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Analysis of signal transduction in cell-free extracts and rafts of Xenopus eggs
Methods 51 (2010) 177–182
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Review Article
Analysis of signal transduction in cell-free extracts and rafts of Xenopus eggs Alexander A. Tokmakov a,b,*, Tetsushi Iwasaki b, Ken-Ichi Sato c, Yasuo Fukami a,b a
Graduate School of Science, Kobe University, Rokkodai 1-1, Nada, Kobe 657-8501, Japan Research Center for Environmental Genomics, Kobe University, Rokkodai 1-1, Nada, Kobe 657-8501, Japan c Department of Biotechnology, Faculty of Engineering, Kyoto Sangyo University, Kamigamo-Motoyama, Kita-ku, Kyoto 603-8555, Japan b
a r t i c l e
i n f o
Article history: Accepted 12 January 2010 Available online 15 January 2010 Keywords: Xenopus egg Egg activation Fertilization Signal transduction Cell-free extract Raft
a b s t r a c t Intracellular signaling during egg activation/fertilization has been extensively studied using intact eggs, which can be manipulated by microinjection of different mRNAs, proteins, or chemical drugs. Furthermore, egg extracts, which retain high CSF activity (CSF-arrested extracts), were developed for studying fertilization/activation signal transduction, which have significant advantages as a model system. The addition of calcium to CSF-arrested extracts initiates a plethora of signaling events that take place during egg activation. Hence, the signaling downstream of calcium mobilization has been successfully studied in the egg extracts. Moreover, despite disruption of membrane-associated signaling compartments and ordered compartmentalization during extract preparation, CSF-arrested extracts can be successfully used to study early signaling events, which occur upstream of calcium release during egg activation/fertilization. In combination with the CSF-arrested extracts, activated egg rafts can reproduce some events of egg activation, including PLCc activation, IP3 production, transient calcium release, MAPK inactivation, and meiotic exit. This becomes possible due to complementation of the sperm-induced egg activation signaling machinery present in the rafts with the components of signal transduction system localized in the extracts. Herein, we describe protocols for studying molecular mechanisms of egg fertilization/activation using cell-free extracts and membrane rafts prepared from metaphase-arrested Xenopus eggs. Ó 2010 Elsevier Inc. All rights reserved.
1. Xenopus eggs 1.1. Metaphase-arrested eggs After the completion of meiosis, mature Xenopus eggs are arrested in the metaphase of the second meiotic division (metaphase II) due to the high activity of the key mitotic regulators, maturation promoting factor (MPF) and cytostatic factor (CSF). MPF was originally defined by Masui and Markert as an activity in the cytoplasm of mature frog oocytes that could induce maturation in frog oocytes [1]. MPF consists of cyclin B and cyclin-dependent protein kinase CDK1 homologous to the yeast cell division control 2 protein (Cdc2). Several enzymes of protein phosphorylation, which represent two signaling pathways, are activated in eggs, supporting high MPF activity. The polo-like protein kinase pathway, mediated by the polo-like kinase Plx1 and Xenopus polo-like kinase kinase xPlkk1, is linked to activation of MPF-activating phosphatase Cdc25C [2,3] (Fig. 1). On the other hand, mitogen-activated protein kinase (MAPK), highly active in the presence of Mos kinase, phosphorylates and activates the downstream Ser/Thr-specific ribo-
* Corresponding author. Address: Research Center for Environmental Genomics, Kobe University, Rokkodai 1-1, Nada, Kobe 657-8501, Japan. Fax: +81 78 803 5951. E-mail address: [email protected] (A.A. Tokmakov). 1046-2023/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2010.01.008
somal s6 kinase Rsk, which down-regulates the Cdc2-inhibitory kinase Myt1, supporting MPF activation [4,5]. CSF, defined as an activity that causes metaphase arrest in frog eggs, first appears in progesterone-treated oocytes at the time of Mos accumulation due to its stabilization by phosphorylation on Ser3 and disappears shortly after fertilization, coincident with Mos degradation [6]. The exact composition of CSF is not established. Injections of Mos, or constitutively active MAPK kinase (MAPKK), or thiophosphorylated MAPK into two-cell blastomeres promote metaphase arrest [7–9], and Mos-induced arrest can be abolished by co-injecting anti-MAPKK antibody [10]. Similarly, the MAPK phosphatase MKP1 can release a MAPK-dependent metaphase arrest induced in Xenopus egg extracts by spindle depolymerization [11]. Also, Rsk has been shown to mediate CSF activity of the MAPK cascade and to induce metaphase arrest in cleaving Xenopus embryos [12]. Immunodepletion of Rsk from egg extracts abolished their capacity to undergo mitotic arrest in response to MAPK cascade activation [13]. Thus, it was concluded that the MAPK pathway represents an important component of CSF. Although immunodepletion of Mos from the cytoplasm of unfertilized eggs leads to the loss of CSF activity [9,14], inhibition of the MAPK cascade with U0126 or PD98059, or depletion of Rsk from CSF-arrested extracts does not release CSF arrest [13,6,15]. It implies that Mos is necessary for both establishing and maintaining metaphase arrest, whereas MAPK and Rsk are required to only
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MOS
MEK
calpain calcineurin
MAPK
Rsk 1,2
Bub1
Myt1
Emi2/Erp1
Wee1
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ActiCa2+ vation
APC/C
Cyclin/Cdc2
Cyclin B
active MPF
Cdc2 inactive preMPF
CaMKII
CaMKII
Emi2/Erp1 xPlkk1
Plx1
Cdc25C
Fig. 1. Signaling pathways in metaphase-arrested and activated eggs.
establish this arrest. Still, the fact, that the fully active MAPK cascade cannot impose metaphase arrest in meiosis I, alongside with the finding that Mos cannot induce meiosis II in the absence of protein synthesis [9], indicates that CSF arrest requires some other components newly synthesized in meiosis II. MAPK-dependent inhibition of the APC/C, the ubiquitin ligase controlling cyclin B degradation at the anaphase/metaphase transition, was proposed to play a major role in maintaining high activity of MPF during CSF arrest [6]. The APC/C inhibitor Emi2/Erp1 has recently been identified as a pivotal CSF component, required to maintain metaphase II arrest [16,17]. It was shown that Rsk can directly phosphorylate Emi2/Erp1 [18,19]. This phosphorylation increases Emi2/Erp1 stability and promotes APC/C inhibition. Evidence has been presented that Bub1, a component of the spindle assembly checkpoint, is activated by Rsk and also inhibits APC/ C [20]. This finding suggests that in CSF-arrested eggs with the intact spindle this checkpoint can be activated independently of kinetochore-microtubule attachment by the constitutively active, due to the unique presence of Mos in meiotic metaphase, MAPK cascade [6]. Active Rsk also phosphorylates and inactivates Cdc2inactivating protein kinase Myt1, directly promoting Cdc2 activity [4,5]. Another Cdc2-inactivating kinase, Wee1, is highly phosphorylated and only weakly active in metaphase-arrested eggs [21]. Some other mechanisms, such as Mos-stimulated polyadenylation of cyclin mRNA [22], may also contribute to maintaining high MPF activity in CSF-arrested eggs and extracts. 1.2. Egg activation The loop of positive feedback comprising the MAPK cascade and MPF assures the stability of CSF arrest in fully matured eggs. Fertilization or parthenogenetic activation disrupts positive feedback, inactivates CSF, and releases metaphase arrest. In contrast to progesterone-induced meiotic initiation, where Mos synthesis and MAPK cascade activation precede and promote MPF activation, fertilization triggers MPF inactivation before Mos degradation and MAPK cascade inactivation. Calcium-dependent degradation of cyclin B in 10–20 min after fertilization is thought to trigger the disruption of positive feedback between MPF and CSF. Then, within 15–30 min after egg activation, Mos protein is specifically degraded by the calcium-dependent cysteine protease calpain [23] and Mos mRNA is deadenylated at its 3’UTR and translationally arrested [24]. Furthermore, MAPK inactivation occurs in 30–40 min after fertilization, simultaneously with the loss of CSF activity in activated egg [25]. The early universal event of fertilization-induced egg activation is a mobilization of intracellular calcium that originates from the sperm entry point and spreads through the entire egg cytoplasm. Recently, this process has been established to require the sequential activation of Src family kinases, PLCc, and IP3 receptor of the endoplasmic reticulum [rewieved in 26,27]. Elevation of intracellu-
lar calcium is necessary and sufficient for egg activation. Calcium ionophores induce complete egg activation, whereas calcium-chelating agents, such as EGTA and BAPTA, prevent sperm-induced egg activation [28,29]. The calcium calmodulin-dependent kinase CaMKII is thought to mediate calcium effects on the cyclin degradation machinery. Addition of constitutively active CaMKII to CSF-arrested egg extracts causes cyclin degradation and CSF release in the absence of calcium, whereas a specific inhibitor of CaMKII prevents cyclin degradation and exit from metaphase arrest after calcium addition [30]. Besides cyclin B degradation, CaMKII also triggers sister chromatid separation, presumably, through the degradation of mitotic regulator proteins that bind sister chromatids together [31]. A direct target of CaMKII related to the protein degradation machinery was shown to be the APC/C inhibitor Emi2/ Erp1. Phosphorylation of Emi2/Erp1 by the calcium-activated CaMKII leads to the recruitment of the polo-like kinase Plx1 via its Polo Box domain. Then, Plx1 phosphorylates Emi2/Erp1 at the site of phosphorylation-dependent degradation signal, which is recognized by the SCFbTrCP complex for ubiquitination and degradation [17,18]. In addition to targeting the protein degradation machinery, CaMKII rapidly stimulates the inhibitory phosphorylation of Cdc25 on Ser287, which is repressed in the unfertilized eggs [32]. On the other hand, calcium induces not only the activation of CaMKII but also the dephosphorylation of the Apc3 component of APC/C. The calcium/calmodulin-dependent protein phosphatase calcineurin and Fizzy/Cdc20, a key regulator of the APC/C, were shown to be involved in this process [33,34]. Further, it was demonstrated that CaMKII and calcineurin activities together allow nuclear envelope formation [33]. Calcium-dependent activation of the APC/C triggers cyclin B ubiquitination and targets the Cdc2/cyclin B complex to the 26S proteasome. Proteasome-dependent dissociation of Cdc2 from cyclin B down-regulates Cdc2 kinase. It is sufficient to inactivate MPF even without following cyclin B degradation [35]. In the absence of MPF activity, Mos dephosphorylation at Ser3, a site of direct phosphorylation by Cdc2 kinase, occurs, leading to accelerated degradation of Mos protein by an ubiquitination pathway [36]. Dephosphorylated Mos cannot support high activity of the MAPK cascade, although Mos rephosphorylation by Cdc2 kinase is able to cause MAPK reactivation [37]. MAPK inactivation disrupts another important loop of positive feedback inside the MAPK cascade itself (between MAPK and Mos), resulting in the complete switchlike shutdown of the MAPK cascade in fertilized eggs. Thereafter, the MAPK cascade plays an important role in the regulation of following early embryo mitotic cell cycles, and Mos protein is completely degraded after fertilization and is not resynthesized again [23,24].
1.3. Egg extracts Intracellular signaling during egg activation/fertilization has been extensively studied using intact eggs, which can be manipulated by microinjection of different mRNAs, proteins, or chemical drugs. However, this technique is rather cumbersome, and microinjection often fails to achieve the desired cytosolic concentration of injected molecules because of variable leakage. Besides, synchrony among different eggs is not sufficient sometimes for studies of fast signaling events. Therefore, cell-free egg extracts has been developed for the purpose of cell-cycle transition studies, which have significant advantages as a model system [38]. Large volumes (several milliliters) of highly synchronized homogenous extract can be produced from the eggs of a single frog, while the exogenous chemical compounds and proteins can be added easily at precisely known concentrations and also efficiently removed by immunodepletion.
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Cell-free extracts of Xenopus eggs can recapitulate a number of physiological processes. The most studied of them are meiotic exit, mitotic cell-cycle transition, and apoptotic process. The procedures of extract preparation vary in details, depending on the nature of considered processes. The egg extracts, which retain high CSF activity (CSF-arrested extracts), are commonly used for studying fertilization/activation signaling. The addition of calcium to CSF-arrested extracts initiates a plethora of signaling events that take place during egg activation, resulting in the meiotic exit. Hence, the signaling downstream of calcium mobilization can be successfully studied in the egg extracts. For instance, it has been shown that proteasomes posses non-proteolytic activity dissociating Cdc2 kinase from cyclins and that MPF inactivation is separated from cyclin B degradation upon activation of CSF extracts in the presence of the proteasome proteolytic activity inhibitor MG115 [35]. Moreover, cyclin B/Cdc2 dissociation precedes Cdc2 Thr161 dephosphorylation upon MPF inactivation in CSF extracts [39]. Another recent example includes establishing the role of the APC/C inhibitor Emi2/Erp1 and the polo-like kinase Plx1 in the calciuminitiated meiotic exit. It was shown that CaMKII and Plx1 sequentially phosphorylate Emi2/Erp1 to trigger its destruction, APC/C activation, cyclin degradation and cell-cycle transition in the CSFarrested extracts [17,18]. The importance of calcium/calmodulindependent protein phosphatase calcineurin for initiation of embryonic development was established based on the fact that calcineurin is required to release CSF-arrested Xenopus egg extracts from M phase [33,34]. Remarkably, despite disruption of membrane-associated signaling compartments and ordered compartmentalization during extract preparation, CSF-arrested extracts can be successfully used to study early signaling events, which occur upstream of calcium release during egg activation/fertilization. It was demonstrated that catalytically active Src kinase can initiate calcium response of a low magnitude in CSF-arrested extracts without releasing them into interphase [40]. The addition of catalytically active Src kinase increases tyrosine phosphorylation of PLCc, induces its activation, and elevates IP3 contents in the extracts, resulting in calcium release from IP3-regulated calcium stores. Listed signaling events faithfully reproduce the early steps of the fertilization-induced egg activation [41] (Fig. 2). These findings helped to estab-
sperm
xSrc
Tyr-P
PP2
PLC IP3
Calcium stores
lish a pivotal role of Src kinase in Xenopus egg fertilization and activation. In yet another study from our group, it was shown that egg rafts incubated with sperm or hydrogen peroxide can promote Src-dependent phosphorylation of PLCc and transient calcium release in the extracts of unfertilized Xenopus eggs [42]. Rafts prepared from peroxide-activated eggs can also promote Srcdependent dephosphorylation of MAPK and transition from metaphase to interphase in egg extracts. In detail, the experimental system allowing reconstitution of these signaling events is described later in the section ‘‘Xenopus egg rafts”. 1.4. Preparation of egg extracts 1.4.1. Isolation of eggs Fully grown female frogs are primed for ovulation by dorsal lymph sac injection of 40 U of pregnant mare serum gonadotropin (PMSG) 5–10 days before ovulation. The frogs are induced to ovulate by injecting 500 U of human chorionic gonadotropin (hCG). To avoid mixing egg batches from different animals, each frog should be placed in a separate container and maintained at 18–20 °C in water containing 0.1 M NaCl. Normally, ovulation starts in 8–10 h after the hCG injection. Egg extracts can be prepared from two types of eggs: overnight laid eggs or eggs squeezed from frogs immediately before extract preparation. Usually, extracts prepared from squeezed eggs have higher quality and more reproducible properties, however the yield of squeezed eggs from each frog is low (1 to 2 ml). Ovulated or squeezed eggs are collected into 100-mm Petri dishes filled with 30 ml of DeBoer’s solution (DB), containing 110 mM NaCl, 1.3 mM KCl, and 0.44 mM CaCl2 titrated to pH 7.2 with NaHCO3. All damaged eggs with abnormal coloring, as well as spontaneously activated eggs should be discarded. Healthy eggs are transferred into a 50-ml Falcon tube and washed with DB solution. Dejellying solution (DB containing 2% w/v cysteine–HCl titrated to pH 7.8 with 10 N NaOH) in a volume equal to the packed egg volume is added. Egg suspension is gently swirled for 3–6 min. Dejellying is complete when the eggs pack tightly without visible separation by their jelly coats. Dejellying solution is removed and the eggs are washed gently several times with DB. Once the eggs have been dejellied, they should not be left for more than a few minutes before beginning extract preparation. 1.4.2. Protocol for CSF extract preparation
membrane raft H2O2
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U-73122 HEPARIN
EGTA
Calcium release Fig. 2. A model for Src-, H2O2-, and sperm-induced calcium release in Xenopus egg extracts.
1. Dejellied eggs are washed four times in the extract buffer (XB), containing 100 mM KCl, 0.1 mM CaCl2, 1 mM MgCl2, 50 mM sucrose, and 10 mM potassium HEPES, pH 7.7, at room temperature. 2. The eggs are washed two more times in XB containing 10 lg/ml each leupeptin, pepstatin, and chymotrypsin. 3. Then, the eggs are transferred to centrifuge tubes containing XB plus 100 lg/ml cytochalasin B and 10 lg/ml each leupeptin, pepstatin, and chymotrypsin. 4. The tubes are centrifuged in a tabletop centrifuge for 30 s at 500g and then for 30 s at 1500g to obtain tight egg packing. 5. All XB buffer is removed from the top of the packed eggs. 6. Eggs are crushed by centrifugation at 15,000g for 15 min at 2 °C. 7. After the centrifugation tube content will be separated into three layers: lipid, cytoplasm, and yolk (from top to bottom). The cytoplasmic layer is slowly collected. Some contamination with lipid and yolk fractions is inevitable at this stage. 8. The cytoplasmic layer is subjected to the second clarifying spin under the same conditions as the crushing centrifugation.
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9. The extract will again separate into lipid, cytoplasmic, and yolk layers. The cytoplasmic layer is slowly collected and placed on ice. 10. Cytochalasin B, leupeptin, pepstatin, and chymotrypsin are added to a final concentration of 10 lg/ml each, as well as 1/20 volume of energy mix stock solution (150 mM creatine phosphate, 20 mM ATP, pH 7.4, 2 mM EGTA, pH 7.7, 20 mM MgCl2). 11. The prepared CSF-arrested egg extract should be kept on ice until use. It is not advisable to freeze the extracts because freezing destroys the extract’s ability to synthesize proteins de novo. Nevertheless, frozen extracts stored at 70 °C are capable of reproducing some steps of the egg activation signaling. The final extract volume does not exceed 20% of initial egg packed volume. When administering pharmacological agents, exogenous proteins, or antibodies to oocyte extracts, it is important to minimize extract dilution and keep a volume of added reagents within 10% of the initial extract volume. The inclusion of EGTA in the extraction buffer at the steps 2 and 3 of the above protocol is crucial for obtaining CSF-arrested extracts that maintain metaphase arrest. It prevents possible initiation of the signaling by the low levels of calcium present in XB buffer and by calcium released from the egg intracellular stores during extract preparation. An alternative method for producing M phase-arrested extracts is based on the use of non-degradable cyclin B lacking the 90 N-terminal residues. However, these extracts are regulated differently from CSF-arrested extracts and usually are referred to as D90 extracts [43,44]. CSF extracts remain arrested at the metaphase with high activities of the MAPK pathway and Cdc2 kinase and high content of cyclin, unless the signaling is initiated to inactivate CSF. Addition of calcium or constitutively active CaMKII mutant induces cyclin degradation and extracts progression to interphase with low MAPK and Cdc2 activities within 30–40 min. However, the extracts often return to metaphase later in 90–100 min. To prevent the transition to metaphase, cycloheximide can be added to the extracts at 100 lg/ml to block cyclin resynthesis before calcium administration. Cell-cycle transition in the extracts can be easily monitored by the change in the morphology of the added demembranated sperm nuclei, according to the following protocol. 1.4.3. Cell-cycle transition assay 1. Demembranated sperm nuclei are prepared at a final concentration of 107/ml according to the protocol described earlier [45]. 2. The nuclei are added to the CSF-arrested extracts on ice prior to the initiation of cell-cycle transition experiment at a final concentration of 105/ml. 3. Signaling in the extracts is initiated at RT. 4. Nuclear morphology is observed and scored by fluorescent microscopy after withdrawing 1 ll of extract at 10–15 min intervals and adding 4 ll of MMR buffer (100 mM NaCl, 1 mM MgCl2, 2 mM CaCl2, 0.1 mM EDTA, 5 mM HEPES, pH 7.8) containing 1 mg/ml Hoechst 33342, 10% formaldehyde, and 50% glycerol). 2. Xenopus egg rafts
nal transduction processes [reviewed in 46,47]. Xenopus egg rafts were shown to contain xSrc, Gqa, Ras, integrins, uroplakins, CD9 protein and other signaling molecules [48]. The egg rafts were demonstrated to play an important role in Xenopus egg fertilization. Disrupting rafts with the cholesterol-depleting drug methylb-cyclodextrin results in the inhibition of sperm-egg interaction, sperm-induced Src phosphorylation, and calcium release. It was shown that the addition of sperm to the egg rafts promotes protein–tyrosine phosphorylation and activation of raft-associated Src kinase [48]. This result supports the possibility that egg rafts may contain sperm-interacting receptor complex. One probable candidate for the sperm receptor component in the egg rafts can be uroplakin III, which is robustly phosphorylated on tyrosine shortly after fertilization [49,50]. Upon the analysis of signaling events downstream of Src activation, it was found that PLCc is translocated from the non-raft fraction to the raft fraction within several minutes of fertilization [42]. Raft translocation of PLCc is transient and cannot be detected in 20 min after fertilization. The molecular mechanism of PLCc translocation to rafts is not established. This translocation is accompanied by tyrosine phosphorylation and activation of PLCc. Using the reconstitution system containing membrane rafts and cell-free extracts of Xenopus eggs, the egg rafts were shown to be capable of inducing a transient calcium release in CSF-arrested extracts in a Src-PLCc-IP3-dependent manner, resulting in MAPK dephosphorylation and cell-cycle transition [42] (Fig. 2). Altogether, these findings suggest that the egg rafts serve as a subcellular microdomain, which triggers sperm-induced egg activation signaling.
2.2. Protocol for egg rafts preparation Unfertilized dejellied Xenopus eggs should be isolated according to the procedure described in Section 1.4.1 of this article. All following manipulations are conducted at 4 °C. 1. Eggs in a packed volume of 1 ml are mixed with fivefold excess of ice-cold raft buffer (20 mM Tris–HCl, 1 mM EDTA, 1 mM EGTA, 10 mM b-mercaptoethanol, pH 7.5) containing 150 mM NaCl, 250 mM sucrose, 1 mM Na3VO4, 10 lg/ml leupeptin, 20 lM APMSF and homogenized in a tight Teflon–glass Dounce homogenizer. 2. The homogenates are centrifuged at 500g for 10 min, then the supernatants are centrifuged at 150,000g for 20 min. 3. The crude membrane fraction (a fluffy layer on the top of the pellet) is collected. 4. Triton X-100 is added to the crude membrane fraction to a final concentration of 1%. 5. The samples are homogenized again, incubated on ice for 10 min, and mixed with equal volumes of ice-cold raft buffer containing 150 mM NaCl and 85% (w/v) sucrose. 6. The mixture (about 5 ml) is layered successively with 18 ml of 30% sucrose and with 12 ml of 5% sucrose in the same buffer. 7. The samples are centrifuged at 144,000g for 20–24 h. 8. Twelve 3-ml fractions are collected from the top to the bottom of the tubes. Fractions 3–6 represent egg raft preparation, whereas fractions 10–12 are detergent-soluble non-raft fractions. 9. For some experiments, pooled raft fractions can be additionally concentrated. In this case, they are fourfold diluted with water and centrifuged at 150,000g for 30 min. The pellet is resuspended in 200 ll of raft buffer containing 150 mM NaCl.
2.1. Rafts as a platform for initiation of egg fertilization signaling Lipid rafts are the cholesterol- and sphingolipid-enriched nanoscale microdomains in the cell plasma membrane. They are also enriched in the signaling molecules and are involved in many sig-
Rafts can also be prepared by the described protocol from fertilized or parthenogenetically activated eggs. In this case, the eggs should be briefly washed with DB buffer after the activation treatment.
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2.3. In vitro reconstitution system composed of egg rafts and CSF extracts In combination with the CSF-arrested extracts, activated egg rafts can reproduce some events of egg activation, including PLCc activation, IP3 production, transient calcium release, MAPK inactivation, and meiotic exit. This becomes possible due to complementation of the sperm-induced egg activation signaling machinery present in rafts with the components of signal transduction system localized in the extracts. For instance, in contrast to Src, PLCc is not present in the rafts of unfertilized Xenopus eggs but it is predominantly localized in the CSF extract [40,48]. The addition of sperm to the raft preparation promotes activation of raft-associated Src, recruitment of PLCc into the raft fraction, and its Src-dependent activation. Furthermore, CSF extracts obtained by a described procedure of low centrifuge centrifugation, evidently, contain IP3-regulated calcium stores, which can be specifically blocked by heparin [40]. This makes possible calcium release in the extracts after stimulation of IP3 production by the activated PLCc (Fig. 2). The level of free calcium in the extracts can be monitored real-time in the presence of a fluorescent calcium indicator. Activation state of Src, PLCc, and MAPK is evaluated by detection of their phosphorylation state with the phosphotyrosine-specific antibodies, whereas the addition of demembranated sperm nuclei to extracts can document cell-cycle transition, according to the protocol described in Section 1.4.3. Below, the protocols for detecting tyrosine phosphorylation of Src, PLCc, and MAPK, IP3 assay, and real-time calcium monitoring are provided.
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4. The extracts are neutralized with 1 M NaHCO3 (2 ll per 20 ll extract). 5. The content of IP3 in the samples is measured with the use of commercially available D-myo-IP3 [3H] assay system (GE Healthcare, Amersham Signal Transduction Assays) according to the manufacturer’s protocol. 2.3.3. Real-time calcium monitoring in the extracts 1. Ratiometric ultraviolet-excitable fluorescent calcium indicator Fura-2 is added to the CSF extracts at a final concentration of 2 lM prior to the signaling initiation. 2. The basal level of fluorescent signal is recorded over several minutes until its stabilization. 3. The level of free calcium in the extracts is continuously monitored at intervals 10–15 s by ratio-imaging microscopy using high-frame digital CCD imaging ARGUS/HISCA system from Hamamtsu Photonics. Excitation wavelengths 340 and 380 nm are used while monitoring emission at 510 nm. It is recommended to take several parallel measurements in the detection field. 4. The signaling in the extracts is initiated by the addition of rafts, preincubated in the presence or absence of different inhibitors and activators. 5. Further monitoring of the fluorescent signal is carried out for about 30 min. 6. Initial time of calcium elevation, peak amplitude and duration are determined as the parameters of calcium response.
2.3.1. Detection of tyrosine phosphorylation of Src, PLCc, and MAPK 3. Concluding remarks 1. Raft fraction, containing the equivalent of 10–20 eggs in a volume of 3 ll, CSF extracts, 27 ll, or a mixture of the both are incubated in the presence or absence of 1.5 ll of activators or inhibitors for 10 min at 30 °C. 2. To perform protein kinase reaction, the mixtures are incubated with 5 mM MgCl2 and 1 mM ATP for 10 min at 30 °C. 3. The reaction is stopped by the addition of 30 ll of 10 mM EDTA on ice. 4. Proteins are solubilized by the addition of 0.1% SDS and 1 mM sodium orthovanadate and incubating at 37 °C for 10 min. 5. Samples are centrifuged at 150,000g, 4 °C for 10 min and supernatant fractions are collected. 6. Aliquots of samples (100–500 lg of total protein) are immunoprecipitated with anti-PLCc antibody (1 lg/ml). 7. Immunoprecipitate and supernatant fractions (10–30 lg of total protein) are run on 10% SDS–PAGE. 8. To access tyrosine phosphorylation of PLCc, immunoprecipitate fractions are analyzed by immunoblotting with either antiphosphotyrosine antibody PY99 (1 lg/ml), or anti-PLCc antibody (0.5 lg/ml). 9. To access tyrosine phosphorylation of Src and MAPK, supernatant fractions are analyzed by immunoblotting with either anti-phosphoY416 Src antibody (1 lg/ml), or with anti-phospho MAPK antibody (0.5 lg/ml), respectively.
Cell-free experimental format provides great advantages for dissecting signal transduction pathways of egg activation. Individual components of signal transduction pathways in the egg extracts can be selectively targeted using specific inhibitors and activators. Also, isolated raft fraction of Xenopus eggs can be easily manipulated in vitro by selective targeting the signaling components present in this fraction. In our hands, the inhibitors of Src kinase, PLCc, blockers of IP3 receptor and calcium release were proved to be useful in dissecting early steps of egg activation. Furthermore, such initiators of egg activation signaling as sperm, hydrogen peroxide, and GTPcS were surprisingly quite effective when applied to in vitro reconstitution system composed of egg rafts and CSF-arrested extracts. Many other interesting results can be expected from the analysis of signal transduction in the cell-free extracts and rafts of Xenopus eggs. References [1] [2] [3] [4] [5] [6] [7]
2.3.2. Extraction and assay of IP3 1. Eighty microliters of 15% trichloracetic acid is added to 20-ll aliquots of the extracts on ice. 2. Mixtures are centrifuged at 7000g, 4 °C for 20 min to remove precipitated materials. 3. The resulting supernatants are extracted four times with 1 ml of water-saturated diethyl ether to remove traces of trichloracetic acid.
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Review Article
Analysis of signal transduction in cell-free extracts and rafts of Xenopus eggs Alexander A. Tokmakov a,b,*, Tetsushi Iwasaki b, Ken-Ichi Sato c, Yasuo Fukami a,b a
Graduate School of Science, Kobe University, Rokkodai 1-1, Nada, Kobe 657-8501, Japan Research Center for Environmental Genomics, Kobe University, Rokkodai 1-1, Nada, Kobe 657-8501, Japan c Department of Biotechnology, Faculty of Engineering, Kyoto Sangyo University, Kamigamo-Motoyama, Kita-ku, Kyoto 603-8555, Japan b
a r t i c l e
i n f o
Article history: Accepted 12 January 2010 Available online 15 January 2010 Keywords: Xenopus egg Egg activation Fertilization Signal transduction Cell-free extract Raft
a b s t r a c t Intracellular signaling during egg activation/fertilization has been extensively studied using intact eggs, which can be manipulated by microinjection of different mRNAs, proteins, or chemical drugs. Furthermore, egg extracts, which retain high CSF activity (CSF-arrested extracts), were developed for studying fertilization/activation signal transduction, which have significant advantages as a model system. The addition of calcium to CSF-arrested extracts initiates a plethora of signaling events that take place during egg activation. Hence, the signaling downstream of calcium mobilization has been successfully studied in the egg extracts. Moreover, despite disruption of membrane-associated signaling compartments and ordered compartmentalization during extract preparation, CSF-arrested extracts can be successfully used to study early signaling events, which occur upstream of calcium release during egg activation/fertilization. In combination with the CSF-arrested extracts, activated egg rafts can reproduce some events of egg activation, including PLCc activation, IP3 production, transient calcium release, MAPK inactivation, and meiotic exit. This becomes possible due to complementation of the sperm-induced egg activation signaling machinery present in the rafts with the components of signal transduction system localized in the extracts. Herein, we describe protocols for studying molecular mechanisms of egg fertilization/activation using cell-free extracts and membrane rafts prepared from metaphase-arrested Xenopus eggs. Ó 2010 Elsevier Inc. All rights reserved.
1. Xenopus eggs 1.1. Metaphase-arrested eggs After the completion of meiosis, mature Xenopus eggs are arrested in the metaphase of the second meiotic division (metaphase II) due to the high activity of the key mitotic regulators, maturation promoting factor (MPF) and cytostatic factor (CSF). MPF was originally defined by Masui and Markert as an activity in the cytoplasm of mature frog oocytes that could induce maturation in frog oocytes [1]. MPF consists of cyclin B and cyclin-dependent protein kinase CDK1 homologous to the yeast cell division control 2 protein (Cdc2). Several enzymes of protein phosphorylation, which represent two signaling pathways, are activated in eggs, supporting high MPF activity. The polo-like protein kinase pathway, mediated by the polo-like kinase Plx1 and Xenopus polo-like kinase kinase xPlkk1, is linked to activation of MPF-activating phosphatase Cdc25C [2,3] (Fig. 1). On the other hand, mitogen-activated protein kinase (MAPK), highly active in the presence of Mos kinase, phosphorylates and activates the downstream Ser/Thr-specific ribo-
* Corresponding author. Address: Research Center for Environmental Genomics, Kobe University, Rokkodai 1-1, Nada, Kobe 657-8501, Japan. Fax: +81 78 803 5951. E-mail address: [email protected] (A.A. Tokmakov). 1046-2023/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2010.01.008
somal s6 kinase Rsk, which down-regulates the Cdc2-inhibitory kinase Myt1, supporting MPF activation [4,5]. CSF, defined as an activity that causes metaphase arrest in frog eggs, first appears in progesterone-treated oocytes at the time of Mos accumulation due to its stabilization by phosphorylation on Ser3 and disappears shortly after fertilization, coincident with Mos degradation [6]. The exact composition of CSF is not established. Injections of Mos, or constitutively active MAPK kinase (MAPKK), or thiophosphorylated MAPK into two-cell blastomeres promote metaphase arrest [7–9], and Mos-induced arrest can be abolished by co-injecting anti-MAPKK antibody [10]. Similarly, the MAPK phosphatase MKP1 can release a MAPK-dependent metaphase arrest induced in Xenopus egg extracts by spindle depolymerization [11]. Also, Rsk has been shown to mediate CSF activity of the MAPK cascade and to induce metaphase arrest in cleaving Xenopus embryos [12]. Immunodepletion of Rsk from egg extracts abolished their capacity to undergo mitotic arrest in response to MAPK cascade activation [13]. Thus, it was concluded that the MAPK pathway represents an important component of CSF. Although immunodepletion of Mos from the cytoplasm of unfertilized eggs leads to the loss of CSF activity [9,14], inhibition of the MAPK cascade with U0126 or PD98059, or depletion of Rsk from CSF-arrested extracts does not release CSF arrest [13,6,15]. It implies that Mos is necessary for both establishing and maintaining metaphase arrest, whereas MAPK and Rsk are required to only
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MOS
MEK
calpain calcineurin
MAPK
Rsk 1,2
Bub1
Myt1
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Wee1
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ActiCa2+ vation
APC/C
Cyclin/Cdc2
Cyclin B
active MPF
Cdc2 inactive preMPF
CaMKII
CaMKII
Emi2/Erp1 xPlkk1
Plx1
Cdc25C
Fig. 1. Signaling pathways in metaphase-arrested and activated eggs.
establish this arrest. Still, the fact, that the fully active MAPK cascade cannot impose metaphase arrest in meiosis I, alongside with the finding that Mos cannot induce meiosis II in the absence of protein synthesis [9], indicates that CSF arrest requires some other components newly synthesized in meiosis II. MAPK-dependent inhibition of the APC/C, the ubiquitin ligase controlling cyclin B degradation at the anaphase/metaphase transition, was proposed to play a major role in maintaining high activity of MPF during CSF arrest [6]. The APC/C inhibitor Emi2/Erp1 has recently been identified as a pivotal CSF component, required to maintain metaphase II arrest [16,17]. It was shown that Rsk can directly phosphorylate Emi2/Erp1 [18,19]. This phosphorylation increases Emi2/Erp1 stability and promotes APC/C inhibition. Evidence has been presented that Bub1, a component of the spindle assembly checkpoint, is activated by Rsk and also inhibits APC/ C [20]. This finding suggests that in CSF-arrested eggs with the intact spindle this checkpoint can be activated independently of kinetochore-microtubule attachment by the constitutively active, due to the unique presence of Mos in meiotic metaphase, MAPK cascade [6]. Active Rsk also phosphorylates and inactivates Cdc2inactivating protein kinase Myt1, directly promoting Cdc2 activity [4,5]. Another Cdc2-inactivating kinase, Wee1, is highly phosphorylated and only weakly active in metaphase-arrested eggs [21]. Some other mechanisms, such as Mos-stimulated polyadenylation of cyclin mRNA [22], may also contribute to maintaining high MPF activity in CSF-arrested eggs and extracts. 1.2. Egg activation The loop of positive feedback comprising the MAPK cascade and MPF assures the stability of CSF arrest in fully matured eggs. Fertilization or parthenogenetic activation disrupts positive feedback, inactivates CSF, and releases metaphase arrest. In contrast to progesterone-induced meiotic initiation, where Mos synthesis and MAPK cascade activation precede and promote MPF activation, fertilization triggers MPF inactivation before Mos degradation and MAPK cascade inactivation. Calcium-dependent degradation of cyclin B in 10–20 min after fertilization is thought to trigger the disruption of positive feedback between MPF and CSF. Then, within 15–30 min after egg activation, Mos protein is specifically degraded by the calcium-dependent cysteine protease calpain [23] and Mos mRNA is deadenylated at its 3’UTR and translationally arrested [24]. Furthermore, MAPK inactivation occurs in 30–40 min after fertilization, simultaneously with the loss of CSF activity in activated egg [25]. The early universal event of fertilization-induced egg activation is a mobilization of intracellular calcium that originates from the sperm entry point and spreads through the entire egg cytoplasm. Recently, this process has been established to require the sequential activation of Src family kinases, PLCc, and IP3 receptor of the endoplasmic reticulum [rewieved in 26,27]. Elevation of intracellu-
lar calcium is necessary and sufficient for egg activation. Calcium ionophores induce complete egg activation, whereas calcium-chelating agents, such as EGTA and BAPTA, prevent sperm-induced egg activation [28,29]. The calcium calmodulin-dependent kinase CaMKII is thought to mediate calcium effects on the cyclin degradation machinery. Addition of constitutively active CaMKII to CSF-arrested egg extracts causes cyclin degradation and CSF release in the absence of calcium, whereas a specific inhibitor of CaMKII prevents cyclin degradation and exit from metaphase arrest after calcium addition [30]. Besides cyclin B degradation, CaMKII also triggers sister chromatid separation, presumably, through the degradation of mitotic regulator proteins that bind sister chromatids together [31]. A direct target of CaMKII related to the protein degradation machinery was shown to be the APC/C inhibitor Emi2/ Erp1. Phosphorylation of Emi2/Erp1 by the calcium-activated CaMKII leads to the recruitment of the polo-like kinase Plx1 via its Polo Box domain. Then, Plx1 phosphorylates Emi2/Erp1 at the site of phosphorylation-dependent degradation signal, which is recognized by the SCFbTrCP complex for ubiquitination and degradation [17,18]. In addition to targeting the protein degradation machinery, CaMKII rapidly stimulates the inhibitory phosphorylation of Cdc25 on Ser287, which is repressed in the unfertilized eggs [32]. On the other hand, calcium induces not only the activation of CaMKII but also the dephosphorylation of the Apc3 component of APC/C. The calcium/calmodulin-dependent protein phosphatase calcineurin and Fizzy/Cdc20, a key regulator of the APC/C, were shown to be involved in this process [33,34]. Further, it was demonstrated that CaMKII and calcineurin activities together allow nuclear envelope formation [33]. Calcium-dependent activation of the APC/C triggers cyclin B ubiquitination and targets the Cdc2/cyclin B complex to the 26S proteasome. Proteasome-dependent dissociation of Cdc2 from cyclin B down-regulates Cdc2 kinase. It is sufficient to inactivate MPF even without following cyclin B degradation [35]. In the absence of MPF activity, Mos dephosphorylation at Ser3, a site of direct phosphorylation by Cdc2 kinase, occurs, leading to accelerated degradation of Mos protein by an ubiquitination pathway [36]. Dephosphorylated Mos cannot support high activity of the MAPK cascade, although Mos rephosphorylation by Cdc2 kinase is able to cause MAPK reactivation [37]. MAPK inactivation disrupts another important loop of positive feedback inside the MAPK cascade itself (between MAPK and Mos), resulting in the complete switchlike shutdown of the MAPK cascade in fertilized eggs. Thereafter, the MAPK cascade plays an important role in the regulation of following early embryo mitotic cell cycles, and Mos protein is completely degraded after fertilization and is not resynthesized again [23,24].
1.3. Egg extracts Intracellular signaling during egg activation/fertilization has been extensively studied using intact eggs, which can be manipulated by microinjection of different mRNAs, proteins, or chemical drugs. However, this technique is rather cumbersome, and microinjection often fails to achieve the desired cytosolic concentration of injected molecules because of variable leakage. Besides, synchrony among different eggs is not sufficient sometimes for studies of fast signaling events. Therefore, cell-free egg extracts has been developed for the purpose of cell-cycle transition studies, which have significant advantages as a model system [38]. Large volumes (several milliliters) of highly synchronized homogenous extract can be produced from the eggs of a single frog, while the exogenous chemical compounds and proteins can be added easily at precisely known concentrations and also efficiently removed by immunodepletion.
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Cell-free extracts of Xenopus eggs can recapitulate a number of physiological processes. The most studied of them are meiotic exit, mitotic cell-cycle transition, and apoptotic process. The procedures of extract preparation vary in details, depending on the nature of considered processes. The egg extracts, which retain high CSF activity (CSF-arrested extracts), are commonly used for studying fertilization/activation signaling. The addition of calcium to CSF-arrested extracts initiates a plethora of signaling events that take place during egg activation, resulting in the meiotic exit. Hence, the signaling downstream of calcium mobilization can be successfully studied in the egg extracts. For instance, it has been shown that proteasomes posses non-proteolytic activity dissociating Cdc2 kinase from cyclins and that MPF inactivation is separated from cyclin B degradation upon activation of CSF extracts in the presence of the proteasome proteolytic activity inhibitor MG115 [35]. Moreover, cyclin B/Cdc2 dissociation precedes Cdc2 Thr161 dephosphorylation upon MPF inactivation in CSF extracts [39]. Another recent example includes establishing the role of the APC/C inhibitor Emi2/Erp1 and the polo-like kinase Plx1 in the calciuminitiated meiotic exit. It was shown that CaMKII and Plx1 sequentially phosphorylate Emi2/Erp1 to trigger its destruction, APC/C activation, cyclin degradation and cell-cycle transition in the CSFarrested extracts [17,18]. The importance of calcium/calmodulindependent protein phosphatase calcineurin for initiation of embryonic development was established based on the fact that calcineurin is required to release CSF-arrested Xenopus egg extracts from M phase [33,34]. Remarkably, despite disruption of membrane-associated signaling compartments and ordered compartmentalization during extract preparation, CSF-arrested extracts can be successfully used to study early signaling events, which occur upstream of calcium release during egg activation/fertilization. It was demonstrated that catalytically active Src kinase can initiate calcium response of a low magnitude in CSF-arrested extracts without releasing them into interphase [40]. The addition of catalytically active Src kinase increases tyrosine phosphorylation of PLCc, induces its activation, and elevates IP3 contents in the extracts, resulting in calcium release from IP3-regulated calcium stores. Listed signaling events faithfully reproduce the early steps of the fertilization-induced egg activation [41] (Fig. 2). These findings helped to estab-
sperm
xSrc
Tyr-P
PP2
PLC IP3
Calcium stores
lish a pivotal role of Src kinase in Xenopus egg fertilization and activation. In yet another study from our group, it was shown that egg rafts incubated with sperm or hydrogen peroxide can promote Src-dependent phosphorylation of PLCc and transient calcium release in the extracts of unfertilized Xenopus eggs [42]. Rafts prepared from peroxide-activated eggs can also promote Srcdependent dephosphorylation of MAPK and transition from metaphase to interphase in egg extracts. In detail, the experimental system allowing reconstitution of these signaling events is described later in the section ‘‘Xenopus egg rafts”. 1.4. Preparation of egg extracts 1.4.1. Isolation of eggs Fully grown female frogs are primed for ovulation by dorsal lymph sac injection of 40 U of pregnant mare serum gonadotropin (PMSG) 5–10 days before ovulation. The frogs are induced to ovulate by injecting 500 U of human chorionic gonadotropin (hCG). To avoid mixing egg batches from different animals, each frog should be placed in a separate container and maintained at 18–20 °C in water containing 0.1 M NaCl. Normally, ovulation starts in 8–10 h after the hCG injection. Egg extracts can be prepared from two types of eggs: overnight laid eggs or eggs squeezed from frogs immediately before extract preparation. Usually, extracts prepared from squeezed eggs have higher quality and more reproducible properties, however the yield of squeezed eggs from each frog is low (1 to 2 ml). Ovulated or squeezed eggs are collected into 100-mm Petri dishes filled with 30 ml of DeBoer’s solution (DB), containing 110 mM NaCl, 1.3 mM KCl, and 0.44 mM CaCl2 titrated to pH 7.2 with NaHCO3. All damaged eggs with abnormal coloring, as well as spontaneously activated eggs should be discarded. Healthy eggs are transferred into a 50-ml Falcon tube and washed with DB solution. Dejellying solution (DB containing 2% w/v cysteine–HCl titrated to pH 7.8 with 10 N NaOH) in a volume equal to the packed egg volume is added. Egg suspension is gently swirled for 3–6 min. Dejellying is complete when the eggs pack tightly without visible separation by their jelly coats. Dejellying solution is removed and the eggs are washed gently several times with DB. Once the eggs have been dejellied, they should not be left for more than a few minutes before beginning extract preparation. 1.4.2. Protocol for CSF extract preparation
membrane raft H2O2
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U-73122 HEPARIN
EGTA
Calcium release Fig. 2. A model for Src-, H2O2-, and sperm-induced calcium release in Xenopus egg extracts.
1. Dejellied eggs are washed four times in the extract buffer (XB), containing 100 mM KCl, 0.1 mM CaCl2, 1 mM MgCl2, 50 mM sucrose, and 10 mM potassium HEPES, pH 7.7, at room temperature. 2. The eggs are washed two more times in XB containing 10 lg/ml each leupeptin, pepstatin, and chymotrypsin. 3. Then, the eggs are transferred to centrifuge tubes containing XB plus 100 lg/ml cytochalasin B and 10 lg/ml each leupeptin, pepstatin, and chymotrypsin. 4. The tubes are centrifuged in a tabletop centrifuge for 30 s at 500g and then for 30 s at 1500g to obtain tight egg packing. 5. All XB buffer is removed from the top of the packed eggs. 6. Eggs are crushed by centrifugation at 15,000g for 15 min at 2 °C. 7. After the centrifugation tube content will be separated into three layers: lipid, cytoplasm, and yolk (from top to bottom). The cytoplasmic layer is slowly collected. Some contamination with lipid and yolk fractions is inevitable at this stage. 8. The cytoplasmic layer is subjected to the second clarifying spin under the same conditions as the crushing centrifugation.
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9. The extract will again separate into lipid, cytoplasmic, and yolk layers. The cytoplasmic layer is slowly collected and placed on ice. 10. Cytochalasin B, leupeptin, pepstatin, and chymotrypsin are added to a final concentration of 10 lg/ml each, as well as 1/20 volume of energy mix stock solution (150 mM creatine phosphate, 20 mM ATP, pH 7.4, 2 mM EGTA, pH 7.7, 20 mM MgCl2). 11. The prepared CSF-arrested egg extract should be kept on ice until use. It is not advisable to freeze the extracts because freezing destroys the extract’s ability to synthesize proteins de novo. Nevertheless, frozen extracts stored at 70 °C are capable of reproducing some steps of the egg activation signaling. The final extract volume does not exceed 20% of initial egg packed volume. When administering pharmacological agents, exogenous proteins, or antibodies to oocyte extracts, it is important to minimize extract dilution and keep a volume of added reagents within 10% of the initial extract volume. The inclusion of EGTA in the extraction buffer at the steps 2 and 3 of the above protocol is crucial for obtaining CSF-arrested extracts that maintain metaphase arrest. It prevents possible initiation of the signaling by the low levels of calcium present in XB buffer and by calcium released from the egg intracellular stores during extract preparation. An alternative method for producing M phase-arrested extracts is based on the use of non-degradable cyclin B lacking the 90 N-terminal residues. However, these extracts are regulated differently from CSF-arrested extracts and usually are referred to as D90 extracts [43,44]. CSF extracts remain arrested at the metaphase with high activities of the MAPK pathway and Cdc2 kinase and high content of cyclin, unless the signaling is initiated to inactivate CSF. Addition of calcium or constitutively active CaMKII mutant induces cyclin degradation and extracts progression to interphase with low MAPK and Cdc2 activities within 30–40 min. However, the extracts often return to metaphase later in 90–100 min. To prevent the transition to metaphase, cycloheximide can be added to the extracts at 100 lg/ml to block cyclin resynthesis before calcium administration. Cell-cycle transition in the extracts can be easily monitored by the change in the morphology of the added demembranated sperm nuclei, according to the following protocol. 1.4.3. Cell-cycle transition assay 1. Demembranated sperm nuclei are prepared at a final concentration of 107/ml according to the protocol described earlier [45]. 2. The nuclei are added to the CSF-arrested extracts on ice prior to the initiation of cell-cycle transition experiment at a final concentration of 105/ml. 3. Signaling in the extracts is initiated at RT. 4. Nuclear morphology is observed and scored by fluorescent microscopy after withdrawing 1 ll of extract at 10–15 min intervals and adding 4 ll of MMR buffer (100 mM NaCl, 1 mM MgCl2, 2 mM CaCl2, 0.1 mM EDTA, 5 mM HEPES, pH 7.8) containing 1 mg/ml Hoechst 33342, 10% formaldehyde, and 50% glycerol). 2. Xenopus egg rafts
nal transduction processes [reviewed in 46,47]. Xenopus egg rafts were shown to contain xSrc, Gqa, Ras, integrins, uroplakins, CD9 protein and other signaling molecules [48]. The egg rafts were demonstrated to play an important role in Xenopus egg fertilization. Disrupting rafts with the cholesterol-depleting drug methylb-cyclodextrin results in the inhibition of sperm-egg interaction, sperm-induced Src phosphorylation, and calcium release. It was shown that the addition of sperm to the egg rafts promotes protein–tyrosine phosphorylation and activation of raft-associated Src kinase [48]. This result supports the possibility that egg rafts may contain sperm-interacting receptor complex. One probable candidate for the sperm receptor component in the egg rafts can be uroplakin III, which is robustly phosphorylated on tyrosine shortly after fertilization [49,50]. Upon the analysis of signaling events downstream of Src activation, it was found that PLCc is translocated from the non-raft fraction to the raft fraction within several minutes of fertilization [42]. Raft translocation of PLCc is transient and cannot be detected in 20 min after fertilization. The molecular mechanism of PLCc translocation to rafts is not established. This translocation is accompanied by tyrosine phosphorylation and activation of PLCc. Using the reconstitution system containing membrane rafts and cell-free extracts of Xenopus eggs, the egg rafts were shown to be capable of inducing a transient calcium release in CSF-arrested extracts in a Src-PLCc-IP3-dependent manner, resulting in MAPK dephosphorylation and cell-cycle transition [42] (Fig. 2). Altogether, these findings suggest that the egg rafts serve as a subcellular microdomain, which triggers sperm-induced egg activation signaling.
2.2. Protocol for egg rafts preparation Unfertilized dejellied Xenopus eggs should be isolated according to the procedure described in Section 1.4.1 of this article. All following manipulations are conducted at 4 °C. 1. Eggs in a packed volume of 1 ml are mixed with fivefold excess of ice-cold raft buffer (20 mM Tris–HCl, 1 mM EDTA, 1 mM EGTA, 10 mM b-mercaptoethanol, pH 7.5) containing 150 mM NaCl, 250 mM sucrose, 1 mM Na3VO4, 10 lg/ml leupeptin, 20 lM APMSF and homogenized in a tight Teflon–glass Dounce homogenizer. 2. The homogenates are centrifuged at 500g for 10 min, then the supernatants are centrifuged at 150,000g for 20 min. 3. The crude membrane fraction (a fluffy layer on the top of the pellet) is collected. 4. Triton X-100 is added to the crude membrane fraction to a final concentration of 1%. 5. The samples are homogenized again, incubated on ice for 10 min, and mixed with equal volumes of ice-cold raft buffer containing 150 mM NaCl and 85% (w/v) sucrose. 6. The mixture (about 5 ml) is layered successively with 18 ml of 30% sucrose and with 12 ml of 5% sucrose in the same buffer. 7. The samples are centrifuged at 144,000g for 20–24 h. 8. Twelve 3-ml fractions are collected from the top to the bottom of the tubes. Fractions 3–6 represent egg raft preparation, whereas fractions 10–12 are detergent-soluble non-raft fractions. 9. For some experiments, pooled raft fractions can be additionally concentrated. In this case, they are fourfold diluted with water and centrifuged at 150,000g for 30 min. The pellet is resuspended in 200 ll of raft buffer containing 150 mM NaCl.
2.1. Rafts as a platform for initiation of egg fertilization signaling Lipid rafts are the cholesterol- and sphingolipid-enriched nanoscale microdomains in the cell plasma membrane. They are also enriched in the signaling molecules and are involved in many sig-
Rafts can also be prepared by the described protocol from fertilized or parthenogenetically activated eggs. In this case, the eggs should be briefly washed with DB buffer after the activation treatment.
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2.3. In vitro reconstitution system composed of egg rafts and CSF extracts In combination with the CSF-arrested extracts, activated egg rafts can reproduce some events of egg activation, including PLCc activation, IP3 production, transient calcium release, MAPK inactivation, and meiotic exit. This becomes possible due to complementation of the sperm-induced egg activation signaling machinery present in rafts with the components of signal transduction system localized in the extracts. For instance, in contrast to Src, PLCc is not present in the rafts of unfertilized Xenopus eggs but it is predominantly localized in the CSF extract [40,48]. The addition of sperm to the raft preparation promotes activation of raft-associated Src, recruitment of PLCc into the raft fraction, and its Src-dependent activation. Furthermore, CSF extracts obtained by a described procedure of low centrifuge centrifugation, evidently, contain IP3-regulated calcium stores, which can be specifically blocked by heparin [40]. This makes possible calcium release in the extracts after stimulation of IP3 production by the activated PLCc (Fig. 2). The level of free calcium in the extracts can be monitored real-time in the presence of a fluorescent calcium indicator. Activation state of Src, PLCc, and MAPK is evaluated by detection of their phosphorylation state with the phosphotyrosine-specific antibodies, whereas the addition of demembranated sperm nuclei to extracts can document cell-cycle transition, according to the protocol described in Section 1.4.3. Below, the protocols for detecting tyrosine phosphorylation of Src, PLCc, and MAPK, IP3 assay, and real-time calcium monitoring are provided.
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4. The extracts are neutralized with 1 M NaHCO3 (2 ll per 20 ll extract). 5. The content of IP3 in the samples is measured with the use of commercially available D-myo-IP3 [3H] assay system (GE Healthcare, Amersham Signal Transduction Assays) according to the manufacturer’s protocol. 2.3.3. Real-time calcium monitoring in the extracts 1. Ratiometric ultraviolet-excitable fluorescent calcium indicator Fura-2 is added to the CSF extracts at a final concentration of 2 lM prior to the signaling initiation. 2. The basal level of fluorescent signal is recorded over several minutes until its stabilization. 3. The level of free calcium in the extracts is continuously monitored at intervals 10–15 s by ratio-imaging microscopy using high-frame digital CCD imaging ARGUS/HISCA system from Hamamtsu Photonics. Excitation wavelengths 340 and 380 nm are used while monitoring emission at 510 nm. It is recommended to take several parallel measurements in the detection field. 4. The signaling in the extracts is initiated by the addition of rafts, preincubated in the presence or absence of different inhibitors and activators. 5. Further monitoring of the fluorescent signal is carried out for about 30 min. 6. Initial time of calcium elevation, peak amplitude and duration are determined as the parameters of calcium response.
2.3.1. Detection of tyrosine phosphorylation of Src, PLCc, and MAPK 3. Concluding remarks 1. Raft fraction, containing the equivalent of 10–20 eggs in a volume of 3 ll, CSF extracts, 27 ll, or a mixture of the both are incubated in the presence or absence of 1.5 ll of activators or inhibitors for 10 min at 30 °C. 2. To perform protein kinase reaction, the mixtures are incubated with 5 mM MgCl2 and 1 mM ATP for 10 min at 30 °C. 3. The reaction is stopped by the addition of 30 ll of 10 mM EDTA on ice. 4. Proteins are solubilized by the addition of 0.1% SDS and 1 mM sodium orthovanadate and incubating at 37 °C for 10 min. 5. Samples are centrifuged at 150,000g, 4 °C for 10 min and supernatant fractions are collected. 6. Aliquots of samples (100–500 lg of total protein) are immunoprecipitated with anti-PLCc antibody (1 lg/ml). 7. Immunoprecipitate and supernatant fractions (10–30 lg of total protein) are run on 10% SDS–PAGE. 8. To access tyrosine phosphorylation of PLCc, immunoprecipitate fractions are analyzed by immunoblotting with either antiphosphotyrosine antibody PY99 (1 lg/ml), or anti-PLCc antibody (0.5 lg/ml). 9. To access tyrosine phosphorylation of Src and MAPK, supernatant fractions are analyzed by immunoblotting with either anti-phosphoY416 Src antibody (1 lg/ml), or with anti-phospho MAPK antibody (0.5 lg/ml), respectively.
Cell-free experimental format provides great advantages for dissecting signal transduction pathways of egg activation. Individual components of signal transduction pathways in the egg extracts can be selectively targeted using specific inhibitors and activators. Also, isolated raft fraction of Xenopus eggs can be easily manipulated in vitro by selective targeting the signaling components present in this fraction. In our hands, the inhibitors of Src kinase, PLCc, blockers of IP3 receptor and calcium release were proved to be useful in dissecting early steps of egg activation. Furthermore, such initiators of egg activation signaling as sperm, hydrogen peroxide, and GTPcS were surprisingly quite effective when applied to in vitro reconstitution system composed of egg rafts and CSF-arrested extracts. Many other interesting results can be expected from the analysis of signal transduction in the cell-free extracts and rafts of Xenopus eggs. References [1] [2] [3] [4] [5] [6] [7]
2.3.2. Extraction and assay of IP3 1. Eighty microliters of 15% trichloracetic acid is added to 20-ll aliquots of the extracts on ice. 2. Mixtures are centrifuged at 7000g, 4 °C for 20 min to remove precipitated materials. 3. The resulting supernatants are extracted four times with 1 ml of water-saturated diethyl ether to remove traces of trichloracetic acid.
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