The cytoskeleton, a structure that is susceptible to the toxic mechanism activated by palytoxins in...

The cytoskeleton, a structure that is susceptible to thetoxic mechanism activated by palytoxins in humanexcitable cellsM. Carmen Louzao1, Isabel R. Ares1, Mercedes R. Vieytes2, Iago Valverde3, Juan M. Vieites3,Takeshi Yasumoto4 and Luis M. Botana1

1 Departamento de Farmacologia, Facultad de Veterinaria, Universidad de Santiago de Compostela, Lugo, Spain

2 Departamento de Fisiologia Animal, Facultad de Veterinaria, Universidad de Santiago de Compostela, Lugo, Spain

3 ANFACO-CECOPESCA, Campus Universitario de Vigo, Vigo, Pontevedra, Spain

4 Japan Food Research Laboratories, Tama Laboratory, Tokyo, Japan

Palytoxin is an extremely toxic product that was first

isolated from the zoanthid Palythoa toxica [1]. This

toxin is a large, water-soluble polyalcohol with a wide

distribution in algae, crabs, herbivorous fish, and the

surgeonfish. Palytoxin enters the food chain and causes

a fatal type of poisoning named clupeotoxism [2,3].

This compound is highly toxic for mammals, with an

LD50 of 33 ngÆkg)1 when administered intravenously

in dogs [4]. Ostreocins are not well-known congeners

of the palytoxin found in Ostreopsis siamensis [5,6].

Likewise, almost nothing is known about a still

unnamed toxin produced by Ostreopsis ovata that may

be related to ostreocins and palytoxins [5].

The putative palytoxin receptor is the Na+ ⁄K+-

ATPase. The membrane enzyme Na+ ⁄K+-ATPase

uses ATP hydrolysis to pump 2 K+ and 3 Na+ in

Keywords

actin cytoskeleton; laser-scanning

cytometry; membrane potential;

ostreocin-D; palytoxin

Correspondence

L. M. Botana, Departamento de

Farmacologia, Facultad de Veterinaria,

27002 Lugo, Spain

Fax ⁄ Tel: +34 982 252 242

E-mail: [email protected]

(Received 31 October 2006, revised 7

February 2007, accepted 14 February 2007)

doi:10.1111/j.1742-4658.2007.05743.x

Palytoxin is a marine toxin responsible for a fatal type of poisoning in

humans named clupeotoxism, with symptoms such as neurologic distur-

bances. It is believed that it binds to the Na+ ⁄K+-ATPase from the extra-

cellular side and modifies cytosolic ions; nevertheless, its effects on internal

cell structures, such as the cytoskeleton, which might be affected by these

initial events, have not been fully elucidated. Likewise, ostreocin-D, an

analog of palytoxin, has been only recently found, and its action on excit-

able cells is therefore unknown. Therefore, our aim was to investigate the

modifications of ion fluxes associated with palytoxin and ostreocin-D activ-

ities, and their effects on an essential cytoskeletal component, the actin

system. We used human neuroblastoma cells and fluorescent dyes

to detect changes in membrane potential, intracellular Ca2+ concentration,

cell detachment, and actin filaments. Fluorescence values were obtained

with spectrofluorymetry, laser-scanning cytometry, and confocal micro-

scopy; the last of these was also used for recording images. Palytoxin and

ostreocin-D modified membrane permeability as a first step, triggering

depolarization and increasing Ca2+ influx. The substantial loss of filamen-

tous actin, and the morphologic alterations elicited by both toxins, are

possibly secondary to their action on ion channels. The decrease in poly-

merized actin seemed to be Ca2+-independent; however, this ion could be

related to actin cytoskeletal organization. Palytoxin and ostreocin-D alter

the ion fluxes, targeting pathways that involve the cytoskeletal dynamics of

human excitable cells.

Abbreviations

bis-oxonol, bis-(1,3-diethylthiobarbituric acid) trimethine oxonol; F-actin, filamentous actin; LSC, laser-scanning cytometry.

FEBS Journal 274 (2007) 1991–2004 ª 2007 The Authors Journal compilation ª 2007 FEBS 1991

opposite directions, thereby creating an ionic

asymmetry that is responsible for the electrochemical

potential difference of Na+ and K+ across the cell

membrane [7]. The marine toxin appears to bind to the

Na+ ⁄K+-ATPase from the extracellular side and elicit

a nonselective cation leak pathway, possibly by dis-

rupting the strict coupling between the pump’s inner

and outer gates, allowing them to both be open [8,9].

Several studies have shown that the palytoxin-induced

channel permits the passage of cations such as Na+

and K+ but not Ca2+ [10–12]. Ouabain, a well-known

inhibitor of the sodium pump, hampers the fluxes

evoked by palytoxin [13] and seems to induce detach-

ment of cells [14]. Likewise, the phenomena of cell–

matrix and cell–cell adhesion have been related to

filamentous actin (F-actin) [15,16].

Until now, the effects of palytoxins on the actin

cytoskeleton of nerve cells, which comprise one of its

main targets in mammals, have not been evaluated.

The ability of excitable cells to generate all-or-none

action potentials in response to depolarizing stimuli is

well known. There are some reports describing the

membrane depolarization induced by palytoxin in

neuronal cells [17–19]. However, surprisingly, this

membrane potential and the increase in intracellular

Na+ and further Ca2+ movements has never been

linked with a possible effect of palytoxin on the cyto-

architecture of excitable cells. On the basis of previous

information, the specific aim of this work was to

investigate and relate the effects of palytoxins (a) on

ion fluxes (shown as changes in membrane potential or

alterations in intracellular Ca2+) and (b) on actin cyto-

skeletal dynamics, morphology and cell attachment, by

using human neuroblastoma cells. Herein, we present

new findings on the mode in which both compounds

induce toxicity in a neuronal model. Knowledge of the

key points involved in the cellular damage is funda-

mental for establishing functional parameters with

practical application in the design and development of

in vitro detection techniques for seafood control pro-

grams. This information is very useful, as it forms the

basis for developing biotoxin detection methods that

could provide an alternative to animal testing.

Results

In this study, we determined the electrophysiologic

consequences of palytoxin action in excitable cells. We

have previously developed an assay to detect changes

in membrane potential by using the sensitive dye

bis-(1,3-diethylthiobarbituric acid) trimethine oxonol

(bis-oxonol) [20]. The negative charge of bis-oxonol

prevents its accumulation in the mitochondria, and

therefore the probe only indicates changes in mem-

brane potential [21]. Process conditions such as con-

centration of dye were taken from our previous studies

[20,22,23]. The dye was allowed to equilibrate with the

cells for 15–20 min before any experiment was begun.

Downward and upward deflections of the fluorescence

tracings represent hyperpolarization and depolari-

zation, respectively. Figure 1 shows that 75 nm

palytoxin or ostreocin-D induced depolarization in

neuroblastoma cells.

The next step was to investigate whether, together

with this depolarization, there was an increase in cyto-

solic Ca2+. It is known that the use of dual indicators

to study intracellular Ca2+ minimizes the contribution

of artefactual changes to the fluorescent signal that are

not related to variations in Ca2+ [24,25]. Taking into

account this fact, we used a combination of Fluo-3

and Fura red to perform this kind of assay. The Fluo-3

and Fura red fluorescence intensities are increased and

reduced, respectively, when the intracellular free Ca2+

rises [26]; in consequence, an elevation in the Fluo-3 ⁄Fura red fluorescence ratio is produced. Figure 2A

shows an increase of cytosolic Ca2+ in neuroblastoma

cells loaded with Fluo-3 and Fura red after addition of

palytoxins to the solution. This ion change might

result from the influx of external Ca2+ or Ca2+ release

from internal stores. To clarify this, the same experi-

ments were carried out in nominally Ca2+-free

1

1.2

1.4

1.6 Toxin

GramicidinOstreocin-D 75 nM

Palytoxin 75 nM

1.8

2

0.80 200 400

Time (s)

Dep

ola

riza

tio

n

Rel

ativ

e F

luo

resc

ence

600 800 1000

Fig. 1. Palytoxin-induced and ostreocin-D-induced changes in mem-

brane potential in human neuroblastoma cells loaded with the

membrane potential dye bis-oxonol. Results are expressed as relat-

ive fluorescence ± SEM (n ¼ 4).

Palytoxin-induced changes to the cytoskeleton M. C. Louzao et al.

1992 FEBS Journal 274 (2007) 1991–2004 ª 2007 The Authors Journal compilation ª 2007 FEBS

medium, and we found that neither palytoxin nor ost-

reocin-D modified the cation concentration within

neuroblastoma cells (Fig. 2B). These results suggest

that toxins induce an extracellular Ca2+ influx.

Modifications of intracellular Ca2+ levels could be

associated with cytotoxic effects. In order to examine

whether palytoxin affected the attachment of cells to

the extracellular matrix, we determined, by additional

fluorimetric microplate assays, the percentage of paly-

toxin-induced cell detachment (Fig. 3). The time course

of cell detachment experiments revealed that less than

10 h of treatment with an effective concentration of

100 nm palytoxin was sufficient to detach more than

60% of cells from the substratum. Because the cyto-

skeleton is involved in cell attachment to the extra-

cellular matrix, we also analyzed the effect of

palytoxin on this structure. Neuroblastoma cells were

incubated with the toxin, and their actin cytoskeletons

were then stained with Oregon green 514 phalloidin, as

described in Experimental procedures. It is important

to point out that a decrease in the fluorescent signal of

this dye indicates F-actin disassembly. Treatment of

cells with palytoxin for 24 h led to dose-dependent dis-

ruption of polymerized actin (Fig. 4), which was

already observed at 0.05 nm. Complete F-actin break-

down was found with palytoxin concentrations ranging

from 0.1 nm to 100 nm.

On the basis of previous results, we investigated whe-

ther 75 nm palytoxin and 75 nm ostreocin-D (concen-

trations that clearly depolarize, increase cytosolic Ca2+,

and cause complete F-actin disruption in 24 h) modified

the actin filament system in a short period of time. The

0 100

A

B

Flu

o-3

/ Fur

a re

dF

luo-

3 / F

ura

red

200 300 400Time (s)

600500 700

0 100 200 300 400Time (s)

600500 700

Ostreocin-D 75 nM

Control3

2

1

0

3

2

1

0

Palytoxin 75 nM

Ostreocin-D 75 nM

ControlPalytoxin 75 nM

Fig. 2. Calcium response of neuroblastoma cells to 75 nM palytoxin

or 75 nM ostreocin-D in Ca2+-containing solution (A) and in nomin-

ally Ca2+-free medium (B). Results are presented as Fluo-3 ⁄ Fura

red fluorescence ratios. The black arrow indicates addition of tox-

ins. Fluorescent signals were collected during scans that were per-

formed every 5 s for up to 10 min. Results are average ± SEM of

at least 50 cells per assay (n ¼ 3).

100 nM palytoxin1 nM palytoxin80

70

60

50

40

30

20

10

00.1 1 10 100

Control

Time (h)

Cel

l det

ach

men

t %

Fig. 3. Time course of cell detachment. Percentage of cell detach-

ment induced by 1 nM and 100 nM palytoxin in neuroblastoma cells.

Results are average ± SEM (n ¼ 4).

0.10.010.001 1 10 100

[Palytoxin] nM

80

100

120

140

60

40

20

–20

0

Per

cen

tag

e o

f C

on

tro

l

Fig. 4. Effect of palytoxin on cytoskeleton architecture of neuro-

blastoma cells. Dose-dependent disruption of the actin filament

system after 24 h of incubation with palytoxin. Results are aver-

age ± SEM (n ¼ 4).

M. C. Louzao et al. Palytoxin-induced changes to the cytoskeleton


fluorescence associated with the microfilaments of neuro-

blastoma cells was measured in a large number of cells

using laser-scanning cytometry (LSC). After 10 min of

incubation with palytoxin or ostreocin-D (Fig. 5), the

signal from cells was decreased with respect to controls.

This loss of fluorescence intensity indicated a

20 ± 6.2% reduction in the actin cytoskeletons of paly-

toxin-treated cells and a 19 ± 2% reduction in ostreo-

cin-D-treated cells. At the same time, images captured

through confocal microscopy allowed us to evaluate the

outcome of F-actin alterations on the morphology of

the cells. Control cells were flat and extended, with mod-

erately long cell processes. In addition, their actin

cytoskeletons were intact and distributed throughout

the cytoplasm. Neuroblastoma cells incubated for

10 min with the toxins did not show relevant changes in

shape. However, the F-actin bands surrounding the cell

margin became less dense, and a small degree of loss of

cytoplasmic fibers was also seen (Fig. 6).

F-actin levels continued to fall in cells exposed for

30 min to the action of the toxins (Fig. 7). In this case,

LSC revealed that palytoxin reduced the amount of

F-actin by 27 ± 1%, whereas ostreocin-D reduced

it by 35 ± 6%. Confocal images showed that

neuroblastoma cells treated for 30 min with palytoxin

were morphologically similar to those incubated for

10 min, although the disappearance of F-actin was

intensified (Fig. 8). On the other hand, 30 min after

addition of ostreocin-D to the solution, the cells began

to lose their normal array of actin fibers. The intense

peripheral bands vanished, being replaced by diffuse

F-actin staining that did not seem to cause modifica-

tions of cell shape (Fig. 8).

Finally, we evaluated the progress of the cytoskeletal

effects caused by the toxins after hours of treatment.

LSC examination (Fig. 9) showed that palytoxin and

ostreocin-D induced, respectively, decreases in poly-

merized actin of 51 ± 6.4% and 45 ± 4% in 4 h.

Visualization of cells showed a substantial reduction of

overall fluorescence, with a few, more intense, patches

of F-actin in the cytoplasm; F-actin staining was rather

widespread, especially in cells treated with palytoxin.

In addition, with this duration of incubation, it was

found that both toxic compounds induced a retraction

of the cellular body, as shown in transmission images

(Fig. 10). This retraction is closely related to the cell

detachment observed in Fig. 3.

On the basis of the data obtained above, it was of

interest to know whether the alterations in micro-

filaments caused by palytoxins were related to their

effects on intracellular Ca2+. Therefore, we decided to

carry out actin cytoskeleton experiments in nominally

10 min

10 min

Control

Control

Green Max Pixel Green Max Pixel Green Max Pixel

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9000 9000 9000

9000 9000

63

50

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25

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20

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60

45

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Green Max Pixel Green Max Pixel

Palytoxin Black: Palytoxin Grey: Control

Black: Ostreocin-D Grey: Control

Ostreocin-D

Fig. 5. Representative diagrams obtained with LSC, showing the fluorescence distribution of control cells (left column) and cells incubated

with palytoxin (upper central) or ostreocin-D (lower central) for 10 min in a Ca2+-containing medium. Spots in the scattergram plot corres-

pond to microfilaments of neuroblastoma cells stained with fluorescent phalloidin. Comparative profiles of fluorescence distribution between

controls and treated cells derived from the selected regions of scattergrams are shown in histograms (right column).



Ca2+-free medium, incubating neuroblastoma cells

with the toxins for 4 h (the time at which the larger

decrease in polymerized actin levels was observed).

Controls analyzed under these conditions showed iden-

tical characteristics as when they were incubated in a

Ca2+-containing medium; however, the response of

treated cells was unexpected. In Fig. 11, scattergrams

and histograms show that after 4 h of incubation of

cells in nominally Ca2+-free medium, the F-actin con-

tent was reduced by 47 ± 4.4% with palytoxin and by

40 ± 2.1% with ostreocin-D. No substantial differ-

ences were observed in the palytoxin effect as com-

pared to incubation with Ca2+-containing medium.

Confocal imaging of samples showed weak cell retrac-

tion but a substantial loss of microfilaments; otherwise,

the marginal F-actin was more resistant, especially to

ostreocin-D (Fig. 12).

Fluorescence values presented in this section are

graphed in Fig. 13.

Discussion

Many phycotoxins responsible for important seafood

poisoning syndromes have been associated with neuro-

logic symptoms in humans. Palytoxin, isolated from

Palythoa spp., is one of the most potent marine toxins

known. We report here the effects of palytoxin on

membrane potential and Ca2+ homeostasis, and how

these events affect cytoskeletal dynamics in an excit-

able cell model. Moreover, we studied ostreocin-D, a

new analog of palytoxin, whose biological activity on

a neuronal model is analyzed here for the first time.

Na+ ⁄K+-ATPase is the suggested palytoxin receptor

[13]. Na+ ⁄K+-ATPase is responsible for generating

and maintaining transmembrane ionic gradients that

are of vital importance for cellular function and activi-

ties such as volume regulation, pH maintenance, gen-

eration of action potentials, and secondary active

transport. In this case, palytoxins may interfere with

the function of Na+ ⁄K+-ATPase. For instance, paly-

toxin, by binding to the pump, may transform it into a

channel and stimulate ion fluxes, as has been previously

suggested [8,27]. We observed that palytoxin and ostreo-

cin-D induced depolarization in human neuroblastoma

cells. It is interesting to point out that we also tested

the effect of a purified extract of Ostreopsis ovata

(kindly provided by T. Yasumoto). This toxic extract,

which has been closely associated with palytoxins, also

Fig. 6. Representative images obtained by confocal microscopy of neuroblastoma cells whose F-actin was stained with Oregon green 514

phalloidin. Fluorescence images show the distribution pattern of microfilaments in control cells (upper left) and cells exposed to palytoxin

(upper central) or ostreocin-D (upper right) for 10 min in a Ca2+-containing medium. Transmission images of the same cells are displayed

(lower). Scale bar ¼ 50 lm.



showed depolarizing activity (data not shown). The

results obtained in this study suggest that depolariza-

tion is due to an increase in cytosolic Na+ as a primary

event in palytoxin and ostreocin-D action. In agree-

ment with this, stimulation of Na+ entry induced by

palytoxin has been described in several cellular models

[11,12,28,29]. We also found an increase in cytosolic

Ca2+ induced by both toxins. It is known that a variety

of cellular processes are regulated by changes in cyto-

solic Ca2+, including the arrangement of the actin cyto-

skeleton [30–32]. Thus, the mode of palytoxin toxicity

in neuroblastoma cells could involve the conversion of

Na+ ⁄K+ pumps into nonselective cation channels,

triggering depolarization, Na+ accumulation with sub-

sequent Ca2+ overload [33], and finally targeting of the

actin cytoskeleton. Our data revealed that palytoxin

modified the cytoskeleton architecture, causing almost

complete F-actin disassembly at 0.1 nm in 24 h. In

addition, changes in the cytoskeleton induced by

1–100 nm palytoxin trigger detachment of cells from

the extracellular matrix after less than 10 h of incuba-

tion. A similar correlation between the two effects has

been found after inhibition of the sodium pump by

ouabain in wild-type canine kidney cells, where the loss

of cellular attachment involved distortion of several

adhesion molecules, including F-actin [14].

It has been reported that the elevation of cytosolic

Ca2+ induced by palytoxin is dependent on extracellu-

lar Ca2+ [12,17,18,34–36]. Our assays using neuroblas-

toma cells also indicated that palytoxin, as well as

ostreocin-D, evoked Ca2+ entry from the external

medium. Later, the possible relationship between Ca2+

movements induced by palytoxins and their effects on

the F-actin cytoskeleton and cellular morphology

were analyzed. The quantitative study with LSC in a

Ca2+-containing medium revealed that palytoxin and

ostreocin-D caused a time-dependent F-actin reduc-

tion; however, clear changes in morphology only

appeared after hours of incubation with the toxins,

indicating that this phenomenon is secondary to the

loss of microfilaments. Interestingly, in assays with

nominally Ca2+-free medium, the decrease in polymer-

ized actin content was almost the same as in Ca2+-

containing medium. This finding seems to suggest that

Ca2+ influx is not involved in F-actin disassembly

caused by the action of both palytoxins in neuronal

cells; interestingly, this behavior was opposite to that

found in intestinal cells, a nonexcitable cellular model

[37]. A different mechanism could be responsible for

these effects, e.g. some signaling pathway triggered

by the sodium pump through intermediary proteins

after interaction with palytoxins. Recent studies on

30 min

30 min

Control

Control


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Palytoxin Black: Palytoxin Grey: Control

Black: Ostreocin-D Grey: Control

Ostreocin-D


with palytoxin (upper central) or ostreocin-D (lower central) for 30 min in a Ca2+-containing medium. Points in the scattergram plot represent

microfilaments of neuroblastoma cells stained with fluorescent phalloidin. Comparative profiles of fluorescence between controls and treated

cells from selected regions of scattergrams are shown in histograms (right column).



Na+ ⁄K+-ATPase have revealed that it not only func-

tions as an ion pump, but also acts as a signal transdu-

cer through interactions with other proteins [38–41].

Events such as increases in tyrosine phosphorylation

of several cellular proteins have been observed as con-

sequence of the interaction of ouabain with Na+ ⁄K+-

ATPase in rat cardiac myocytes and independently of

any changes in intracellular Na+ and Ca2+ concentra-

tions [39–41]. However, the possibility must be consid-

ered that F-actin alterations induced at later times by

palytoxins could be due to slow Ca2+ influx and sub-

sequent accumulation in the cytoplasm in the long

term.

On the other hand, Ca2+ influx could be involved in

microfilament distribution. We found, with confocal

microscopy, that the polymerized actin organization in

cells treated with toxins was different in nominally

Ca2+-free solution than when 1.5 mm CaCl2 was pre-

sent in the medium. In the first case, F-actin appeared

to be diffusely spread throughout the whole cell, and it

even seemed to be increased in the cellular margins.

Taking into account that the quantity of actin cytoske-

leton is almost the same, the density of actin filaments

in these localities might be visibly enhanced by rein-

forcement, bundling, and crosslinking of pre-existing

filaments by F-actin-binding proteins, as has been

established in other studies [42,43]. In agreement with

our results, it was previously observed that microfila-

ment relocation did not affect the amount of F-actin

[42,43]. Also, the influence of Ca2+ on the actin cyto-

skeleton disposition has already been shown in experi-

ments where a rise in cytosolic Ca2+ generated a

displacement of peripheral actin to the inner cell

regions without changes in F-actin content in human

monocytic THP1 cells [44]. It is possible that the level

of cytosolic Ca2+ needs to reach a threshold in the

cells to trigger F-actin reorganization. This could be a

plausible explanation for the faster relocalization of

F-actin observed in neuroblastoma cells treated with

ostreocin-D (between 10 and 30 min) in comparison

with those incubated with palytoxin. Ostreocin-D

seems to increase cytosolic Ca2+ more rapidly,

although the concentration reached was rather similar

to that induced by palytoxin.


phalloidin. The fluorescence images show the distribution patterns of microfilaments in untreated cells (upper left) and cells exposed to paly-

toxin (upper central) or ostreocin-D (upper right) for 30 min in a Ca2+-containing medium. Transmission images of the same cells are also dis-

played (lower). White arrows indicate the substantial loss of F-actin in the margins of cells treated with ostreocin-D, which is still visible in

cells incubated with palytoxin. Scale bar ¼ 50 lm.



In summary, palytoxin and ostreocin-D generate a

sequence of events, including rapid depolarization and

increase of Ca2+ levels with subsequent F-actin disas-

sembly and cytomorphologic changes. Concerning the

quantitative effect of the toxins on the actin filament

system, it seems to be independent of external Ca2+.

Also, a concentration-dependent loss of adhesion was

found. In agreement with this last effect, Contreras

et al. [7] found that once palytoxin acted on the

Na+ ⁄K+-ATPase of epithelial cells, it could trigger a

cascade of cellular signals, resulting in the removal of

membrane-attaching molecules, followed by exclusion

of the cell from the monolayer. Additionally, the data

obtained in the present study have also provided useful

information from structure–activity viewpoint. The

chemical composition of palytoxin and ostreocin-D dif-

fers in that two methyls, two hydroxyls and one proton

present in the first molecule are substituted by four pro-

tons and one hydroxyl in the second molecule [45].

However, these changes do not seem to effect essen-

tially their biological potencies, as both compounds dis-

played very similar effects in the assays performed here.

Recently, several lines of evidence have opened a new

perspective on the functional importance of ion trans-

port proteins as cytoskeletal regulators. It is known that

many ion channels, pumps and exchangers anchor the

actin-based cytoskeleton to the plasma membrane

through cytoskeletal linker proteins [46]. Thus, whereas

anchoring would maintain the position of ion transport

proteins in specific membrane domains, localized

anchoring and ⁄or ion transport activities might, in turn,

modulate the F-actin dynamics. In this respect, the lat-

est studies carried out in fibroblasts have demonstrated

that anchoring by the Na+–H+ exchanger NHE1 could

control the actin filament system [47]. As has been sug-

gested for other marine biotoxins, such as okadaic acid

and pectenotoxin-6, in different cellular models [48–51],

we have also found that the F-actin cytoskeleton is one

of the internal structures affected by the action of both

palytoxins in neuroblastoma cells. This finding is partic-

ularly interesting in the case of palytoxins, taking into

account that their activity has been linked to membrane

transport proteins.

Experimental procedures

Reagents

T. Yasumoto (Japan Food Research Laboratories) provided

ostreocin-D. Veratridine, gramicidin and palytoxin were

4 h

4 h

Control

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Palytoxin Black: PalytoxinGrey: Control

Black: Ostreocin-DGrey: Control

Ostreocin-D


with palytoxin (upper central) or ostreocin-D (lower central) for 4 h in a Ca2+-containing medium. Spots in the scattergram plot correspond to

microfilaments of neuroblastoma cells stained with fluorescent phalloidin. Comparative profiles of fluorescence between controls and treated

cells derived from selected regions of scattergrams are shown in histograms (right column).



obtained from Sigma (Madrid, Spain). The dyes bis-oxonol,

Fluo-3, Fura red and Oregon green 514 phalloidin were

obtained from Molecular Probes (Leiden, the Netherlands).

All other chemicals were of reagent grade and purchased

from Sigma and Merck (Darmstadt, Germany).

Neuroblastoma cells [human neuroblastoma cell

line BE(2)-M17]

The excitable cells were grown in EMEM ⁄Ham’s F12

(1 : 1) supplemented with glutamine, 1% nonessential

amino acids, 10% fetal bovine serum, 50 lgÆmL)1 genta-

micin, and 50 ngÆmL)1 amphotericin B. Cells were grown

in 25 cm2 tissue culture flasks at 37 �C in 5% CO2, and

were subcultured weekly by transferring cells released with

trypsin ⁄EDTA.

Measurement of changes in membrane potential

Changes in membrane potential in BE(2)-M17 cells exposed

to palytoxin or ostreocin-D were evaluated at 37 �C by

using the fluorescent probe bis-oxonol. Suspensions of

0.5 · 106 cells were placed in a stirred quartz microcuvette

in the thermostated cell holder in a Shimadzu (Kyoto,

Japan) RF-500 spectrofluorometer. Bis-oxonol, whose dis-

tribution across the membrane is potential-dependent, was

added directly to the suspension, and neuroblastoma cells

were incubated with the dye for 5–10 min. Fluorescence

was continuously registered at wavelengths of 530 nm (exci-

tation) and 590 nm (emission). After stabilization of the

baseline (equilibrium distribution of the probe), phycotox-

ins were added to the cells. At the end of each experiment

10 lgÆmL)1 gramicidin was added to induce complete cell

depolarization.

Ca2+ measurements

Cytosolic Ca2+ levels were analyzed with two visible-wave-

length Ca2+-sensitive dyes, Fluo-3 and Fura red. Cells

grown on coverslips in culture medium were loaded with a

mixture of 10 lm Fluo-3 and 10 lm Fura red acetoxymethyl

esters plus 0.04% pluronic F-127 for 30 min at 37 �C and

5% CO2. Before fluorescence measurements, the culture

medium was changed, and cells were washed for 30 min at

room temperature with dye-free standard salt solution con-

taining: 150 mm NaCl, 6 mm KCl, 1.5 mm CaCl2, 1 mm


phalloidin. The fluorescence images show the distribution patterns of microfilaments in control cells (upper left) and cells exposed to paly-

toxin (upper central) or ostreocin-D (upper right) for 4 h in a Ca2+-containing medium. Treated cells show diffuse staining of the F-actin cyto-

skeleton throughout the cytoplasm. Small F-actin spots towards the central region are observed in almost all of the cells. Note the

condensation in soma of cells exposed to toxins in the transmission images (lower). Scale bar ¼ 50 lm.



MgCl2, 10 mm Hepes, and 10 mm glucose (pH 7.4). In

nominally Ca2+-free solution, CaCl2 was not added. After

loading, the coverslips were mounted in a chamber, which

was subsequently refilled with standard solution for per-

forming each experiment. The chamber was placed on the

stage of a Nikon (Tokyo, Japan) Eclipse TE300 inverted

microscope equipped with a Nikon oil objective (magnifica-

tion · 60, numerical aperture 1.4) that was connected to a

confocal laser-scanning imaging system (MRC-1024;

Bio-Rad, Hemel Hempstead, UK). The 488 nm wavelength

line of the argon ion laser was used to excite Fluo-3 and

Fura red at intervals of 5 s. The ratios were computed using

time course kinetic imaging software (Bio-Rad).

Cell detachment

The time course of cell detachment was evaluated by ana-

lyzing total DNA content in the supernatant and in cells

growing in microplates following 15 min, 30 min, 60 min,

6 h and 24 h of exposure of cells to 1 nm and 100 nm paly-

toxin as previously described [52]. Total DNA was mea-

sured using the CyQuant-Cell Proliferation Assay Kit

(Molecular Probes). Fluorescence was measured in an LS-

50B microplate reader (Perkin-Elmer, Beaconsfield, UK) set

at 480 nm (excitation) and 520 nm (emission). The sum of

average fluorimetric values obtained for both attached and

detached cells at each time interval was used to calculate

the relative percentage of detached cells.

F-actin depolymerization in microplates

Depolymerization of F-actin was carried out following 24 h

of incubation of cells in microplates by using Oregon green

514 phalloidin, as previously described [50]. Triplicate fluori-

metric values of each sample were expressed as a percentage

of controls. Fluorescence was measured in an LS50B fluori-

metric microplate reader set a 511 nm (excitation) and

528 nm (emission).

F-actin determination by LSC and confocal laser

microscopy

Staining of F-actin

Cells were washed with NaCl ⁄Pi and fixed for 10 min in

4% paraformaldehyde solution. Following two washing

steps with NaCl ⁄Pi, cells were permeabilized with 0.1% Tri-

ton X-100 for 5 min. Triton X-100 was removed by brief

washing in NaCl ⁄Pi, and fixed cells were incubated with

1% BSA for 30 min to reduce nonspecific staining. Then,

F-actin was labeled specifically with the fluorescent dye

Oregon green 514 phalloidin for 20 min. Finally, cells were

4 h

4 h

Control

Control


Co

un

t

Green Max Pixel

90 s

q. µ

Are

a89

0 sq

. µ

90 s

q. µ

9000 9000 9000

9000 9000

111

88

66

44

0

22

Co

un

t

75

60

45

30

0

15

9000

Are

a89

0 sq

. µ

90 s

q. µ

Are

a89

0 sq

. µ

90 s

q. µ

Are

a89

0 sq

. µ


Palytoxin Black: PalytoxinGrey: Control

Black: Ostreocin-DGrey: Control

Ostreocin-D


with palytoxin (upper central) or ostreocin-D (lower central) for 4 h in nominally Ca2+-free medium. Points in the scattergram plot correspond

to microfilaments of neuroblastoma cells stained with fluorescent phalloidin. Comparative profiles of fluorescence between controls and trea-

ted cells derived from selected regions of scattergrams are shown in histograms (right column).



washed twice with NaCl ⁄Pi, and mounted on a slide with

a mixture of NaCl ⁄Pi and glycerol (1 : 1). Fluorescence

corresponding to stained F-actin was measured at

511 nm (excitation) and 528 nm (emission) using the LSC

technique.

LSC to quantify F-actin

The laser-scanning cytometer provides the technology for

solid-phase cytometry. A laser-scanning cytometer (Compu-

Cyte, Cambridge, MA) equipped with a BX50 Olympus

(Melville, NY, USA) microscope and a 488 nm argon laser

was used. After staining F-actin with Oregon green 514

phalloidin the fluorescence of neuroblastoma cells mounted

on slides was quantified with LSC. Moreover, the threshold

was optimized to avoid the loss of fluorescence information.

For each contoured cell, the parameters measured were the

area and the maximum pixel. Between 2000 and 3000 cells

were studied per slide.

Imaging analysis of F-actin

Fluorescence and transmission images of neuroblastoma

cells fixed and stained with Oregon green 514 phalloidin

were recorded using the confocal laser microscopy system

previously described. All images were captured as optical

sections of a Z-series at 0.5-lm intervals and collected in a

mode format of 512 · 512 pixels.

Data analysis

All experiments were carried out at least three times in

duplicate or triplicate. The results of changes in mem-

brane potential are expressed as the mean of relative

fluorescence ± SEM. The results of Ca2+ measurements are

expressed as the mean of fluorescence ratio ± SEM. For the

F-actin assays, data are presented as percentage decrease in

relative fluorescence of cells treated with toxins in relation to

controls (mean ± SEM).

Acknowledgements

This work was funded with grants from the following

agencies: Ministerio de Ciencia y Tecnologı́a, Spain,

grant numbers SAF2003-08765-C03-02, REN2001-

2959-C04-03, REN2003-06598-C02-01, and AGL2004-

08268-02-O2 ⁄ALI; Xunta de Galicia, Spain, grant


phalloidin. The fluorescence images show microfilament distribution in control cells (upper left) and cells exposed to palytoxin (upper central)

or ostreocin-D (upper right) for 4 h in a nominally Ca2+-free medium. Transmission images of the same cells are also displayed (lower). A

substantial fall in cytoplasm F-actin, although not at the edges of the cell, was seen. Scale bar ¼ 50 lm.



numbers PGIDT99INN26101, PGIDIT03AL26101PR,

and PGIDIT04TAL261005PR; Fondo de Investigaci-

ones Sanitarias, Spain, grant number FISS REMA-G03-

007; and EU VIth Frame Program, grant numbers IP

FOOD-CT-2004-06988 (BIOCOP), STREP FOOD-CT-

2004-514055 (DETECTOX), and CRP 030270-2 (SPIES-

DETOX).

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