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 Ostreopsis are the most probable origin of palytoxin [4,5]. In fact, several toxins with palytoxin-like charac- teristics have been described and named according to Keywords actin filament; cytoskeleton; ostreocin-D; Ostreopsis; ovatoxin-a; palytoxin Correspondence M. C. Louzao, Departamento de Farmacologı ´ a, Facultad de Veterinaria, Universidad de Santiago de Compostela, Campus de Lugo, 27002 Lugo, Spain Fax: +34 982 252 242 Tel: +34 982 252 242 E-mail: mcarmen.louzao@usc.es (Received 7 July 2008, revised 12 September 2008, accepted 16 September 2008) doi:10.1111/j.1742-4658.2008.06712.x Palytoxin is a marine toxin first isolated from zoanthids (genus Palythoa), even though dinoflagellates of the genus Ostreopsis are the most probable origin of the toxin. Ostreopsis has a wide distribution in tropical and sub- tropical areas, but recently these dinoflagellates have also started to appear in the Mediterranean Sea. Two of the most remarkable properties of paly- toxin are the large and complex structure (with different analogs, such as ostreocin-D or ovatoxin-a) and the extreme acute animal toxicity. The Na + ⁄ K + -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 palytoxin analog with a LD 50 value of 0.75 lgÆkg )1 when given intraperitoneally [4], Ostreopsis lenticularis produces the neurotoxic ostreotoxins, Ostreopsis mascarenensis produces mascarenotoxins, and recently, Ostreopsis ovata has been identified as the producer of ovatoxin-a [7]. Ostreopsis species are important components of tropical and subtropical reef environments, but recently these dinoflagellates also started to appear in temperate waters, such as those from the Mediter- ranean Sea, even producing toxic outbreaks [7,8]. Human toxicity due to palytoxin has been reported as food poisoning (named clupeotoxism) and respira- tory intoxications. Palytoxin enters the food chain and accumulates mainly in fishes such as sardines, herrings and anchovies from tropical seas, causing neurological and gastrointestinal disturbances asso- ciated with clupeotoxism [1]. Symptoms include a bitter and metallic taste followed by nausea, vomit- ing and diarrhea, with mild to acute lethargy. Several hours later, burning sensations around the mouth and in the extremities, impairment of sensa- tion, muscle spasms and tremor myalgia, dyspnea and dysphonia occur, possibly leading to death due to myocardial injury. Recently, in Italy, exposure to marine aerosols has been reported that may also cause human illness, with fever being associated with 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 gradient is essential in maintaining electrolyte homeo- stasis. The Na + ⁄ K + -ATPase is also the specific target for heart glycosides such as ouabain, which bind to the pump and block it when the Na + -binding sites are open to the extracellular side [10]. Nevertheless, paly- toxin seems to convert the pump into a nonspecific ion channel, allowing Na + and K + fluxes [9,10]. Despite having a different interaction with the Na + ⁄ K + - ATPase, ouabain blocks palytoxin effects in several systems, and palytoxin inhibits ouabain binding as well. It is known that changes in ion fluxes are the imme- diate effects of palytoxin on the cells. In particular, increasing the Na + permeability leads to depolari- zation and a secondary Ca 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 + influx and membrane depolarization; and (c) an as yet unidentified pathway that is independent of changes in membrane potential or changes in pH [9]. The actin cytoskeleton as a selective target for palytoxins Actin filaments or microfilaments are polymers of actin that, together with a large number of actin-binding and associated proteins, constitute the actin cytoskeleton [13]. For a long time, it was believed that the role of this system was mainly structural, providing the sup- port needed to maintain the shape and organization of the cell and the scaffolding for the actions of catalytic molecules such as motor proteins. Nowadays, the actin cytoskeleton is widely recognized as an enormously dynamic structure that undergoes constant reconstruc- tion and reorganization, which is possible because of its ability to switch rapidly between a filamentous actin (F-actin) polymeric form and a monomeric globular actin (G-actin) form [14]. This dynamism enables it to quickly remodel its structure as a consequence of cellu- lar stimuli, and to participate in localized responses to external agents or regional events within a cell. Many natural compounds that have been isolated from mar- ine sources exert their cytotoxicity by modulating cytoskeletal properties, in particular those concerning actin filaments [15–17]. Although this phenomenon could seem surprising, it is not so surprising if one con- siders the pivotal role of this structure in many cellular 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. Interestingly, a purified extract of O. ovata that con- tained a putative palytoxin-like compound was tested under the same experimental system, and identical results were obtained [6]. As previously described, the actin filament system seems to be closely linked to morphological cell characteristics, and therefore it could be reasonable to expect alterations in shape. Nevertheless, they were not observed here. Instead of this, enterocytes retained their typical columnar mor- phology after losing many of their microfilaments. Similar cases using cultured cells have been reported in the literature [31], and other cytoskeletal components could also be participating in maintenance of the cytoarchitecture [32,33]. 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 6069 The above assays were performed by incubating tox- ins and intestinal cells in suspension. After treatment, they were attached to the substratum with poly- l-lysine and later analyzed. To ensure that floating cells were sensitive to morphological modifications by cytotoxic agents, latrunculin-A was tested with an identical procedure to that used for palytoxin and ostreocin-D [28]. Latrunculin-A is a toxic compound extracted from the Red Sea sponge Negombata magni- fica that inhibits actin polymerization and induces morphological changes in living cells [34,35]. Latruncu- lin-A-treated cells lost one-third of their total actin content (reduction by 33 ± 6.7%) and underwent rounding (Fig. 2). This fact helps to confirm that 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- ment, the cellular actin cytoskeleton was specifically stained with fluorescent phalloidin. Right: transmission images recorded by confocal microscopy show the morphological differences between palytoxin (75 n M) and latrunculin-A (10 lM) treatments after 4 h of incubation. Arrows indicate the alterations on microvilli induced in latrunculin-A-treated cells. Palytoxin activity against the cytoskeleton M. C. Louzao et al. 6070 FEBS Journal 275 (2008) 6067–6074 ª 2008 The Authors Journal compilation ª 2008 FEBS rapid depolarization and cytosolic Ca 2+ increase to cytoarchitectural restructuring. Time-dependent studies using a 75 nm concentration of palytoxin and ostreo- cin-D provided interesting insights into how these toxins modify the cytoskeleton of neuroblastoma cells: (a) the distortion of the actin cytoskeleton begins at early stages, being detectable after 10 min of toxin incubation; and (b) both microfilament reorganization and morphological alterations are subsequent to the start of F-actin disruption [12]. In agreement with the studies using the CaCo-2 cell line, palytoxin also elicited loss of cellular adhesion and dose- and time-dependent F-actin disassembly in neuroblastoma cells, leading to its entire collapse in 24 h after toxin doses of 1 nm. Evidence from a number of systems suggests that influx of Na + and ⁄ or Ca 2+ is associated with many palytoxin-induced responses, including muscle contrac- tion, neurotransmitter release, and oncotic death [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 2+ in nominally Ca 2+ -free conditions [12]. On the other hand, the presence or absence of extracellular Ca 2+ was associated with a different F-actin organization in toxin-treated cells, which seems to suggest a new role for this cation in palytoxin action against the actin cytoskeleton (Fig. 3). At present, in spite of the impor- tance of the unpolymerized actin pool for the mainte- nance of the F-actin system, the cellular response Fig. 3. Response induced by palytoxin and ostreocin-D on the actin cytoskeleton of neuroblastoma cells after 4 h of incubation under differ- ent extracellular Ca 2+ conditions. Fluorescent phalloidin was utilized for labeling the cellular actin cytoskeleton before quantitative analysis with laser-scanning cytometry and imaging with confocal microscopy. 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 6071 triggered by palytoxin and ostreocin-D activity at this level has not yet been reported. Recent studies per- formed by Ares et al. have revealed that these marine toxins also induce alterations in G-actin of neuroblas- toma cells (I. R. Ares, E. Cagide, M. C. Louzao, B. Espin ˜ a, M. R. Vieytes, T. Yasumoto and L. M. Botana, unpublished results). Conclusions and perspectives 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 and an inhibitor of palytoxin action, have pro- vided interesting findings. Through the partial inhibition of Na + ⁄ K + -ATPase, and regardless of changes in intra- cellular ion concentrations, ouabain-induced signaling pathways have been recently found in several cellular models [44–47]. It has been proposed that the ouabain- bound Na + ⁄ K + -ATPase is capable of recruiting and activating protein tyrosine kinases through specific protein–protein interactions [45]. On the other hand, it should be not forgotten that the Na + pump could inter- act with cytoskeletal components. Specific cytoskeletal proteins thought to interact with Na + ⁄ K + -ATPase, either directly or indirectly, include spectrin, actin and ankyrin [48,49], and even regulation of 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- istry (Botana LM, ed.), pp. 95–118. Blackwell Publish- ing, Ames, IA. 7 Ciminiello P, Dell’Aversano C, Fattorusso E, Forino M, Tartaglione L, Grillo C & Melchiorre N (2008) Putative palytoxin and its new analogue, ovatoxin-a, in Ostreopsis ovata collected along the Ligurian coasts dur- ing the 2006 toxic outbreak. J Am Soc Mass Spectrom 19, 111–120. 8 Gallitelli M, Ungaro N, Addante LM, Procacci V, Silver NG & Sabba ` C (2005) Respiratory illness as a reaction to tropical algal blooms occurring in a temper- ate climate. JAMA 293, 2599–2600. 9 Scheiner-Bobis G (1998) Ion-transporting ATPases as ion channels. Naunyn Schmiedebergs Arch Pharmacol 357, 477–482. 10 Hilgemann DW (2003) From a pump to a pore: how palytoxin opens the gates. Proc Natl Acad Sci USA 100, 386–388. 11 Louzao MC, Vieytes MR, Yasumoto T, Yotsu-Ya- mashita M & Botana LM (2006) Changes in membrane potential: an early signal triggered by neurologically active phycotoxins. Chem Res Toxicol 19, 788–793. 12 Louzao MC, Ares IR, Vieytes MR, Valverde I, Vieites JM, Yasumoto T & Botana LM (2007) The cytoskele- ton, a structure that is susceptible to the toxic mecha- nism activated by palytoxins in human excitable cells. FEBS J 274, 1991–2004. 13 dos Remedios CG, Chhabra D, Kekic M, Dedova IV, 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) Molecular model of the contractile ring. Phys Rev Lett 95, 98–102. 22 Gerthoffer WT (2005) Actin cytoskeletal dynamics in smooth muscle contraction. Can J Physiol Pharmacol 83, 851–856. 23 Posern G & Treisman R (2006) Actin’ together: serum response factor, its cofactors and the link to signal transduction. Trends Cell Biol 16, 588–596. 24 Ishida Y, Satake N, Habon J, Kitano H & Shibata S (1985) Inhibitory effect of ouabain on the palytoxin- induced contraction of human umbilical artery. J Pharmacol Exp Ther 232, 557–560. 25 Nakanishi A, Yoshizumi M, Morita K, Murakumo Y, Houchi H & Oka M (1991) Palytoxin: a potent stimula- tor of catecholamine release from cultured bovine adre- nal chromaffin cells. Neurosci Lett 121, 163–165. 26 Contreras RG, Flores-Maldonado C, Lazaro A, Shosh- ani L, Flores-Benitez D, Larre I & Cereijido M (2004) Ouabain binding to Na+,K+-ATPase relaxes cell attachment and sends a specific signal (NACos) to the nucleus. J Membr Biol 198, 147–158. 27 Oku N, Sata NU, Matsunaga S, Uchida H & Fusetani N (2004) Identification of palytoxin as a principle which causes morphological changes in rat 3Y1 cells in the zoanthid Palythoa aff. margaritae. Toxicon 43, 21–25. 28 Ares IR, Louzao MC, Vieytes MR, Yasumoto T & Botana LM (2005) Actin cytoskeleton of rabbit intesti- nal cells is a target for potent marine phycotoxins. J Exp Biol 208, 4345–4354. 29 Drenckhahn D & Dermietzel R (1988) Organization of 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- ity and cell death in intestinal cells. J Appl Toxicol 28, 294–302. 37 Chantret I, Barbat A, Dussaulx E, Brattain MG & Zweibaum A (1988) Epithelial polarity, villin expres- sion, and enterocytic differentiation of cultured human colon carcinoma cells: a survey of twenty cell lines. Cancer Res 48, 1936–1942. 38 Louzao MC, Cagide E, Vieytes MR, Sasaki M, Fuwa H, Yasumoto T & Botana LM (2006) The sodium channel of human excitable cells is a target for gambier- ol. Cell Physiol Biochem 17, 257–268. 39 Ares IR, Louzao MC, Espina B, Vieytes MR, Miles CO, Yasumoto T & Botana LM (2007) Lactone ring of pectenotoxins: a key factor for their activity on cyto- skeletal dynamics. Cell Physiol Biochem 19, 283–292. 40 Cagide E, Louzao MC, Ares IR, Vieytes MR, Yotsu- Yamashita M, Paquette LA, Yasumoto T & Botana LM (2007) Effects of a synthetic analog of polycaverno- side A on human neuroblastoma cells. Cell Physiol Biochem 19, 185–194. 41 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. 42 Karaki H, Nagase H, Ohizumi Y, Satake N & Shibata S (1988) Palytoxin-induced contraction and release of endogenous noradrenaline in rat tail artery. Br J Phar- macol 95, 183–188. 43 Schilling WP, Snyder D, Sinkins WG & Estacion M (2006) Palytoxin-induced cell death cascade in bovine 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 inhibition of Na+-K+-ATPase activity requires integ- rity of actin cytoskeleton in opossum kidney cells. Acta Physiol Scand 175, 93–101. Palytoxin activity against the cytoskeleton M. C. Louzao et al. 6074 FEBS Journal 275 (2008) 6067–6074 ª 2008 The Authors Journal compilation ª 2008 FEBS . MINIREVIEW Marine toxins and the cytoskeleton: a new view of palytoxin toxicity M. Carmen Louzao, Isabel R. Ares and Eva Cagide Departamento de Farmacologia,. system was mainly structural, providing the sup- port needed to maintain the shape and organization of the cell and the scaffolding for the actions of catalytic molecules