A MAPK pathway is involved in the control of mitosis after ...NFSW was replaced by Ca2+-free...

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Developmental Biology 2

A MAPK pathway is involved in the control of mitosis after

fertilization of the sea urchin egg

Wen Ling Zhanga, Philippe Huitorelb, Rainer Glassa, Montserra Fernandez-Serrad,

Maria I. Arnoned, Sandrine Chiria, Andre Picardc, Brigitte Ciapaa,e,*

aUMR 7622 CNRS, Universite Paris 6, 9 Quai St Bernard, Bat C, 5e etage, case 24, 75252 Paris cedex 05, FrancebUMR 7009 CNRS, Universite Paris 6, Station Zoologique, Observatoire Oceanologique, 06234 Villefranche/Mer cedex, France

cUMR 7628 CNRS, Observatoire Oceanologique de Banyuls, Universite Paris 6/CNRS/INSU, BP 44, F-66651, Banyuls-sur-mer cedex, FrancedStazione Zoologica A. Dohrn, 80121, Italy

eGDR N- 2688

Received for publication 26 January 2005, revised 26 January 2005, accepted 12 March 2005

Available online 19 April 2005

Abstract

Activation and role of mitogen-activated protein (MAP) kinase (MAPK) during mitosis are still matters of controversy in early embryos.

We report here that an ERK-like protein is present and highly phosphorylated in unfertilized sea urchin eggs. This MAPK becomes

dephosphorylated after fertilization and a small pool of it is transiently reactivated during mitosis. The phosphorylated ERK-like protein is

localized to the nuclear region and then to the mitotic poles and the mitotic spindle. Treatment of eggs after fertilization with two different

MEK inhibitors, PD 98059 and U0126, at low concentrations capable to selectively induce dephosphorylation of this ERK-like protein, or

expression of a dominant-negative MEK1/2, perturbed mitotic progression. Our results suggest that an ERK-like cascade is part of a control

mechanism that regulates mitotic spindle formation and the attachment of chromosomes to the spindle during the first mitosis of the sea

urchin embryo.

D 2005 Elsevier Inc. All rights reserved.

Keywords: MAP kinase; Egg; Sea urchin; ERK; Mitosis; Fertilization; Mitotic spindle

Introduction

Activation of mitogen-activated protein kinase (MAPK)

is generally triggered after exit from a G0 or G2 quiescent

state. In somatic cells, stimulation by various extracellular

signals that regulate morphology, proliferation, differentia-

tion or survival often leads to the activation of at least one

member of the MAPK family, for instance the extracellular

signal-regulated kinases (ERK1/2) (reviews by Peysonnaux

and Eychene, 2001; Volmat and Pouyssegur, 2001). More

than 10 years ago, it was shown that microinjection of the

proto-oncogene mos, a MAPK kinase kinase (Sagata et al.,

0012-1606/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.ydbio.2005.03.008

* Corresponding author.

E-mail address: [email protected] (B. Ciapa).

1989), or of thiophosphorylated MAP kinase (Haccard et

al., 1993) into one blastomere of a two-cell Xenopus embryo

induced cell cycle arrest. This suggested that a MAPK

pathway could also regulate the cell cycle in early embryos.

It is well accepted that an ERK cascade, implying mos,

acts at different steps during maturation and regulates

meiosis in oocytes of various species (Abrieu et al., 2001;

Karaiskou et al., 2001). This pathway has been implicated in

regulating the meiotic spindle itself (Lefebvre et al., 2002;

Verlhac et al., 1996). However, whether MAPK is stimu-

lated and plays a role during the following mitotic cycles in

living embryos still remains a matter of controversy. In

intact Xenopus oocytes, Gotoh et al. (1991) observed that a

42/44-kDa protein, which phosphorylated preferentially

MBP or MAP2 (microtubule associated protein) rather than

histone H1, was activated at the time of first mitosis.

82 (2005) 192 – 206

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W.L. Zhang et al. / Developmental Biology 282 (2005) 192–206 193

Activation of MAPK at time of mitosis was indeed

measured in Xenopus cycling extracts (Guadagno and

Ferrell, 1998), but since the reports by Ferrell et al.

(1991), it was admitted for many years that this activity

decreased at exit of meiosis II and was not further

reactivated in early Xenopus embryos (Tunquist and Maller,

2003). This dogma was broken very recently by Yue and

Ferrell (2004) who detected, in dividing Xenopus embryos,

activation of p42 in MAPK that could be due to low levels

of Mos remaining after fertilization. In clam oocytes,

fertilization that occurs during meiosis I triggers a decrease

in MAPK activity, and no further reactivation of MAPK

during the following cell cycles has been reported so far

(Shibuya et al., 1992). We then set about this work to clarify

the situation in sea urchin eggs, where meiosis is completed

prior to fertilization, and since activation of MAPK activity

during the first mitosis following fertilization has been

detected (Chiri et al., 1998; Philipova and Whitaker, 1998)

or not (Kumano et al., 2001). We show in the present report

that an ERK-like protein, highly phosphorylated in the sea

urchin unfertilized egg, is dephosphorylated soon after

fertilization. We found that a small proportion of the

ERK-like protein is transiently rephosphorylated at the time

of mitosis, and that this pool of phosphorylated ERK is

localized to the nuclear region and then to the mitotic

apparatus.

In various types of somatic cells, several data have

shown the association of ERK1/2 and/or of upstream

partners such as MEK or RISK with the mitotic spindle

(Cha and Shapiro, 2001; Shapiro et al., 1998; Wang et al.,

1997; Willard and Crouch, 2001; Zecevic et al., 1998). It

has also been suggested that a MAPK pathway plays a role

in the spindle checkpoint, a mechanism that delays anaphase

onset in eukaryotic somatic cells when all chromosomes are

not aligned or under proper tension and that is expressed as

a negative signal produced by the Mad2/Bub1 machinery

active at unattached kinetochores (Hardwick, 1998; Hoyt,

2001; Sadler and do Carmo Avides, 2000). This hypothesis

came from results obtained in somatic cells showing that

active MAPK associates with kinetochores (Shapiro et al.,

1998; Zecevic et al., 1998) and that this checkpoint does not

operate properly after inhibition of the MAPK pathway with

PD 98059 (Cross and Smythe, 1998), anti-MAPK anti-

bodies (Takenaka et al., 1997) or injection of XCL100, a

MAPK phosphatase (Wang et al., 1997). In cycling Xenopus

extracts, various data indicate that MAP kinase is required

for the spindle assembly checkpoint (Chen, 2004; Chung

and Chen, 2003; Minshull et al., 1994; Takenaka et al.,

1997). However, it is accepted that such a mechanism

depends on the nucleus/cytoplasm ratio, ruling out its

existence in early dividing eggs where the ratio nucleus/

cytoplasm is too low. The question was then whether the

phosphorylated ERK that we detected at time of mitosis in

sea urchin early embryos could regulate mitotic events such

as the formation of the mitotic spindle. We found that

treatment of fertilized eggs with two different MEK

inhibitors, PD 98059 and U0126, at low concentrations that

selectively trigger dephosphorylation of the ERK-like

protein, or expression of a dominant-negative MEK1/2,

induced alterations in the formation of the mitotic spindle

and of the attachment of chromosomes to the spindle during

the first mitosis of the sea urchin embryo. Our results

strongly suggest that an ERK-like protein is an important

regulator of mitosis and might be part of a spindle

checkpoint mechanism in the early sea urchin embryo.

Materials and methods

Handling of gametes

Gametes collected from the sea urchin Paracentrotus

lividus were prepared and fertilized in artificial sea water

(ASW, Reef Crystals Instant Ocean) as described in our

previous papers (Chiri et al., 1998; De Nadai et al., 1998).

Eggs were treated or not before or after fertilization with

PD 98059 (Calbiochem, 15 mM stock in DMSO), U0124 or

U0126 (Calbiochem, 15 mM stock in DMSO).

Western blot analysis

Samples of eggs were prepared as previously described

(Chiri et al., 1998; De Nadai et al., 1998). In all experi-

ments, 10 Ag of proteins was separated by 12.5% SDS–

PAGE and transferred on PVDF membranes. After transfer,

the membrane was rinsed in distilled water for 5 min, dried

in air for 15 min in order to fix proteins and then incubated

for one h in blocking buffer (10 mM Tris–Cl, pH 7.4, 150

mM NaCl, 1 mM EDTA, 0.1% Tween-20, 3% BSA). The

membrane was incubated with primary antibody for 2 h at

room temperature and then washed three times for 5 min in

washing buffer (10 mM Tris–Cl, pH 7.4, 150 mM NaCl,

0.5% NP40). The membrane was incubated with secondary

antibody for 1 h, rinsed three times for 5 min in washing

buffer, and proteins were revealed by enhanced chemo-

luminescence (ECL, Amersham).

Whenever necessary, band intensities were quantified by

scanning radiographic films followed by densitometry

analysis using Adobe Photoshop.

All the antibodies were diluted in blocking buffer.

Primary antibodies and dilutions were as following: mouse

anti-phospho-MAPK42/44 (Thr202/Tyr204, Cell Signal-

ling) at 1/2000, rabbit anti-phospho-MEK1/2 (ser217/221,

Cell Signalling, 9121) at 1/1000, rabbit anti-phospho-cdc2

(Tyr15, Cell Signalling) at 1/500 and rabbit anti-cdk1/cdc2

(PSTAIR, UBI) at 1/1000. The anti-mouse HRP-conjugated

goat IgG and the anti-rabbit HRP-conjugated goat IgG

secondary antibodies (ICN) were diluted 1/1000 and 1/

10,000, respectively.

In order to verify that the anti-phospho-MAPK42/44

antibody recognized only the active form of a MAPK-like

protein, samples of unfertilized eggs were treated by

W.L. Zhang et al. / Developmental Biology 282 (2005) 192–206194

alkaline phosphatase before SDS–PAGE analysis as follow-

ing. A dry pellet of eggs (Chiri et al., 1998) containing 250

Ag proteins was incubated for 2 h at 37-C in 65 Albicarbonate buffer (100 mM NaHCO3, pH 10.5) containing

84 U/ml alkaline phosphatase. Incubation was stopped by

adding 65 Al denaturation buffer (Chiri et al., 1998) and

boiling for 10 min at 95-C. 10 Ag of proteins was then

separated on SDS–PAGE and analyzed by Western blot.

Cytochemistry

Eggs were fertilized in the presence of 1 mM ATAZ (3-

amino-1,2,4-triazole, Sigma) to prevent hardening of the

fertilization membrane. 2 min after fertilization, eggs were

diluted 10 times in normal filtered sea water (NFSW) and

quickly filtered several times through a 85-Am filter to

remove fertilization membranes. Decanted eggs were rinsed

twice in NFSW and cultured in NFSW, treated or not with

PD 98059 or U0126 and taken for fixation and labeling at

various times post-fertilization.

Fig. 1. Identification of a MEK/ERK-like pathway. (A) Detection of an ERK-like

MAPK42/44 antibody (arrow). Phosphorylation of the 42-kDa ERK-like protein

extracts with alkaline phosphatase (+AP) or after treatment of intact unfertilized

phosphorylation after fertilization. Samples were separated on two equivalent gels

transferred and blotted in parallel and in the same solutions. A very strong signa

become undetectable 20 min after fertilization. (C) Reactivation of the ERK-like p

detected around 60 min, time corresponding to the first mitosis in both experime

second experiment. (D) Time course of ERK-like and MEK activities. For ERK-

seven different experiments, indicated by seven different black or white symbols,

obtained in the next panel (E) were quantified after scanning and densitometry, and

of signal intensity measured in unfertilized eggs and that was arbitrarily taken a

fertilization analyzed by SDS–PAGE and Western blot analysis using an anti-ph

upper panel and later times on lower panel) that were run and then transferred and

were loaded on both gels to ascertain that both gels could be compared.

At a given time point, an aliquot of eggs (typically 1 ml)

was quickly decanted twice in an Eppendorf tube, and

NFSW was replaced by Ca2+-free artificial sea water

(CaFSW: 484 mM NaCl, 10 mM KCl, 27 MgCl2, 29 mM

MgSO4, 2.4 mM NaHCO3, pH 8.2) just before fixation.

Actin filaments and chromatin were labeled using a

method described by Mabuchi (1994) that was modified as

follows. Embryos were fixed for 0.5–1 h at room temper-

ature with 5% (v/v) formaldehyde in F-buffer (0.1 M KCl, 2

mM MgCl2, 1 mM EGTA, 10 mM MOPS buffer, pH 7.4).

They were then incubated for 30 min in F-buffer containing

5% formalin, 0.8 M glucose and 0.2% NP40. Eggs were

then washed with F-buffer containing 0.8 M glycerol

(glycerol-F-buffer) and stuck on 0.1% polylysine or prot-

amine-treated slide. Eggs were incubated for 30 min in

blocking buffer (1% BSA in PBS–0.1% Triton X-100

(PBS-T)), and then stained for 3 min with 0.3 AMrhodamine-conjugated phalloidin (Rh-ph, Sigma) and 0.5

Ag/ml Hoechst 33258 dissolved in PBS-T containing 0.1

mM h-mercaptoethanol in order to label actin filaments and

protein by Western blot after SDS–PAGE analysis using the anti-phospho-

detected in unfertilized control eggs (C) was lost after treatment of egg

eggs with 1 AM PD 98059 for 20 min (+PD). (B) Decrease of ERK-like

(early times in left panel and later times on right panel) that were cast, run,

l observed in unfertilized eggs decreased progressively after fertilization to

rotein at time of mitosis. Two different experiments are shown. A band was

nts, and at 92 min, time corresponding to the second mitosis shown in the

like activity, signals obtained after immunoblots obtained as in above from

were quantified after scanning and densitometry. For MEK activity, bands

results are shown in a dotted line. All values were calculated as percentages

s 100 in each experiment. (E) Changes in phosphorylation of MEK after

ospho-MEK antibody. Samples were separated on two gels (early times in

blotted at the same time. Unfertilized samples (time 0) and times 50 and 51


chromosomes, respectively. Eggs were finally washed three

times in PBS-T and mounted in Immuno-Mount (Shandon).

For microtubule labeling (De Nadai et al., 1998), fertilized

eggs deprived of fertilization membrane were taken at

different times after sperm addition and fixed for 1 h at room

temperature with 2% (v/v) formaldehyde, 20 mM PIPES, 5

mM EGTA, 0.5 mM MgSO4 and 0.1% Triton X-100. Eggs

were then rinsed in TBS-T, stuck on 0.1% polylysine coated

slide, incubated for 1 h in blocking buffer and then overnight

with the monoclonal rat anti-tubulin antibody YL1/2

(Chemicon) diluted 1/1000 in blocking buffer containing

1% donkey serum. After three rinses in TBS-T, eggs were

incubated for 1 h with FITC- or TRITC-conjugated-donkey-

anti-rat IgG 1/250 (Jackson ImmunoResearch), rinsed again

three times and treated for 2 min with 0.5 Ag/ml Hoechst

33258. The eggs were finally rinsed three times in PBS-Tand

mounted between slide and coverslip in a homemade

mounting medium containing an anti-bleach reagent.

A similar protocol was followed for anti-phospho-

MAPK42/44 labeling, except that 20 mM beta-glycero-

phosphate, 2 mM Sodium Ortho-Vanadate and 4 mM NaF

were added in the fixation buffer.

For conventional fluorescence microscopy, all micro-

tubule and actin filaments observations were performed on a

Nikon Eclipse TE300, using a �20 plan fluor or a �40 S

Fig. 2. Effect of U0126 and PD 98059 on ERK-like phosphorylation. Western blot

44 antibody. (A) Effect of increasing concentrations of U0126 in unfertilized eggs

Effect of 1 or 10 AM U0124 or U0126 on ERK-like phosphorylation. Unfertilized

U0126 on ERK-like reactivation during mitosis. Eggs were treated or not (contro

fertilization. (D) Effect of increasing concentrations of PD 98059 in unfertilized egg

PD 98059 in fertilized eggs analyzed 6 min after fertilization or at time of mitos

fluor Nikon objectives. Pictures were taken on Kodak Gold

100 Asa films that were developed and then scanned using

an Epson Perfection 2400 scanner. Final images were

treated using Adobe Photoshop. Confocal microscopy was

done on a Leica Laser Scanning Confocal Microscope (SP2)

using a �63 lens. 1024 � 1024 images were captured using

the Leica Lite Software and edited with Adobe Photoshop.

H1 kinase activity

H1 kinase activity was measured on egg extracts

following the same protocol as that described previously

(Chiri et al., 1998; De Nadai et al., 1998). Histone H1

(Sigma type III-S) was used as substrate.

Video microscopy

In order to estimate the timing of mitosis, fertilized eggs

treated or not with U0126 or PD 98059 were observed under

bright-field illumination on an inverted microscope Leica

DM IRB and images taken every 30 s. Eggs were maintained

at 18-C under a slight and constant flow of refrigerated ASW.

The time from nuclear envelope breakdown (NEB) to

beginning of cytokinesis was referred as the duration of

mitosis.

analysis was performed after SDS–PAGE using the anti-phospho-MAPK42/

after different treatment duration. The inhibitor was added at time zero. (B)

eggs were treated during 25 or 60 min with the drug. (C) Effect of 1 AMl) 30 min after fertilization with U0126 and arrested at various times after

s treated for 30 min with the drug. (E) Effect of increasing concentrations of

is (62 min). The inhibitor was added prior to sperm addition.


MEK constructs, mRNA preparation and microinjection

We used a Mekneg expression plasmid containing the

entire coding sequence of the K96M human MEK mutant

(Mansour et al., 1994). The plasmid was constructed, and

mRNA purified and injected in the presence of 0.12 M KCl

as previously described (Fernandez-Serra et al., 2004).

Fig. 3. Localization of phospho-MAPK during mitosis by confocal imaging. (A)

(b), later prophase (c), metaphase (d) or telophase (e). Eggs were double labeled w

an anti-tubulin antibody (aV1, bV1, cV1, dV1, eV1) or an anti-phospho-MAPK42/44 (

unfertilized egg (aV2), very weak at early prophase (bV2), then localized in the nucleaLabeling disappeared before telophase (eV2). Scale bar: 50 Am. (B) Effect of U0126

eggs (b and c) were treated at 20 min post-fertilization and observed at metaphase

or with the anti-phospho-MAPK42/44 (aV, bV, cV).

Results

A MEK/ERK pathway is transiently activated at time of

mitosis

In order to detect changes after fertilization in the active

form of MAPK, we used a commercial monoclonal

Control eggs. Eggs were observed before fertilization (a), at early prophase

ith Hoechst 33258 (a1, a2, b1, b2, c1, c2, c3, d1, d2, e1, e2) and with either

aV2, bV2, cV2, cV3, dV2, eV2). PhosphoMAPK42/44 labeling was intense in the

r region (cV2), to centrosomes (cV3) and finally to the spindle at mitosis (dV2).. Unfertilized eggs (a) were treated by 5 AMU0126 for 30 min, and fertilized

(b) or at telophase (c), respectively. Eggs were labeled with Hoechst (a, b, c)

Fig. 3 (continued).


antibody raised against the dual phosphorylated form of

human ERK1 and ERK2. On a Western blot of sea urchin

extracts, it labeled a single band of 42 kDa that likely

corresponds to the phosphorylated form of an ERK-like

protein, since this signal was lost after treatment of the egg

extracts either by alkaline phosphatase or by PD 98059, an

inhibitor of MEK (Fig. 1A). The high level of this

phosphorylated protein contained in the unfertilized egg

progressively decreased after fertilization to become unde-

tectable 20 min after fertilization (Figs. 1B and C). A slight

increase in the amount of the phosphorylated form of this

ERK-like protein occurred at the time of first and second

mitosis (Fig. 1C). Results obtained from several experi-

ments (Fig. 1D) establish that the level of transient

phosphorylation of ERK at mitosis could reach in some

experiments 20% of the level observed in the unfertilized

egg, the mean increase T SEM of 18 values obtained

between 55 and 68 min was 10.0 T 1.9%.

We investigated whether the activity of MEK, the

MAPK kinase that is the ERK1/2 activator in most

systems, was subject to the same variations after fertiliza-

tion as that of the ERK-like protein described above. The

anti-phospho-MEK antibody raised against the dual phos-

phorylated form of a mammalian MEK1/2 recognized a

single band migrating around 45 kDa (Fig. 1E). This

MEK-like protein was progressively dephosphorylated

after fertilization to become poorly phosphorylated 20

min after fertilization (Fig. 1E). An increase of this signal

was then observed 50 min after fertilization, that remained

elevated during mitosis and then decreased again after

cleavage (Fig. 1E). The quantification of the MEK signal

(Fig. 1E) shows that these variations correspond to those

observed for the ERK-like protein (Fig. 1D).

Effect of MEK inhibitors on ERK activation during the first

cell cycle

In order to analyze the role of the MEK-MAPK cascade

during mitosis, we used two different MEK inhibitors,

U0126 which inhibits both the active and the inactive forms

of MEK1/2, and PD 98059 which only inhibits activation of

inactive MEK (Davies et al., 2000). We determined the

concentration necessary to cancel ERK-like phosphoryla-

tion. 1 AM U0126 was sufficient to rapidly turn this activity

off in unfertilized eggs (Fig. 2A) and during mitosis (Fig.

2C). The inactive analog U0124 did not trigger dephos-

phorylation of the ERK-like protein, even when used at 10

AM, suggesting that the effects observed with U0126 are

specific (Fig. 2B). 20-min treatment with 0.5 AM PD 98059

could also abolish this activity in unfertilized eggs (Fig.

2D). However, 15 AM (Fig. 2E) of PD 98059, depending on

the batch of eggs, was necessary to obtain a complete loss of

the activity in fertilized eggs.

Localized activation of ERK in the fertilized sea urchin egg

We then checked whether ERK activation by phosphor-

ylation occurred homogeneously throughout the sea urchin

embryo. Eggs were fixed at different times following

fertilization and immunostained using the anti-phospho-

MAPK42/44 antibody (Fig. 3A). Unfertilized eggs (Fig.

3Aa) were highly fluorescent, the cytoplasmic labeling

appearing granular and the intranuclear space brightly

stained (Fig. 3AaV2).30 min after fertilization, eggs reached prophase (Figs.

3Ab and Ac). Control eggs started to polymerize micro-

tubules (Figs. 3AbV1 and cV1) and to condense chromatin in

the nucleus (Figs. 3Ab1, b2, c1, c2 and c3). The anti-

phospho-MAPK signal was highly reduced at early pro-

phase (Fig. 3AbV2) compared to that observed in unfertilized

eggs. At later prophase (Fig. 3Ac), the cytoplasmic signal

appeared less granular and faint, while a higher labeling

accumulated close to nuclei (Fig. 3AcV2a and h) and then in

the vicinity of the centrosomes (Figs. 3AcV2h and AcV3g).Control eggs were at metaphase 40 min after fertilization,

showing alignment of condensed chromosomes (Figs. 3Ad1

and d2) on the bipolar mitotic spindle (Fig. 3AdV1). Thecentral spindle and asters were labeled by the anti-phospho-

MAPK antibody, whereas the periphery of the central

spindle, which also contains a high density of microtubules

in sea urchin embryos, was negative (Fig. 3AdV2). The

cytoplasm surrounding the spindle at that stage still

contained a low but significant granular staining.

Finally, control eggs reached the telophase stage 60 min

after fertilization (Fig. 3Ae). A very low and diffuse

Fig. 4. Time course during the first two cell cycles of MPF activity after

fertilization of eggs treated with PD 98059 or U0126. The inhibitors were

added 30 min after fertilization. In the inset is shown a Western blot

analysis performed after SDS–PAGE using the anti-phospho-Tyr cdc2

antibody of eggs arrested at time of mitosis and treated or not (control) with

15 AM PD 98059.


staining was obtained with the anti-phospho-MAPK anti-

body (Fig. 3eV2).Unfertilized eggs treated for 30 min by U0126 showed a

much reduced and diffuse fluorescent signal throughout the

eggs even though some faintly stained granules could still be

seen (Fig. 3BaV). Only a very low and diffuse phospho-

MAPK signal was seen in eggs treated by U0126 and

observed 30 min (Fig. 3BbV) or 60 min after fertilization (Fig.

3BcV), times when eggs were at prophase (Fig. 3Bb) and

telophase (Fig. 3Bc), respectively. Similar results were

obtained using U0126 or PD 98059 (not shown). It is

important to note that the intensity of general staining

obtained in control eggs with the anti-phospho-MAPK

antibody followed that obtained after Western blotting using

this same antibody (Figs. 1B and C), that is, high in

unfertilized eggs, very low at prophase, slightly increased

during mitosis and undetectable at telophase. Taken together,

our results show that ERK is transiently activated during sea

urchin egg mitosis and that this activation mostly takes place

at the spindle poles during prophase and prometaphase.

Effect of MEK inhibition on MPF activation

We analyzed whether the MEK inhibitors affected MPF

activity. The MPF activity due to cdc2/cyclin B is often

referred to as histone H1 kinase, since MPF shows a

pronounced activity toward this substrate and is the main

source of histone H1 kinase activity in eggs during mitosis

(Meijer et al., 1989). We observed that H1 kinase activity

during the first two mitosis was slightly increased when

eggs were treated with 15 AM PD 98059 or with 5 AMU0126, concentrations that inhibited the ERK-like activity

(Fig. 4). This increase in H1 activity corresponded to a

greater decrease in the tyrosine phosphorylation status of

cdc2 during mitosis (Fig. 4 inset), a decrease in phosphor-

ylation of cdc2 corresponding to an increase in MPF activity

(Ohi and Gould, 1999).

Role of MEK/ERK on mitotic spindle formation and cell

division

The role of MAPK cascade during the first two division

cycles was tested by treating eggs 30 min after sperm

addition with concentrations of U0126 or PD 98059 that

induce ERK-like dephosphorylation (Fig. 2). Similar

altered morphologies were observed after both treatments.

Compared to controls, treated embryos showed abnormal

metaphase plates or incomplete cytokinesis, and formed

two or three daughter cells of different sizes or with altered

shapes (Fig. 5). Proportions of altered embryos are

reported in Table 1. The proportion of altered embryos

increased with the concentration of PD 98059. A very high

proportion of embryos were abnormal after treatment with

15 AM PD 98059; almost all eggs divided (Fig. 5A) and

formed morula, although with very altered morphologies,

that finally died before reaching the gastrula stage (not

shown). Treatment with 1 AM U0126 induced abnormal

divisions (Fig. 5B) or mitotic arrest in a number of eggs

(Table 1). Increasing U0126 concentration to 5 AM led to

the increase in the proportion of non-divided eggs (Fig. 5B

and Table 1). Video microscopy experiments allowed us to

determine the timing of the first cell cycle. We observed

that 15 AM PD 98059 or 5 AM U0126 significantly

delayed entry in mitosis but slightly accelerated exit of

mitosis (Table 2).

The abnormal divisions described above led us to

investigate whether U0126 and PD 98059 altered the

formation of the cleavage furrow. Examples of alterations

are shown in Fig. 6. We observed that treated eggs

normally formed a contractile ring of actin filaments

between two sets of chromosomes that were either not as

widely separated (Fig. 6B) as in the control experiment

(Fig. 6A) or not separated at all (Figs. 6C and D). This

probably led to the situation where chromosomes remained

close enough to the cleavage plane and did not separate

properly in a number of cases. Several furrows were also

observed in a number of treated eggs that formed three or

four sets of chromosomes. Those eggs with multiple

furrows seemed to form lobes or cells of different sizes

(Figs. 6E–G). No clear difference in the timing of

formation of the actin ring was observed, suggesting that

these alterations were not due to an accelerated or

premature formation of the cleavage furrow.

We tested whether the smaller gap between the two sets

of chromosomes seen at the time of furrow formation in PD

98159 or U0126 treated eggs (Fig. 6) was due to an altered

mitotic spindle. Eggs were treated in the presence of these

inhibitors 30 min after fertilization and fixed for micro-

tubule and chromatin staining at the normal time of

anaphase in control eggs. In those conditions, we observed

the same percentage of treated eggs as that reported above

showing chromosomes that were not equally distributed in

two sets and several types of aberrant mitotic spindles (Fig.

Fig. 5. Effect of increasing concentrations of PD 98059 (A) or U0126 (B) on the first two mitotic cycles. Each inhibitor was added 30 min after fertilization.

Embryos treated with PD 98059 were observed at different times after fertilization and those treated with U0126 were observed 90 min after fertilization.

U0126-treated eggs or eggs treated with 2.5 AM 98059 PD 98059 showing altered shapes are shown with a star. Almost all eggs treated with 15 AM PD 98059

in this shot had altered morphologies. No alteration was observed when eggs were treated with the inactive analog U0124 (not shown).


7). Some eggs showed two distinct sets of chromosomes,

but one of the two sets appeared separated (Figs. 7B1 and

C1), which could lead to the formation of a tripolar spindle

(Fig. 7C2). Some other eggs contained four sets of

chromosomes (Fig. 7D1), separated on a tetrapolar mitotic

spindle (Fig. 7D2). Finally, a number of eggs showed one or

a few chromosomes lagging out of the normal sets of

chromosomes (Figs. 7D1, E1, F1 and G1). In some cases,

bipolar spindles were formed but were made either of

unequal half spindles and with a poorly defined main axis,

suggesting a loose connection between half spindles (Figs.

7E2 and F2), or with an extra lobe containing extra

microtubules (Fig. 7G2). Similar morphological alterations

were obtained with U0126 and PD 98059, at a higher

frequency in PD 98059-treated eggs, and no alteration was

observed when eggs were treated with the inactive analog

U0124 (not shown) compared to control eggs (Fig. 7A).

Higher resolution micrographs, obtained after confocal

imaging, of some alterations seen after fertilization in

U0126 treated eggs, are shown in Fig. 8. Two very altered

Table 1

Effect of two different concentrations of PD 98059 or U0126 on division

PD 98059 U0126

2.5 AM,

n = 6

15 AM,

n = 7

1 AM,

n = 5

5 AM,

n = 6

Abnormal divisions (%) 15 T 2 67 T 7 22 T 4 21 T 3

Non-divided eggs (%) 3 T 2 3 T 2 6 T 2 13 T 3

Mortality (%) / / / 4 T 2

Total (%) 18 68 28 38

Eggs were treated with MEK inhibitors 30 min after fertilization and

observed under light microscopy. Results are mean T SEM; 15–57 eggs

were counted in each experiment, n representing the number of

experiments.

Table 2


metaphase are shown in Figs. 8a and b. In these eggs,

chromosomes are condensed and well individualized (Figs.

8a and b), but microtubules are severely disorganized (Figs.

8aV and bV). Eggs observed at telophase (Fig. 8c) often

showed decondensed chromosomes that were dispersed in

the cytoplasm (Figs. 3Bc and Figs. 8c), which was never

seen in control eggs. All these results show that ERK

inactivation induced various anomalies such as mitotic

spindle defects or lagging chromosomes and lead to the

conclusion that a MEK/ERK pathway is involved in mitotic

spindle formation and function.

In order to confirm the defects caused by PD 98059 and

U0126, we overexpressed an inactive form of MEK

(MEKneg) that acts as a dominant-negative protein (Mansour

et al., 1994). The high degree of sequence homology

between human and sea urchin MEK (around 90% in the

catalytic domain, Rizzo and Arnone, unpublished) sug-

gested that these mutants could be used to activate or to

block the MEK/ERK pathway in sea urchin, and expression

of MEKneg indeed induces dephosphorylation of ERK-like

in the sea urchin embryo (Fernandez-Serra et al., 2004).

Injection of MEKneg mRNA just before fertilization induced

aberrant first and second divisions in a very high percentage

of eggs and inhibition of mitosis in a few eggs, in

comparison with control non-injected eggs or eggs injected

with KCl buffer only (Figs. 9A and B). Similar alterations to

those observed after PD 98059 or U0126 treatment were

observed, such as incomplete cytokinesis, formation of

daughter cells of different sizes or with altered shapes (Fig.

9A). Altogether, these results strongly suggest that an MEK/

ERK-like pathway controls the first mitotic divisions in sea

urchin.
Effect of U0126 and PD 98059 on the timing of mitosis entry
Control

n = 14

5 AM U0126

n = 6

15 AM PD 98059

n = 5

Mean time to NEB

(minutes after fertilization)

45 T 2 52 T 4 51 T 4

Duration of mitosis (min) 30 T 1 26 T 2 27 T 2

Eggs were treated with MEK inhibitors 30 min after fertilization and

observed under video microscopy. Results are mean T SEM. n represents

the number of eggs. A Student’s t test shows that all values obtained in

U0126 or PD 98059-treated eggs are significantly different ( P < 0.01) from

those of control eggs.

Discussion

The data provided here in sea urchin embryos show that a

MEK/ERK-like cascade is transiently activated at the time

of mitosis. Moreover, we show that the activated ERK pool

is located close to the nucleus then to the mitotic spindle.

The specific inhibition of this cascade leads to mitotic

defects that strongly implicates a MEK-ERK cascade in the

formation of the mitotic spindle and in the attachment of

chromosomes during the early embryo mitotic cell cycles.

The unfertilized sea urchin egg contains a highly

phosphorylated ERK-like protein

The fact that unfertilized eggs contain a highly phos-

phorylated ERK-like protein that was gradually dephos-

phorylated during the first minutes following fertilization

(this paper and Carroll et al., 2000; Chiri et al., 1998;

Kumano et al., 2001) fits with the idea that maintaining

MAPK activity at a high level in the unfertilized egg blocks

DNA synthesis. Indeed, a decrease in this activity with

MEK inhibitors leads to progression into S phase in starfish

oocytes (Abrieu et al., 1997; Fisher et al., 1998; Tachibana

et al., 1997) and in the sea urchin eggs of S. purpuratus

(Carroll et al., 2000). In contrast, Philipova and Whitaker

(1998) observed a low MAPK activity in unfertilized L.

pictus eggs that was followed by an increase after

fertilization. These authors may not have detected MAPK

contained in the nucleus (Carroll et al., 2000) or in the

cortex (Walker et al., 1997) of sea urchin eggs since they

followed a protocol described by Shibuya et al. (1992) and

used soluble fractions that may lack nuclei and insoluble

cytoskeleton elements for Western blots and immunopreci-

pitations. This could also be explained if these authors have

detected another MAPK in their study. As a matter of fact,

these authors reported very recently that a ERK1-like

MAPK, which should then be different from the ERK that

we detect here, was required for S phase onset in sea urchin

embryos (Philipova et al., 2005).

A small pool of ERK-like protein is activated during mitosis

Only a minor fraction of total ERK-like activity is

transiently activated at time of mitosis and is located in close

vicinity to the mitotic spindle in the sea urchin egg of P.

lividus (this paper) and in some other cell types (Cha and

Shapiro, 2001; Shapiro et al., 1998; Wang et al., 1997;

Willard and Crouch, 2001; Zecevic et al., 1998). This could

explain why MAPK activity has not been detected at the time

of mitosis in oocytes of clam (Shibuya et al., 1992) or even

other species of sea urchin (Carroll et al., 2000; Kumano et

Fig. 6. Effect U0126 or PD 98059 on chromosomes separation and actin ring formation. 5 AM U0126 or 15 AM PD 98059 was added 30 min after fertilization

and eggs were taken 30 min later for fixation and labeling as described in Materials and methods. Chromosomes (upper picture) and actin (lower picture) were

labeled with Hoechst and rhodamine phalloidin, respectively. (A) An anaphase in control eggs. (B–G) Different phenotypes that were commonly observed in

U0126 (B, C, E) or PD 98059 (D, F, G) treated eggs. Scale bar: 100 Am.


al., 2001). The changes in levels ofMAPK activity just before

or during mitosis could be a way to control exit from mitosis

during the first cell cycles of the early embryo, at least in sea

urchin, starfish and Xenopus. We observed that treatment of

fertilized eggs with 20 AMwortmannin, which leads to arrest

in prometaphase-like state (De Nadai et al., 1998), inhibited

ERK-like dephosphorylation normally seen at exit of mitosis

(results not shown and Chiri et al., 1998). This sustained

MAPK phosphorylation can then be correlated with arrest at

mitosis during the first embryonic cell cycle in sea urchin as

reported inXenopus (Chau and Shibuya, 1998; Haccard et al.,

1993; MacNicol et al., 1995; Sagata et al., 1989) and starfish

(Abrieu et al., 1997).

Specificity of PD 98059 or U0126

We believe that PD 98059 or U0126, used at concen-

trations as low as 1 AM and never exceeding 15 AM or 5 AM,

respectively, inhibited specifically the MEK/ERK-like path-

way while U0124 had no effect, considering the data

reported by Davies et al. (2000). These concentrations are

in the same range as those used by Carroll et al. (2000), and

very low if compared to the 50-AM U0126 used in somatic

cells (Horne and Guadagno, 2003) or the 200-AM PD 98059

used in Xenopus extracts (Guadagno and Ferrell, 1998). The

fact that only a very low percentage of cells was blocked in

mitosis by PD 98059 or U0126 corroborates the data

reported by Kumano et al. (2001). Increasing the concen-

trations of PD 98059 or U0126 at levels as high as 50 AMcould indeed lead either to eggs arrested at pseudo-

metaphase showing one or a few unaligned chromosomes

and an unusually small mitotic spindle, or to divided eggs

showing alterations very similar to our present observations

(Pesando et al., 1998, 1999). Indeed, 100 AM U0126 was

necessary to inhibit DNA synthesis in sea urchin embryos

(Philipova et al., 2005). However, such high concentrations

of PD 98059 or U0126 might act on other MAPK cascades

or act non-specifically on other proteins that may have an

MBP kinase activity, since several myc or MBP kinases can

be detected in the sea urchin egg and are activated during

mitosis (Chiri et al., 1998; Walker et al., 1997). Finally, we

observed that dominant-negative MEK human constructs

had a greater effect than the inhibitors, comparing the

percentage of altered eggs. This could be due to the fact that

MEK inhibitors were added 30 min after fertilization to a

population of eggs, where diffusion in eggs, access to MEK,

and sensitivity to the drugs may differ between eggs. These

limiting factors cannot be taken into account in dominant-

negative MEK experiments, where all eggs were injected

before fertilization. However, we cannot rule out non-

specific effects of human proteins in the sea urchin

embryo.

Fig. 7. Effect of PD 98059 or U0126 on mitotic spindle formation and chromosome separation. A1 to A6 show control eggs that were taken at time of early

anaphase (1,2), late anaphase (3,4) and early telophase (5,6), labeled with Hoechst (1,3,5) or with an anti-tubulin antibody (2,4,6), and observed with a light

microscope. Eggs were treated 30 min after fertilization with either 10 AM PD 98059 (B, F, G) or with 5 AM U0126 (C, D, E) and taken at different times of

mitosis for labeling with Hoechst (1) or with anti-tubulin antibody (2). Scale bar: 50 Am.


Fig. 8. Examples of alterations in U0126 treated eggs observed by confocal imaging. 5 AM U0126 was added 20 min after fertilization and eggs taken for

fixation at metaphase (a/aV and b/bV) or at telophase (c/cV). Eggs were labeled with Hoechst (a, b, c) or with an anti-tubulin antibody (aV, bV and cV) and observed

by confocal imaging. Very altered spindles were seen at metaphase (aV and bV), while the two daughter cells observed at telophase showed lots of decondensed

and dispersed chromosomes (c). Scale bar: 50 Am.

Fig. 9. Effect of injection of Mekneg on first mitosis. Embryos are observed 2 or 3 h after injection with Mekneg or not (control eggs non-injected or injected

with KCl only). (A) Different types of altered divisions seen in eggs injected with MEK mutants. (B) The percentage of altered embryos. 27, 43 and 56

embryos were counted after 2 h in control embryos, embryos injected with KCl buffer, or injected with Mekneg, respectively. 22, 21 and 62 embryos were

counted after 3 h in control embryos, embryos injected with KCl or injected with Mekneg, respectively.



Role of ERK-like in mitotic events

The association of the phosphorylated ERK-like with

the centrosomes and the mitotic spindle in the early sea

urchin embryo corroborates various data obtained in

somatic cells (Shapiro et al., 1998; Wang et al., 1997;

Willard and Crouch, 2001). The different defects induced

by the MEK inhibitors, such as poorly defined mitotic

axis, anarchic microtubule organization within the spin-

dle, extra arrays of microtubules and poor chromosome

separation, suggest that an ERK-like cascade acts at

different levels of mitotic spindle formation. By being

associated with the cortex (Walker et al., 1997), this

ERK-like could regulate the mitotic spindle–cortex

interactions important for spindle orientation and ana-

phase B. This could explain why we frequently observed

spindles that were not properly centered and formation of

two daughter cells of different sizes in eggs treated by

MEK inhibitors. Moreover, daughter cells might contain

an abnormal number of chromosomes, since a few

chromosomes were not properly aligned on the metaphase

plate in many cases, which could lead to the delay of cell

division that accumulated as the embryos developed and

to asynchronous divisions within embryos, giving rise to

stages three or five (Fig. 5). The occurrence of multipolar

spindles (Fig. 6) also contributed to the formation of

three or four cells of unequal sizes and of unequal

content in chromosomes. The association of phosphory-

lated ERK-like with the centrosomes suggests a role of

this MAPK in the regulation of centrosome activity.

MEK inhibitors could induce centrosomes/spindle poles

to split, which would explain the formation of tri- or

tetrapolar spindles in these conditions. Finally, almost all

eggs divided, even though the number of actin rings was

variable and a function of the number of microtubule

asters (spindle poles or extra microtubule arrays). These

results suggest that the ERK-like activity is not involved

in the induction of the cleavage furrow or of the actin

ring formation. Other proteins that also have an MBP

kinase activity and have been identified in the cortex of

the sea urchin egg (Walker et al., 1997) could be

involved in the triggering of cleavage furrow formation

(Shuster and Burgess, 2002a,b).

Relations MPF/MAPK during mitosis

All these results strongly suggest that an ERK-like

contributes to regulate mitotic spindle structure and

function in the early embryo similarly to somatic cells.

These effects cannot be due to inhibition of MPF activity

normally stimulated during mitosis, since treatment with

U0126 or PD 98059 did not inhibit MPF but on the

contrary led to a slight increase in this activity during

mitosis. This is in agreement with the results obtained by

Murakami et al. (1999) and Walter et al. (2000) who

showed that the MAPK pathway regulates cdc2 phos-

phorylation and thus MPF activation through Wee1. The

activation of ERK-like seems then to be necessary to

ensure normal progression through mitosis in the early

embryo, but not essential to entry or exit mitosis. This

fits with results obtained in Xenopus extracts where

MAPK activity is not needed to enter or exit mitosis but

is required for normal mitotic progression (Guadagno and

Ferrell, 1998; Takenaka et al., 1997). Finally, similar

alteration of the length of mitosis and no arrest of mitosis

were observed in tissue culture cells where M phase ERK

activity was blocked (Roberts et al., 2002).

Is an ERK-like part of a spindle checkpoint mechanism in

the early sea urchin embryo?

Our results strongly suggest that an ERK-like cascade

is part of a mechanism that controls the formation of the

mitotic spindle and the attachment of chromosomes to the

mitotic spindle during the first mitosis of the sea urchin

embryo. In somatic cells and in cycling Xenopus extracts,

various data show a role of a MEK/ERK pathway in the

spindle checkpoint mechanism (review by Maller et al.,

2002). Several points argue for the existence of a similar

mechanism in the early sea urchin embryo. Firstly,

phosphorylated ERK-like associates with the centrosomes

and the spindle at mitosis. Secondly, alterations of the

mitotic spindle and chromosomes attachment in PD

98059 or U0126-treated eggs that we report here look

like those observed in somatic cells lacking the mitotic

checkpoint proteins Mad2 (Dobles et al., 2000) or Bub1

(Taylor and McKeon, 1997). Preliminary Western blotting

analysis using anti-Bub1 and -Bub3 polyclonal antibodies

showed that these proteins are present in P. lividus

embryos (unpublished results), and it would be interesting

to investigate whether these proteins are active during

cleavage stages or not, and whether other checkpoint

proteins are expressed in the early sea urchin embryo.

Thirdly, only a minority of mitoses were abnormal when

ERK-like was inhibited, which indicates that this MAPK

might only be required in case of mitosis defects, as

opposed to being an intrinsic part of mitosis. However,

the existence itself of a spindle checkpoint in the sea

urchin early embryo has been questioned. Mitosis is

prolonged when the zygotes are induced to form

monopolar spindles or when the spindle is cut into two

half spindles (Sluder and Begg, 1983), which is in favor

of this hypothesis. On the contrary, unattached or

misoriented chromosomes do not induce arrest of division

(Rieder et al., 1995; Sluder et al., 1994), and duration of

second mitosis is not altered in embryos containing

tripolar or tetrapolar spindles (Sluder et al., 1997), which

rather suggests that no spindle checkpoint was functional.

One possibility is that an ERK-like is part of a

mechanism that is not a ‘‘spindle checkpoint’’ stricto

sensus, since it is not able to stop division as in somatic

cells in case of a single misoriented chromosome or other


spindle defects, and some component of this checkpoint

could be either absent or inactive. Another possibility is

that a real spindle checkpoint mechanism exists in the

early sea urchin embryo, but the inhibitory activity

produced by an unattached kinetochore either remains

local or diffuses in a large cytoplasmic volume containing

large asters and a high microtubule density, which would

render inefficient this activity. This is in agreement with

the hypothesis where the spindle checkpoint is regulated

by a mechanism that depends on the nucleo-cytoplasmic

ratio (Maller et al., 2002), ratio that then allows an active

mechanism in somatic cells or in Xenopus egg extracts

supplemented with a high concentration of nuclei per

microliter of cytoplasm (Guadagno and Ferrell, 1998;

Minshull et al., 1994; Takenaka et al., 1997). It would be

interesting to investigate whether the inhibition of the

mos/MEK/ERK pathway leads in dividing Xenopus

embryos to the same alterations of mitotic events than

those we report here. However, the dependency on the

nucleo-cytoplasmic ratio of the spindle checkpoint mech-

anism in Xenopus embryos was questioned a few years

ago by Clute and Masui (1997).

Acknowledgments

This work was supported by the Association pour la

Recherche sur le Cancer (ARC), the CNRS, The University

Pierre et Marie Curie and the French Ministere de la

Recherche. S. Chiri acknowledges receipt of an ARC

fellowship, and W.L. Zhangh a Ministere de la Recherche

and a KC Wong fellowships. We thank C. Sunkel for the

anti-Bub1-Bub3 antibodies and G. Pruliere for the Bub1 and

Bub3 experiment. We dedicate this work to our dearest

friend and colleague Andre Picard, who was a strong

supporter of the sea urchin model in France.

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A MAPK pathway is involved in the control of mitosis after ...NFSW was replaced by Ca2+-free...

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