Background information. The IP3R (inositol 1,4,5-trisphosphate receptor) is a tetrameric channel that accounts for a large part of the intracellular Ca2+ release in virtually all cell types. We have previously demonstrated that caspase-3-mediated cleavage of IP3R1 during cell death generates a C-terminal fragment of 95 kDa comprising the complete channel domain. Expression of this truncated IP3R increases the cellular sensitivity to apoptotic stimuli, and it was postulated to be a constitutively active channel.
Results. In the present study, we demonstrate that expression of the caspase-3-cleaved C-terminus of IP3R1 increased the rate of thapsigargin-mediated Ca2+ leak and decreased the rate of Ca2+ uptake into the ER (endoplasmic reticulum), although it was not sufficient by itself to deplete intracellular Ca2+ stores. We detected the truncated IP3R1 in different cell types after a challenge with apoptotic stimuli, as well as in aged mouse oocytes. Injection of mRNA corresponding to the truncated IP3R1 blocked sperm factor-induced Ca2+ oscillations and induced an apoptotic phenotype.
Conclusions. In the present study, we show that caspase-3-mediated truncation of IP3R1 enhanced the Ca2+ leak from the ER. We suggest a model in which, in normal conditions, the increased Ca2+ leak is largely compensated by enhanced Ca2+-uptake activity, whereas in situations where the cellular metabolism is compromised, as occurring in aging oocytes, the Ca2+ leak acts as a feed-forward mechanism to divert the cell into apoptosis.
Many physiological processes are regulated by changes in free [Ca2+]i (intracellular Ca2+ concentration). Various Ca2+ channels, pumps, exchangers and buffers are responsible for the accurate control of [Ca2+]i (Berridge et al., 2000). Perturbations in the [Ca2+]i homoeostasis are considered to be of great importance in initiating and modulating both apoptotic (programmed) and necrotic cell death (Berridge et al., 1998, 2003; Hajnoczky et al., 2003; Orrenius et al., 2003; Rizzuto et al., 2003). The caspase family of cysteine proteases represents one of the key players in apoptotic cell-death processes (Degterev et al., 2003; Yuan et al., 2003). The two major signalling pathways of caspase activation, the death-receptor pathway (extrinsic pathway) (Curtin and Cotter, 2003; Micheau and Tschopp, 2003) and the intrinsic or mitochondria-mediated pathway (Schafer and Kornbluth, 2006), eventually lead to the activation of so-called effector caspases, including caspase 3. The latter mediates the proteolytic processing of a wide variety of cellular targets, including many that directly control or affect intracellular Ca2+ homoeostasis (Verbert et al., 2007). Cleavage by caspase 3 can induce a gain- or a loss-of-function of these substrates, possibly leading to the stimulation of aberrant Ca2+ signalling that can influence the execution of the apoptotic process.
The IP3R (inositol 1,4,5-trisphosphate receptor), a large tetrameric intracellular Ca2+-release channel mainly located in the ER (endoplasmic reticulum) and represented by three isoforms (types 1, 2 and 3), is one of the targets of caspase 3. There is increasing evidence that the IP3R plays a pivotal role in apoptotic programmed cell-death pathways (Joseph and Hajnoczky, 2007). IP3R-knockout cells are less sensitive to apoptotic stimuli (Sugawara et al., 1997; Assefa et al., 2004), and anti-sense knockout of IP3R1 blocks cell death in Jurkat T-lymphoma cells (Jayaraman and Marks, 1997). IP3R activity is also shown to be regulated by cytochrome c (Boehning et al., 2005), GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (Patterson et al., 2005) and members of the Bcl-2 family of proteins (Chen et al., 2004; Oakes et al., 2005; White et al., 2005; Pinton and Rizzuto, 2006), which are all well-known key players in cell-death processes. Moreover, IP3R1 is specifically cleaved by caspase 3 in Jurkat T-lymphoma cells upon induction of apoptosis by STS (staurosporin) or anti-Fas IgM (Hirota et al., 1999), thereby generating a truncated 95-kDa protein containing all of the transmembrane domains and the channel pore. The consensus site for caspase-3-dependent cleavage of mouse IP3R1 was identified at D1888EVD* (* indicates cleavage site) (Hirota et al., 1999). The role of IP3R1 truncation during apoptosis has been investigated in DT40 cells deficient in all three IP3R isoforms [IP3RKO (IP3R knockout) cells]. IP3RKO cells expressing a caspase-3-non-cleavable IP3R1 mutant were less sensitive to apoptotic stimuli, whereas cells expressing full-length IP3R1 or the truncated IP3R1 displayed an increased propensity to undergo apoptosis. In addition, we have found previously that caspase 3 cleavage of IP3R1 was related to an increase in [Ca2+]i during the execution phase of apoptosis (Assefa et al., 2004). Until now, the functional properties of this truncated IP3R1 have not been elucidated. Therefore, the aim of the present study was to further investigate the formation and the Ca2+-leak properties of this channel in different cell types and in well-defined conditions. Our results demonstrate that stable or transient expression of the truncated IP3R1 induced a modest Ca2+ leak from the ER. However, this Ca2+ leak was not sufficient to deplete the ER Ca2+ stores in normal conditions. Nevertheless, our results emphasize the physiological importance of the generation of a truncated IP3R, as it was consistently found in several cell types undergoing apoptotic cell death, including aged mouse oocytes. Furthermore, expression of the C-terminal fragment in fresh oocytes led to severe perturbations in the Ca2+ signalling and resulted in cellular fragmentation. Proteolytic cleavage of IP3R1 and generation of a long-lived channel domain may therefore represent a feed-forward pathway in the execution of cell death, particularly when intracellular Ca2+-removal mechanisms become inadequate.
Increased ER Ca2+ leak in cells stably expressing the truncated IP3R1
We have first investigated the Ca2+-leak properties of the truncated IP3R1 independently of plasma-membrane fluxes. Therefore, we have performed unidirectional Ca2+ fluxes in permeabilized DT40 cells. IP3RKO cells, deficient in all three IP3Rs, were stably transfected with the caspase 3-truncated channel domain (Δ1–1891)IP3R1 as described previously (Assefa et al., 2004). We measured the [Ca2+] in a permeabilized cell suspension with fluo 3 and monitored the leak after inhibition of the SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) by TG (thapsigargin; 4 μM) (Figure 1A). After permeabilization of the cells with digitonin, Ca2+ was actively sequestered into the ER. Once a steady-state [Ca2+] was reached, we monitored the increase in [Ca2+] in the cuvette upon addition of TG. The total Ca2+ store content was estimated by the application of the Ca2+ ionophore A23187. To directly compare the Ca2+-leak properties in IP3RKO cells and (Δ1–1891)IP3R1 cells, we measured the Ca2+-leak rate (d[Ca2+]/dt) as a function of time (Figure 1B). The rate of Ca2+ leak was significantly higher in (Δ1–1891)IP3R1 compared with IP3RKO cells during a large part of the release process (P<0.05). Hence, these data indicate that expression of (Δ1–1891)IP3R1 enhances the Ca2+ leak from the ER in response to TG. (Δ1–1891)IP3R1 therefore represents a leaky channel. Furthermore, the A23187-releasable Ca2+ was not significantly affected by the presence of the truncated IP3R1. This finding indicates, in contrast with previous observations (Nakayama et al., 2004), that the presence of (Δ1–1891)IP3R1 was not sufficient to reduce or deplete the total amount of stored Ca2+. This observation was confirmed in intact DT40 cells using the ratiometric Ca2+ probe fura 2/AM (fura 2 acetoxymethyl ester), where no significant differences in TG-releasable store content were detected (Figure 1C).
Altered kinetics of ER Ca2+ uptake and release in transiently transfected cells
Since stable cell lines might have developed compensatory mechanisms for a reduced store content, we determined the free [Ca2+]ER (Ca2+ concentration in the ER) in HEK-293 cells (human embryonic kidney cells) transiently transfected with the truncated IP3R1 channel. HEK-293 were chosen as they express low levels of endogenous IP3R1 and have a high efficiency of transfection. The cells were co-transfected with the bioluminescent protein erAEQ (ER-targeted aequorin) and an empty vector as a control or with erAEQ and the truncated (Δ1–1891)IP3R1. Co-transfection conditions, using a cDNA ratio for (Δ1–1891)IP3R1/erAEQ of 3:1, were checked by immunocytochemistry using an antibody against the HA (haemagglutinin) tag of the erAEQ (Brini et al., 1995), and also an antibody against the C-terminal part of IP3R1 (Rbt04) to detect the truncated channel. More than 90% of the erAEQ-expressing cells also expressed the truncated channel (data not shown). AEQ reconstitution was carried out in Ca2+-depleting conditions. Ca2+ uptake in the ER was measured by perfusion of the cells with a KRB (Krebs—Ringer Buffer) supplemented with 1 mM CaCl2 (Figure 2A). Average curves (n=9–10) representing uptake of Ca2+ into the ER were plotted as [Ca2+]ER against time. Expression of the C-terminal fragment in HEK-293 cells significantly decreased the initial rate of Ca2+ uptake into the ER (P<0.05) as compared with control cells transfected with an empty vector (Figure 2B). However, steady-state [Ca2+]ER values were slightly, but not significantly, lower in the cells transfected with the truncated channel (163±15 μM, n=10, compared with 183±21 μM, n=9, in control conditions) (Figure 2A), indicating that the stores could be adequately refilled, even in the presence of the truncated channel.
Alternatively, using cytAEQ (cytosol-targeted AEQ), we observed a significantly faster increase in [Ca2+]i upon application of TG (1 μM) in (Δ1–1891)IP3R1-expressing HEK-293 cells (Figure 3A). During the first 30 s, the rate of TG-induced Ca2+ release was significantly higher (P<0.05) than in control cells (Figure 3B), indicating an enhanced Ca2+ leak from the ER. In addition, the small, but not significant, difference in maximal amplitude between (Δ1–1891)IP3R1-expressing cells and control cells (1.62±0.05 μM, n=18, compared with 1.35±0.14 μM, n=16, in control cells), may be caused by an enhanced store-operated Ca2+ influx, which is known to be stimulated by an enhanced Ca2+ release from the ER.
Taken together, these data establish that the ER Ca2+ store was not fully depleted upon expression of the caspase 3-cleaved IP3R1 C-terminus.
IP3R1 truncation in apoptotic cells
We have shown previously that a truncated IP3R1 channel is formed in IP3RKO cells expressing full-length IP3R1 after treatment with STS or anti-IgM to activate apoptotic pathways (Assefa et al., 2004). We now further investigated this phenomenon in other cell types to elucidate whether the proteolytic cleavage of IP3R1 is of more general importance. Expression levels of IP3R1 and its caspase 3-cleavage product were assayed in SHSY-5Y, HeLa, L15 and Lvec cells by Western-blot analysis (Figure 4A). In all cell types, we observed high levels of the 95-kDa C-terminal fragment after treatment with 1 μM STS for 8 h. As shown in Figure 4(B), there was a concomitant increase in caspase 3 activity upon STS treatment. It was remarkable that the increase in caspase 3 activity was much higher in L15 cells that have a very high [approx. 8-fold compared with control Lvec cells (Mackrill et al., 1996)] overexpression of IP3R1. These data reveal that IP3R1 cleavage by caspase 3 during apoptosis occurred both in cells overexpressing this type of receptor (L15 cells), as well as in cells that endogenously expressed IP3R1 at various levels (HeLa, Lvec and SHSY-5Y cells) (Wojcikiewicz, 1995; Missiaen et al., 2004; Nadif Kasri et al., 2005).
(Δ1–1891)IP3R1 is present in aged mouse MII (second meiotic division)-stage eggs and leads to perturbed Ca2+ signals
Apoptosis is a process that is often linked with neurodegeneration and aging. Hence, we investigated if (Δ1–1891)IP3R1 formation plays a role in the well-defined conditions of oocyte aging. Indeed, in crude lysates from aged mouse MII-stage eggs [34 h post-hCG (human chorionic gonadotropin)] we detected a 95-kDa fragment that was absent in lysates from fresh MII-stage eggs (14 h post-hCG), as assessed by Western blotting (Figure 5A). To investigate further the effect of a truncated IP3R1 with respect to Ca2+ signalling and apoptosis, (Δ1–1891)IP3R1 mRNA (1 μg/μl) was injected into fresh MII-stage eggs. Expression level of the truncated channel was verified on Western blot (Figure 5B). These eggs displayed an increased fragmentation rate 6 h after (Δ1–1891)IP3R1 mRNA injection (Figures 5C and 5D), a phenomenon that resembles apoptotic blebbing. Ca2+ oscillations in response to sperm factor (0.5 μg/μl) were measured with the ratiometric fluorescent Ca2+ probe fura 2/AM, and plotted as ratios of the fluorescence intensity at 340 nm/380 nm wavelengths as a function of time (Figures 5E and 5F). In contrast to control fresh MII-stage eggs (Figure 5E), eggs expressing (Δ1–1891)IP3R1 failed to establish persistent oscillatory responses (Figure 5F). Furthermore, the TG-induced Ca2+ leak into the cytosol in the absence of extracellular Ca2+ was significantly decreased (Figure 5G). The Ca2+-store content thus was decreased, but not completely depleted. Together, these data confirm that the formation of the truncated IP3R1 represents a leaky Ca2+ channel that disturbs cellular Ca2+ signalling.
In the present study we have performed a detailed characterization of the properties of the caspase-3-truncated IP3R1. On the basis of data obtained from different expression systems [stable transfection of DT40 cells lacking all three IP3Rs, transient transfection, mRNA injection of (Δ1–1891)IP3R1 in oocytes], different techniques (fluo 3 in permeabilized cells; erAEQ, cytAEQ and fura 2 in intact cells) and several cell types, we conclude that the truncated IP3R1 creates a modest leak, but, by itself, does not lead to an empty ER Ca2+ store. These data are in contrast with the study by Nakayama et al. (2004), in which it was shown that overexpression of an EGFP (enhanced green fluorescent protein)-tagged form of the caspase-3-truncated IP3R1 in fura-2-loaded COS and HeLa cells almost completely depleted ER Ca2+ stores, as no increase in [Ca2+]i was observed in response to TG. The latter findings are also not compatible with our previous study (Assefa et al., 2004), in which a stable cell line expressing the truncated IP3R showed normal store filling and where an additional apoptotic trigger (STS) was required to induce an increased Ca2+ response over a prolonged time period. The reason for these discrepancies is not clear.
We experienced technical difficulties with the use of EGFP in combination with an UV-excitable Ca2+ indicator, such as fura 2. Since EGFP and fura 2 have partially overlapping spectral properties, as documented by Bolsover et al. (2001), we observed a significant decrease of the fura 2 ratio signal as a function of EGFP expression in COS cells, as well as in HEK-293 cells (data not shown). We therefore choose to evaluate the role of the (Δ1–1891)IP3R1 expression on Ca2+ signalling in HEK-293 cells co-transfected with the Ca2+-sensitive bioluminescent protein AEQ. This technique avoids the possible fluorescence artifacts and, furthermore, also allows us to study [Ca2+]i as well as [Ca2+]ER changes. Using erAEQ, we found that the steady-state [Ca2+]ER was only slightly, but not significantly, decreased in cells expressing the truncated channel. Nonetheless, the lower initial rate of Ca2+ refilling clearly indicated the presence of a significant additional Ca2+-leak pathway. Hence, in transiently transfected HEK-293 cells, the active Ca2+ pumps could, to a large extent, counteract the Ca2+ leak through the truncated channel. These data are entirely consistent with our observations using cytAEQ, where inhibition of the Ca2+ pumps with TG revealed a significantly faster Ca2+ leak into the cytosol in cells co-transfected with (Δ1–1891)IP3R1. Similar effects were observed for the TG-induced Ca2+ leak in cell suspensions of a DT40 IP3RKO cell line stably expressing the (Δ1–1891)IP3R1.
Our findings of only small effects on ER Ca2+ content in cells that have undergone caspase cleavage of IP3R1 or that overexpress a truncated version of IP3R1 are compatible with several studies. For example, Hirota et al. (1999) reported previously that a significant reduction in IP3 (inositol 1,4,5-trisphosphate)-induced Ca2+-release activity required caspase-3-mediated cleavage of at least 80% of the IP3R1. As shown in Figure 4(A), and as described in Assefa et al. (2004), STS treatment of different cell types did not yield more than 35% of truncated channels in endogenously IP3R1-expressing cells and up to 60% in IP3R1-overexpressing cells. This finding indicates that, even under apoptotic conditions, it is likely that the intact IP3R1 is more abundant than the 95-kDa channel and that the Ca2+-leak properties of the truncated channel are not preponderant. In addition, in most cell types, IP3R2 and IP3R3 could also contribute to normal IP3-induced Ca2+-release function. Furthermore, it has been shown that IP3Rs can exist as heterotetramers (Monkawa et al., 1995). Since the transmembrane regions and the C-terminal part of the IP3R are the main determinants for dimerization (Boehning and Joseph, 2000; Galvan and Mignery, 2002), it is conceivable that heterotetramers are formed containing one or more truncated IP3R1 monomers. The regulatory suppressor/coupling N-terminal domains of the other intact subunits, whether from isoform 1, 2 or 3, could then still control the opening of the channel (Devogelaere et al., 2007). Collectively, our results support the view that the insertion of a caspase-3-truncated C-terminus of IP3R1 could introduce a constitutive (i.e. IP3-independent), albeit modest, leak from the ER. Conceivably, given the previous findings demonstrating that a limited elevation of mitochondrial [Ca2+] activates ATP production which enhances Ca2+-pump activity (Visch et al., 2006; Giacomello et al., 2007), the presumed decrease in the Ca2+-store content may be compensated by this activation. This presumption is confirmed by findings that cytosolic ATP levels were increased for up to 6 h after STS treatment. Longer incubation times with STS are needed before ATP levels drop below original levels, which coincides with the massive activation of caspase 3 and reduction of cell viability (Zamaraeva et al., 2005). This may explain why the effects of expression of the C-terminal fragment on intracellular Ca2+ handling and cell death were only observed after prolonged incubation with STS (Assefa et al., 2004). Apparently, an additional trigger is required, creating a double-hit mechanism to increase the [Ca2+]i, leading to cell death. The feed-forward effect exerted by the truncated IP3R1 was particularly marked in L15 cells, probably because of the high level of IP3R1 in these cells. Hence, the fact that the caspase-3-truncated C-terminus of IP3R1 is a constitutively open channel should be interpreted as an IP3-independent Ca2+-leak pathway that under normal conditions is largely compensated by an enhanced SERCA-mediated Ca2+-uptake activity, but that in situations in which the cellular metabolism is compromised may lead to the execution of the apoptotic cascade.
The physiological relevance of our observations was demonstrated in aging mouse oocytes. Aged oocytes endogenously expressed higher levels of the IP3R1 C-terminal 95-kDa fragment. Strikingly, injection of (Δ1–1891)IP3R1 mRNA in fresh mouse oocytes prematurely terminated sperm factor-induced Ca2+ oscillations, decreased the TG-releasable Ca2+-store content and induced morphological changes that resemble apoptotic blebbing. In aged oocytes, the presence of the leaky C-terminal 95-kDa IP3R1 fragment, in combination with the decreased SERCA activity as a result of the decreased intracellular [ATP] (Takahashi et al., 2000; Gordo et al., 2002; Igarashi et al., 2005), may lead to the observed disturbed Ca2+ homoeostasis (Fissore et al., 2002; Gordo et al., 2002), with fertilization often resulting in the activation of caspases and fragmentation, rather than the normal event of oocyte activation and embryo development (Gordo et al., 2002).
There is as yet no consensus about the exact mechanisms by which Ca2+ signals and particularly IP3-independent Ca2+ signals contribute to the cell-death pathways (Rizzuto et al., 2003; Joseph and Hajnoczky, 2007). On the basis of our findings, we hypothesize that caspase-3-mediated truncation of IP3R1 creates a modest Ca2+ leak from the ER. This Ca2+ leak becomes an important part of the cell-death programme, if accompanied by a decrease in the activity of ATP-dependent Ca2+-removal mechanisms. These changes will result in depletion of the ER Ca2+ store that evokes a persistent increase in store-operated Ca2+ entry, leading to a prolonged cellular Ca2+ overload that diverts the normal cellular functions towards cell death. Caspase-3-mediated cleavage of IP3R1 can thereby act as a feed-forward mechanism in the execution of the apoptotic process.
Materials and methods
The polyclonal antibody (Rbt04) raised against amino acids 2735–2749 of mouse IP3R1 has been described previously (Parys et al., 1995). STS, TG and A23187 were purchased from Sigma.
Cells and culture conditions
IP3R1-overexpressing L15 and control Lvec fibroblasts were a gift from Dr K. Mikoshiba (Division of Molecular Neurobiology, University of Tokyo, Tokyo, Japan). DT40 chicken B-lymphocytes lacking all three IP3Rs (IP3RKO) were a gift from Dr T. Kurosaki (Laboratory for Lymphocyte Differentiation, RIKEN Research Center for Allergy and Immunology, Kanagawa, Japan). IP3RKO cells were stably transfected with pcDNA(+)-IP3R1 (wild-type IP3R1) or pcDNA(+)-(Δ1–1891)IP3R1 [(Δ1–1891)IP3R1], as described previously (Assefa et al., 2004). DT40-derived cells were maintained in RPMI 1640 medium (Gibco) containing 10% (v/v) FCS (fetal calf serum) (Sigma), 1% (v/v) chicken serum (Sigma), 50 μM 2-mercaptoethanol, 85 units/ml penicillin, 85 μg/ml streptomycin and 3.5 mM L-alanyl-L-glutamine (Glutamax™, Gibco) in a humidified incubator at 5% CO2 and 37°C. The culture medium of the transfected IP3RKO cells was supplemented with 1.5 mg/ml G418 for selection.
HEK-293, HeLa, Lvec and L15 cells were grown in DMEM (Dulbecco's modified Eagle's medium) (Gibco) containing 10% (v/v) FCS, 4 mM Glutamax™, 100 units/ml penicillin, 100 μg/ml streptomycin and MEM (minimal essential medium) with NEAAs (non-essential amino acids) (Gibco) in a humidified incubator at 10% (v/v) CO2 and 37°C. For L15 cells this medium was supplemented with G418 (400 μg/ml). SHSY-5Y cells were grown in DMEM/Ham's F12 medium containing 15% FCS, 4 mM Glutamax™, 100 units/ml penicillin, 100 μg/ml streptomycin and MEM with NEAAs.
[Ca2+] measurements in DT40 cell suspensions
Cell pellets were resuspended in intracellular medium (120 mM KCl, 30 mM Hepes, pH 7.4, and 1 mM MgCl2) in the presence of 1 mM ATP, 25 mM phosphocreatine, 50 units/ml creatine kinase and 5 μM fluo 3 (Molecular Probes) and transferred into a 4 ml fluorescence quartz cuvette that was thermostatically maintained at 37°C. Cell density was 5×107 cells/ml.
Mild treatment of the cells with digitonin (50 μM) disrupted the plasma membrane. The fluo 3 fluorescence (λexcitation=503 nm and λemission=530 nm) was measured with an Aminco-Bowman® Series 2 spectrometer (Spectronic Unicam). A23187 (8 μM) was added at the end of each experiment to measure the total releasable Ca2+. The fluorescence signal (F) was calibrated by addition of 0.5 mM CaCl2 (Fmax) followed by 5 mM EGTA (Fmin). The free [Ca2+] was calculated using the following equation: [Ca2+]=Kd×(F−Fmin)/(Fmax−F) (Grynkiewicz et al., 1985), with a Kd value of 864 nM, as determined in cytosol-like medium at 37°C (Missiaen et al., 1991). Ca2+-leak rate (d[Ca2+]/dt) was determined by adding TG (4 μM).
Free [Ca2+]i was also measured in intact cells (1.5×107 cells/ml) loaded with fura 2/AM (Teflabs). After de-esterification, cells were resuspended in modified KRB (135 mM NaCl, 5.9 mM KCl, 1.2 mM MgCl2, 11.6 mM Hepes, pH 7.3, and 11.5 mM glucose) containing 1.5 mM CaCl2. The [Ca2+]i was monitored at excitation wavelengths of 340 and 380 nm and emission wavelength of 510 nm.
The constructs for erAEQ and cytAEQ were provide by Dr R. Rizzuto (Department of Experimental and Diagnostic Medicine, University of Ferrara, Ferrara, Italy). HEK-293 cells, seeded on to 13 mm (diameter) gelatine-coated coverslips, were transfected with the constructs using the GeneJuice® transfection reagent (Novagen). Experiments were performed 2 days after transfection with a confluent cell layer at 37°C.
Prior to measuring erAEQ signals, the culture medium was replaced with KRB (135 mM NaCl, 5 mM KCl, 1 mM MgSO4, 20 mM KH2PO4, 20 mM Hepes and 5.5 mM glucose, pH 7.4) containing 600 μM EGTA. Cells were then incubated for 1 h with 5 μM ionomycin (Sigma) for Ca2+ depletion, and with 5 μM coelenterazine-n (Molecular Probes) for reconstitution of the active AEQ at 4°C. The cells were then washed extensively with KRB supplemented with 2% BSA (Sigma) and 1 mM EGTA. After superfusion of the cells with KRB supplemented with 100 μM EGTA, the stores were loaded by perfusion with a KRB solution containing 1 mM CaCl2. Finally, digitonin (100 μM in water containing 10 mM CaCl2) was added to lyse the cells and to take up the remaining AEQ.
Reconstitution of cytAEQ with coelenterazine-w (Molecular Probes) was performed in culture medium for 1 h at 37°C. The cells were washed with KRB containing 1 mM CaCl2. After a 2 min perfusion with the same solution, TG-induced Ca2+ release was measured (1 μM TG in KRB supplemented with 1 mM CaCl2). Calibration was performed in a similar manner to erAEQ measurements.
The light signal was collected by a low-noise photomultiplier tube with a built-in amplifier—discriminator, and conversion into [Ca2+] values was carried out as described previously (Brini et al., 1995; Rizzuto et al., 1995).
Preparation of cell lysates
Suspension cells were harvested (400 g for 5 min at 4°C) and lysed in RIPA buffer (25 mM Hepes, pH 7.5, 0.3 mM NaCl, 1.5 mM MgCl2, 0.5 mM dithiothreitol, 20 mM β-glycerolphosphate, 10% glycerol, 1 mM Na3VO4, 1% Triton X-100, 1 mM PMSF, 20 μg/ml aprotinin and 20 μg/ml leupeptin). After 15 min of incubation on ice, the lysates were cleared by centrifugation (400 g for 15 min at 4°C). Adherent cells were first scraped with a rubber policeman into ice-cold PBS before lysis with RIPA. Protein concentrations were determined according to Lowry et al. (1951).
Whole-cell lysates (25 μg) from the various cell types were subjected to electrophoresis on Tris/acetate SDS polyacrylamide gradient gels (3–8% gel) (Invitrogen), transferred on to a PVDF membrane. The blot was blocked with PBS containing 0.1% Tween 20 and 5% skimmed dried milk and subsequently incubated with the primary antibody Rbt04 against the C-terminus of IP3R1 (dilution, 1:3000). Detection of the immunoreactive bands was performed by exposure to Hyperfilm ECL (GE healthcare) using a horseradish-peroxidase-conjugated secondary antibody (dilution, 1:2000) and ECL substrate (GE healthcare). The Hyperfilm was developed using an X-Omat 1000 (Kodak).
Evaluation of caspase activity
Caspase 3 activity in STS-treated cells was measured in whole-cell lysates using a colorimetric CaspACE™ Assay System (Promega). Briefly, 50 μg of protein was incubated with 200 μM Ac-DEVD-p-nitroaniline (N-acetyl-Asp-Glu-Val-Asp-p-nitroaniline; caspase 3 substrate) for 4 h at 37°C. A405 was measured using a 96-well plate reader (Corning Incorporated).
Recovery of eggs and culture conditions
MII-stage eggs were collected from the oviducts of 6- to 8-week-old CD-1 female mice. Females were superstimulated with an injection of 5 units of PMSG (pregnant mare serum gonadotropin; Sigma). In vivo MII-stage eggs were recovered in TL-Hepes (Hepes-buffered tyrode/lactate solution), supplemented with 5% heat-treated FCS (Gibco), 12–14 h after injection of 5 units of hCG, which was administered 45–48 h after PMSG stimulation. After the removal of cumulus cells, eggs were transferred into 50 μl drops of KSOM (potassium simplex optimized medium; Speciality Media) and cultured under paraffin oil at 36.5°C in a humidified atmosphere containing 6% CO2.
Preparation of mouse MII-stage egg crude lysates and Western blotting
Cell lysates from 50 MII-stage eggs were mixed with 15 μl of 2×Laemmli sample buffer, boiled and loaded on to NuPAGE Novex Tris/acetate 3–8% gels (Invitrogen). After electrophoresis, proteins were transferred on to nitrocellulose membranes (Micron Separations) and blocked with PBS containing 0.05% Tween 20 supplemented with 6% skimmed dried milk and incubated overnight with Rbt04 antibody (dilution, 1:3000). The membranes were subsequently washed and incubated for 1 h with a goat anti-rabbit secondary antibody coupled to horseradish peroxidase. Membranes were washed and incubated for 1 min in chemiluminescence reagent (NEN Life Science Products) and developed according to the manufacturer's instructions. Each nitrocellulose membrane was digitally captured and quantified using an Imaging Station 440 CF (Kodak).
Fluorescence recordings and [Ca2+]i determinations in mouse MII-stage eggs
Measurements of [Ca2+]i were performed using fura 2/AM (Molecular Probes) as reported previously (Kurokawa et al., 2005). Eggs were monitored in drops of TL-HEPES under mineral oil. Up to 10 eggs were monitored simultaneously using the software SimplePCI (C-Imaging System), which controls a high-speed filter wheel rotating between excitation wavelengths of 340 and 380 nm. Illumination was provided by a 75 W xenon arc lamp, and the light emitted above 510 nm was collected by a cooled Photometrics SenSys CCD (charge-coupled-device) camera (Roper Scientific). Fluorescence ratios of 340/380 nm were obtained every 20–30 s.
The results are expressed as means±S.E.M. Individual groups were compared by Student's unpaired t test. Statistics was performed with Origin 7.0 software (OriginLab Corporation).
We are grateful for excellent technical assistance from Tomas Luyten, Irène Willems and Marina Crabbé, and fruitful discussions with Benoit Devogelaere, Dr Nael Nadif Kasri (Leuven, Belgium) and Dr Fabrizio de Mattia (Nijmegen, The Netherlands). This work was supported by grants G.0604.07 and G.0210.03 from the Fund for Scientific Research—Flanders (F.W.O. – Vlaanderen to J.B.P. and H.D.S.), grant G.O.A. 2004/07 from the Concerted Actions of the K.U. Leuven (to J.B.P., L.M., G.C. and H.D.S.), grant P6/28 (to J.B.P., L.M. and H.D.S.) of the Belgian State (Belgian Science Policy), and grants from the USDA (United States Department of Agriculture) and the NIH/NICHD (National Institutes of Health/National Institute of Child Health and Human Development) (to R.A.F.). G.B. is a postdoctoral fellow of the Fund for Scientific Research—Flanders (F.W.O. – Vlaanderen).