MINIREVIEW
Marine toxins and the cytoskeleton: okadaic acid
and dinophysistoxins
Carmen Vale and Luis M. Botana
Departamento de Farmacologı
´
a, Facultad de Veterinaria, USC, Lugo, Spain
Introduction
The syndrome diarrheic shellfish poisoning (DSP) was
first recognized in Japan 30 years ago. Although fatali-
ties associated with DSP-contaminated shellfish have
not been reported, this intoxication has become a seri-
ous problem for public health and for the economy of
aquaculture industries in several parts of the world.
Symptoms of DSP poisoning are mainly gastrointesti-
nal problems such as diarrhea, nausea, vomiting, and
abdominal pain. The major toxin involved in DSP is a
polyether derivative named dinophysistoxin-1 (DTX1).
Another previously identified polyether fatty acid com-
pound, named okadaic acid (OA), was found to be
one of the toxic components of DSP [1]. OA was first
isolated from the marine sponges Halichondria okadaii
and Halichondria melanodocia, and it was subsequently
shown to be produced by marine dinoflagellates of the
genera Dinophysis and Prorocentrum [2,3]. DTX1 was
confirmed to be 35S-methylokadaic acid [1]. The
molecular structures of OA and its analogs are shown
in Fig. 1.
Molecular and cellular effects of
diarrheic shellfish toxin exposure
The Ser/Thr protein phosphatases

Neither methyl okadaate (a weak phosphatase inhibitor) nor OA modify
the total amount of F-actin, but both toxins cause similar changes in the
F-actin cytoskeleton, with strong retraction and rounding, and in many
cases cell detachment. OA and dinophysistoxin-1 (35S-methylokadaic acid)
cause rapid changes in the structural organization of intermediate fila-
ments, followed by a loss of microtubules, solubilization of intermediate
filament proteins, and disruption of desmosomes. The detailed pathways
that coordinate all these effects are not yet known.
Abbreviations
AD, Alzheimer’s disease; DSP, diarrheic shellfish poisoning; DTX1, dinophysistoxin-1; F-actin, filamentous actin; FAK, focal adhesion kinase;
IF, intermediate filament; OA, okadaic acid; TPA, 12-O-tetradecanoylphorbol-13-acetate.
6060 FEBS Journal 275 (2008) 6060–6066 ª 2008 The Authors Journal compilation ª 2008 FEBS
phorylation of phosphoserine or phosphothreonine
residues. In mammalian cells, four major classes of
Ser ⁄ Thr protein phosphatases, termed PP1, PP2A,
PP2B (calcineurin) and PP2C, have been identified.
The number of physiological processes in which the
Ser ⁄ Thr protein phosphatases are involved is immense,
including regulation of glycogen metabolism and coor-
dination of the cell cycle and gene expression. The crit-
ical importance of phosphatases in cell metabolism is
underlined by the fact that they are targets of natural
toxins such as OA. Protein phosphorylation and
dephosphorylation events have been established as key
factors in the regulation of cytoskeletal structure and
function [4]. In this minireview, we focus on how diar-
rheic shellfish toxins (OA, dinophysistoxins and ana-
logs) affect Ser ⁄ Thr protein phosphatases and their
effects on cell adhesion and cytoskeletal dynamics, the
disruption of which is linked to loss of cell polarity,

protein phosphatase activity observed in human carci-
noma, metastatic and melanoma BL6 cells is associated
with increased cell motility and invasiveness. OA-medi-
ated PP2A inhibition also enhances cell motility. In
these cells, altered PP2A activity induced by pharmaco-
logical treatment with OA is accompanied by decreased
cell adhesion and cytoskeletal reorganization [10].
Interaction of diarrheic shellfish toxins
with cytoskeletal dynamics and
organization
Although there have been numerous studies employing
DSPs, and mainly OA, to examine the role of protein
phosphatases in cytoskeletal dynamics and cytoskeletal
organization, most of the reports were focused on the
role of phosphatases in the maintenance of cell cyto-
skeleton integrity. With this purpose, DSP toxins have
been widely employed, with OA being one of the most
useful tools. As the only known targets for OA are
Ser ⁄ Thr protein phosphatases, its toxic effects have
been usually related to the inhibition of PP1 and
PP2A, which are considered to account for most of the
Ser ⁄ Thr protein phosphatase activity in mammalian
cells. However, the fact that nonphosphatase targets
are not known for OA does not mean that they do not
exist. Below we review some of the findings related to
the interaction of DSP toxins with cytoskeleton ele-
ments, effects that are attributed almost exclusively to
their action as phosphatase inhibitors.
Toxin
R

OO
O
OH
RR
OCH
Fig. 1. Molecular structures of OA and
analogs.
C. Vale and L. M. Botana DSPs and the cytoskeleton
FEBS Journal 275 (2008) 6060–6066 ª 2008 The Authors Journal compilation ª 2008 FEBS 6061
Diarrheic shellfish toxins and cell adhesion
The adherence of cells to each other and to the elabo-
rate mesh that comprises the extracellular matrix is
mediated by multiprotein complexes. Taking advantage
of the effects of DSP toxins on protein phosphatase
activity, different studies have employed these toxins
(mainly OA) to examine cell–matrix interactions and
cell–cell contacts, either directly by controlling struc-
tural adhesion proteins, or indirectly by affecting pro-
teins involved in the signaling pathways that regulate
cell adhesion.
Cell adhesion to the extracellular matrix leads to the
formation and stabilization of focal adhesions, special-
ized sites of convergence for the actin cytoskeleton,
integrins and several interconnection protein com-
plexes. Thus, focal adhesions provide sites of signal
transduction that play a pivotal role in several cellular
functions, including cytoskeletal tension. At the center
of this transduction pathway is the focal adhesion
kinase (FAK), which, through interactions with several
other proteins, regulates cell motility. In endothelial

mediate filaments (IFs) to sites of strong adhesion.
The OA-induced desmosome dissasembly was presum-
ably regulated by extracellular Ca
2+
via reversible pro-
tein phosphorylation involving both protein kinases
and protein phosphatases. Inhibition of endothelial cell
PP2A by treatment with OA stimulated endothelial cell
motility through mechanisms related to the focal adhe-
sion proteins. Thus, it was found that OA inhibition
of PP2A caused hyperphosphorylation of the paxillin
Ser residues and dephosphorylation of its Tyr residues,
causing dissolution of FAK–Src–paxillin that will
eventually increase cell motility through increases in
the activities of accessory protein complexes [11].
DSP toxins and cytoskeletal dynamics
Cytoskeletal function and integrity rely on the inter-
play of three filament systems, microtubules, microfila-
ments, and IFs, which are integrated in a complex
network regulated by associated proteins. Cytoskeletal
structures play key roles in the maintenance of cell
architecture, adhesion, migration, differentiation, divi-
sion, and organelle transport. Cytoskeletal function is
directly regulated by DSP toxins, presumably trough
their interaction with Ser ⁄ Thr protein phosphatases, as
assumed in numerous studies employing DSP toxins to
examine cytoskeletal dynamics and integrity [17,18].
Diarrheic shellfish toxins and actin
As Ser ⁄ Thr protein phosphatases are some of the main
cytosolic enzymes involved in actin dynamics, numer-

number. This observation raised the question of
whether OA-induced cytoskeletal changes can be exclu-
sively attributed to its inhibition of protein phosphatase
activity, as methyl okadaate has been reported not to
inhibit PP1 and is a very poor inhibitor of PP2A
in vitro [25]. However, both toxins showed similar levels
of Ser ⁄ Thr phosphorylation on neuroblastoma cells
[18]. This observation might support the idea that
OA- and methyl okadaate-induced cytoskeletal changes
could be due to their effect on phosphatases, although
the effect of methyl okadaate on protein phosphatase
inhibition might have been underestimated previously.
In spite of the well-documented effect of OA on the
actin cytoskeleton, the exact pathway leading to OA-
induced cytoskeletal changes has not been elucidated.
As rearrangement of the actin cytoskeleton can be
induced by increases in the cytosolic Ca
2+
concentra-
tion, the effects of OA and methyl okadaate on cyto-
solic Ca
2+
have been also evaluated in neuroblastoma
cells. None of the toxins modified the intracellular
Ca
2+
concentration, indicating that Ca
2+
influx is not
responsible for OA-induced F-actin reorganization

2+
but might be related to the increases in the levels
of Ser ⁄ Thr phosphorylation of several cellular pro-
teins. However, in view of the reported inhibition of
PP2A and PP1 by methyl okadaate in vitro, it is possi-
ble that more cellular targets for this OA derivative
could exist.
DSPs and microtubules
In eukaryotic cells, microtubules form a well-organized
network that is highly regulated both spatially and
temporally. The microtubule is a dynamically regulated
structure composed of a- and b-tubulins. Microtubules
are stabilized by specific factors, including micro-
tubule-associated proteins, such as tau, and post-trans-
lational modifications (a-tubulin acetylation and
detyrosination), and destabilized by dissociation of tau
from microtubules or a-tubulin tyrosination. Accumu-
Fig. 2. Time course of the effects of OA and methyl okadaate on
the cytosolic Ca
2+
concentration in primary cultures of cerebellar
granule cells. Intracellular Ca
2+
was monitored in neurons loaded
with Fura-2. Mean ± SEM of three experiments.
Fig. 3. Effects of different concentrations of OA and methyl okada-
ate on cell viability in primary cultures of cerebellar granule cells.
Cellular viability was assessed by the 3-(4,5-dimethylthiazole-2-yl)-
2,5-diphenyltetrazolium bromide assay after 24 h of exposure
of the cells to different concentrations of the toxins. Values are

time. These observations are in accordance with recent
studies indicating an increase in tyrosinated tubulins in
primary cortical neurons after treatment with OA [17].
As the regulation of microtubules by phosphatase
activity also plays an important role during morpho-
genesis and tumorigenesis, the effect of OA has also
been investigated in human carcinoma cells. As PP2A
is associated with microtubules, in these cells OA treat-
ment results in increased cell motility and invasiveness
[29].
DSP toxins and intermediate filaments
IF proteins, a large family of tissue-specific proteins,
undergo several post-translational modifications, with
phosphorylation being the most studied of these. IFs
maintain cell shape and the structural integrity of cell
contents, and provide protection against various types
of stress. The mechanism of action of OA as a potent
tumor promoter and the biological significance of
Ser ⁄ Thr and Tyr protein phosphatases have been
extensively investigated in cancer-related research. The
hyperphosphorylation of IFs is one of the early
biochemical changes induced by OA-class tumor pro-
moters. The hyperphosphorylation of keratins induced
by OA treatment resulted in the reorganization of the
keratin filament network, which collapsed into large
perinuclear aggregates [30]. The effects of DSP toxins
on IF integrity were revealed after treatment of BHK-
21 fibroblasts with DSP toxins. OA and DTX1 caused
rapid changes in the structural organization of IFs, fol-
lowed by a loss of microtubules [4]. In a similar way,

study on the effect of methyl okadaate on the cyto-
skeleton, and its reported IC
50
for Ser ⁄ Thr protein
phosphatases in vitro, indicated that some other cellu-
lar targets for this particular compound could exist.
Taking advantage of new available markers to assess
cytoskeletal dynamics in living cells, further detailed
studies should be performed to investigate the effects
of DSP toxins on the cytoskeleton as well as the intra-
cellular mechanisms involved in the cytoskeletal dis-
organization caused by DSP toxins.
References
1 Vale P (2007) Chemistry of diarrhetic shellfish poison-
ing toxins. In Phycotoxins Chemistry and Biochemistry
(Botana LM, ed.), pp. 211–221. Blackwell, Ames, IA.
2 Dickey RW, Bobzin SC, Faulkner DJ, Bencsath FA &
Andrzejewski D (1990) Identification of okadaic acid
from a Caribbean dinoflagellate, Prorocentrum concav-
um. Toxicon 28, 371–377.
3 Cembella A (1989) Occurrence of okadaic acid, a major
diarrheic shellfish toxin, in natural populations of Din-
ophysis spp. from the eastern coast of North America.
J Appl Phycol 1, 307–310.
4 Eriksson JE, Brautigan DL, Vallee R, Olmsted J, Fujiki
H & Goldman RD (1992) Cytoskeletal integrity in
interphase cells requires protein phosphatase activity.
Proc Natl Acad Sci USA 89, 11093–11097.
5 Takai A, Bialojan C, Troschka M & Ru
¨

13 Tripuraneni J, Koutsouris A, Pestic L, De Lanerolle P
& Hecht G (1997) The toxin of diarrheic shellfish poi-
soning, okadaic acid, increases intestinal epithelial para-
cellular permeability. Gastroenterology 112, 100–108.
14 Okada T, Narai A, Matsunaga S, Fusetani N & Shi-
mizu M (2000) Assessment of the marine toxins by
monitoring the integrity of human intestinal Caco-2 cell
monolayers. Toxicol In Vitro 14, 219–226.
15 Serres M, Grangeasse C, Haftek M, Durocher Y, Duclos
B & Schmitt D (1997) Hyperphosphorylation of beta-
catenin on serine–threonine residues and loss of cell–cell
contacts induced by calyculin A and okadaic acid in
human epidermal cells. Exp Cell Res 231, 163–172.
16 Pasdar M, Li Z & Chan H (1995) Desmosome assembly
and disassembly are regulated by reversible protein
phosphorylation in cultured epithelial cells. Cell Motil
Cytoskeleton 30, 108–121.
17 Yoon SY, Choi JE, Choi JM & Kim DH (2008) Dynein
cleavage and microtubule accumulation in okadaic acid-
treated neurons. Neurosci Lett 437 , 111–115.
18 Vilarino N, Ares IR, Cagide E, Louzao MC, Vieytes
MR, Yasumoto T & Botana LM (2008) Induction of
actin cytoskeleton rearrangement by methyl okadaate –
comparison with okadaic acid. FEBS J 275
, 926–934.
19 Yano Y, Sakon M, Kambayashi J, Kawasaki T, Senda
T, Tanaka K, Yamada F & Shibata N (1995) Cytoskel-
etal reorganization of human platelets induced by the
protein phosphatase 1 ⁄ 2 A inhibitors okadaic acid and
calyculin A. Biochem J 307, 439–449.

Evaluation of the use of two human cell lines for okada-
ic acid and DTX-1 determination by cytotoxicity assays
and damage characterization. Nat Toxins 6, 197–209.
27 Nuydens R, Dispersyn G, Van Den Keiboom G, de
Jong M, Connors R, Ramaekers F, Borgers M &
Geerts H (2000) Bcl-2 protects against apoptosis-related
microtubule alterations in neuronal cells. Apoptosis 5,
43–51.
28 Gong CX, Lidsky T, Wegiel J, Zuck L, Grundke-Iqbal
I & Iqbal K (2000) Phosphorylation of microtubule-
associated protein tau is regulated by protein phospha-
tase 2A in mammalian brain. Implications for neurofi-
brillary degeneration in Alzheimer’s disease. J Biol
Chem 275, 5535–5544.
29 Meisinger J, Patel S, Vellody K, Bergstrom R, Benefield
J, Lozano Y & Young MR (1997) Protein phosphatase-
2A association with microtubules and its role in restrict-
ing the invasiveness of human head and neck squamous
cell carcinoma cells. Cancer Lett 111, 87–95.
30 Kasahara K, Kartasova T, Ren XQ, Ikuta T, Chida K
& Kuroki T (1993) Hyperphosphorylation of keratins
by treatment with okadaic acid of BALB ⁄ MK-2 mouse
keratinocytes. J Biol Chem 268, 23531–23537.
31 Yatsunami J, Fujiki H, Suganuma M, Yoshizawa S,
Eriksson JE, Olson MO & Goldman RD (1991) Vimen-
tin is hyperphosphorylated in primary human fibro-
blasts treated with okadaic acid. Biochem Biophys Res
Commun 177, 1165–1170.
32 Lee WC, Yu JS, Yang SD & Lai YK (1992) Reversible
hyperphosphorylation and reorganization of vimentin