [38–41]. Here, palytoxin and ostreocin-D
triggered a cascade of cytotoxic events, ranging from a
Fig. 2. Left: histograms obtained with laser-scanning cytometry, displaying a reduction in the fluorescence associated with F-actin of freshly
isolated rabbit intestinal cells incubated with palytoxin (upper) or latrunculin-A (lower) in comparison to control cells. Previous to measure-

[25,42,43]. A connection has also been found between
Ca
2+
influx and palytoxin and ostreocin-D effects on
the actin cytoskeleton. This phenomenon seems to
occur in different ways in the different cell types inves-
tigated. Ares et al. [28] found that in epithelial cells
from the rabbit duodenum–jejunum, omission of extra-
cellular Ca
2+
(nominally Ca
2+
-free medium) halved
the effect of palytoxins on F-actin disassembly. Those
data led to the idea that these toxins modified the actin
filament system of intestinal cells not only by modulat-
ing some signaling pathway activated by external
Ca
2+
, but also by acting on another, still unknown,
element. Results obtained with neuroblastoma cells
indicated that palytoxin and ostreocin-D stimulated
similar decreases in F-actin quantity, independently of
extracellular Ca
2+
entry. This effect is not related to
Ca
2+
being released from internal stores, as palytoxin
and ostreocin-D do not induce increases of Ca

This minireview compiles the latest findings on how
palytoxin and ostreocin-D are able to act on the actin
filament system within cells. A signaling pathway involv-
ing Ca
2+
influx is partially related to that activity. How-
ever, as Ca
2+
influx is not responsible for some of the
effects elicited by palytoxins on the cellular actin cyto-
skeleton, more factors must be involved. Assuming that
the Na
+
pump is the target of palytoxin, one interesting
option would be that Na
+
⁄ K
+
-ATPase per se activates
some mechanism connected to the actin cytoskeleton
after its interaction with palytoxin (or ostreocin-D, if
they share the same target). This possibility arise from
several recent studies, where has been suggested that in
addition to pumping ions, Na
+
⁄ K
+
-ATPase also acts
as a signal transducer [44,45]. In this respect, studies
carried out with ouabain, a natural blocker of the Na

pump
activity might depend on its linkage to the actin filament
system [50,51]. In any case, the study of the possible
existence of some Na
+
⁄ K
+
-ATPase-mediated signaling
mechanism involved in the modulation of actin cytoskel-
etal dynamics by palytoxins opens a new line to follow
in the future in this field of investigation.
References
1 Onuma Y, Satake M, Ukena T, Roux J, Chanteau S,
Rasolofonirina N, Ratsimaloto M, Naoki H & Yasum-
oto T (1999) Identification of putative palytoxin as the
cause of clupeotoxism. Toxicon 37, 55–65.
2 Moore RE & Bartolini G (1981) Structure of palytoxin.
J Am Chem Soc 103, 2491–2494.
3 Mebs D (1998) Occurrence and sequestration of toxins
in food chains. Toxicon 36, 1519–1522.
4 Usami M, Satake M, Ishida S, Inoue A, Kan Y &
Yasumoto T (1995) Palytoxin analogs from the dino-
flagellate Ostreopsis siamensis. J Am Chem Soc 117,
5389–5390.
5 Taniyama S, Arakawa O, Terada M, Nishio S,
Takatani T, Mahmud Y & Noguchi T (2003) Ostreopsis
sp., a possible origin of palytoxin (PTX) in parrotfish
Scarus ovifrons. Toxicon 42, 29–33.
6 Vale C & Ares IR (2007) Biochemistry of palytoxins
and ostreocins. In Phycotoxins Chemistry and Biochem-

Tsubakihara M, Berry DA & Nosworthy NJ (2003)
Actin binding proteins: regulation of cytoskeletal micro-
filaments. Physiol Rev 83, 433–473.
14 Pollard TD, Blanchoin L & Mullins RD (2000) Molecu-
lar mechanisms controlling actin filament dynamics in
nonmuscle cells. Annu Rev Biophys Biomol Struct 29,
545–576.
15 Fiorentini C, Matarrese P, Fattorossi A & Donelli G
(1996) Okadaic acid induces changes in the organization
of F-actin in intestinal cells. Toxicon 34, 937–945.
16 Twiner MJ, Hess P, Dechraoui MY, McMahon T,
Samons MS, Satake M, Yasumoto T, Ramsdell JS &
Doucette GJ (2005) Cytotoxic and cytoskeletal effects
Palytoxin activity against the cytoskeleton M. C. Louzao et al.
6072 FEBS Journal 275 (2008) 6067–6074 ª 2008 The Authors Journal compilation ª 2008 FEBS
of azaspiracid-1 on mammalian cell lines. Toxicon 45,
891–900.
17 Allingham JS, Klenchin VA & Rayment I (2006) Actin-
targeting natural products: structures, properties and
mechanisms of action. Cell Mol Life Sci 63, 2119–2134.
18 Calderwood DA, Shattil SJ & Ginsberg MH (2000)
Integrins and actin filaments: reciprocal regulation of cell
adhesion and signaling. J Biol Chem 275, 22607–22610.
19 Papakonstanti EA, Vardaki EA & Stournaras C (2000)
Actin cytoskeleton: a signaling sensor in cell volume
regulation. Cell Physiol Biochem 10, 257–264.
20 Pollard TD & Borisy GG (2003) Cellular motility dri-
ven by assembly and disassembly of actin filaments.
Cell 112, 453–465.
21 Biron D, Alvarez-Lacalle E, Tlusty T & Moses E (2005)

the actin filament cytoskeleton in the intestinal brush
border: a quantitative and qualitative immunoelectron
microscope study. J Cell Biol 107, 1037–1048.
30 Ito E, Ohkusu M & Yasumoto T (1996) Intestinal
injuries caused by experimental palytoxicosis in mice.
Toxicon 34, 643–652.
31 Patel K, Harding P, Haney LB & Glass WF (2003)
Regulation of the mesangial cell myofibroblast
phenotype by actin polymerization. J Cell Physiol 195,
435–445.
32 Domnina LV, Rovensky JA, Vasiliev JM & Gelfand
IM (1985) Effect of microtubule-destroying drugs on
the spreading and shape of cultured epithelial cells.
J Cell Sci 74, 267–282.
33 Goldman RD, Khuon S, Chou YH, Opal P & Steinert
PM (1996) The function of intermediate filaments in cell
shape and cytoskeletal integrity. J Cell Biol 134, 971–
983.
34 Coue
´
M, Brenner SL, Spector I & Korn E (1987) Inhi-
bition of actin polymerization by latrunculin A. FEBS
Lett 213, 316–318.
35 Cai S, Liu X, Glasser A, Volberg T, Filla M, Geiger B,
Polansky JR & Kaufman PL (2000) Effect of latruncu-
lin-A on morphology and actin-associated adhesions of
cultured human trabecular meshwork cells. Mol Vis 6,
132–143.
36 Valverde I, Lago J, Vieites JM & Cabado AG (2008)
In vitro approaches to evaluate palytoxin-induced toxic-

aortic endothelial cells. Am J Physiol Cell Physiol 291,
C657–C667.
44 Liu J, Tian J, Haas M, Shapiro JI, Askari A & Xie Z
(2000) Ouabain interaction with cardiac Na+ ⁄ K+-
ATPase initiates signal cascades independent of changes
in intracellular Na+ and Ca2+ concentrations. J Biol
Chem 275, 27838–27844.
M. C. Louzao et al. Palytoxin activity against the cytoskeleton
FEBS Journal 275 (2008) 6067–6074 ª 2008 The Authors Journal compilation ª 2008 FEBS 6073
45 Xie Z & Askari A (2002) Na(+) ⁄ K(+)-ATPase as a
signal transducer. Eur J Biochem 269, 2434–2439.
46 Aizman O, Uhlen P, Lal M, Brismar H & Aperia A
(2001) Ouabain, a steroid hormone that signals with
slow calcium oscillations. Proc Natl Acad Sci USA 98,
13420–13424.
47 Aydemir-Koksoy A, Abramowitz J & Allen JC (2001)
Ouabain-induced signaling and vascular smooth muscle
cell proliferation. J Biol Chem 276, 46605–46611.
48 Koob R, Kraemer D, Trippe G, Aebi U & Drenckhahn
D (1990) Association of kidney and parotid
Na+,K(+)-ATPase microsomes with actin and analogs
of spectrin and ankyrin. Eur J Cell Biol 53, 93–100.
49 Devarajan P, Scaramuzzino DA & Morrow JS (1994)
Ankyrin binds to two distinct cytoplasmic domains of
Na,K-ATPase alpha subunit. Proc Natl Acad Sci USA
91, 2965–2969.
50 Cantiello HF (1997) Changes in actin filament organiza-
tion regulate Na+,K(+)-ATPase activity. Role of actin
phosphorylation. Ann NY Acad Sci 834, 559–561.
51 Gomes P & Soares-da-Silva P (2002) Dopamine-induced