ABSTRACT
CaMKII is a crucial oligomeric enzyme in neuronal and cardiac signaling, fertilization and immunity. Here, we report the construction of a novel, substrate-based, genetically-encoded sensor for CaMKII activity, FRESCA (FRET-based Sensor for CaMKII Activity). Currently, there is one biosensor for CaMKII activity, Camui, which contains CaMKII. FRESCA allows us to measure all endogenous CaMKII variants, while Camui can track a single variant. Since there are ~40 CaMKII variants, using FRESCA to measure aggregate activity allows a fresh perspective on CaMKII activity. We show, using live-cell imaging, FRESCA response is concurrent with Ca2+ rises in HEK293T cells and mouse eggs. In eggs, we stimulate oscillatory patterns of Ca2+ and observe the differential responses of FRESCA and Camui. Our results implicate an important role for the variable linker region in CaMKII, which tunes its activation. FRESCA will be a transformative tool for studies in neurons, cardiomyocytes and other CaMKII-containing cells.
INTRODUCTION
Calcium-calmodulin dependent protein kinase II (CaMKII) is a serine/threonine kinase that plays critical signaling roles in multiple mammalian tissues (Backs et al., 2010; Rokita & Anderson, 2012; Shonesy, Jalan-Sakrikar, Cavener, & Colbran, 2014) and is implicated in a number of diseases (Mollova, Katus, & Backs, 2015; Robison, 2014; Steinkellner et al., 2012; Tu, Okamoto, Lipton, & Xu, 2014). CaMKII plays a key role in all electrically coupled cells, such as neurons and cardiomyocytes, and even cells that are not – such as lymphocytes and eggs – all of which communicate using Ca2+. Depending on the stimulus, the Ca2+ response leads is either a single Ca2+ rise or a more complex responses such as oscillations (Cuthbertson, Whittingham, & Cobbold, 1981; Eisner, Caldwell, Kistamas, & Trafford, 2017; Rutecki, 1992; Swann & Lai, 2013). Absence of Ca2+ signals causes severe defects in cell functionality, such as memory deficits in the case of neurons (Herring & Nicoll, 2016), or in the case of fertilization, failure to conceive (Escoffier et al., 2016; Yoon et al., 2008). CaMKII is responsible for reacting to Ca2+ oscillations and transducing this signal to downstream molecules. Indeed, it has been shown that neuronal CaMKII has a threshold frequency for activation (Chao et al., 2011; De Koninck & Schulman, 1998).
CaMKII has a unique oligomeric structure among the protein kinase family (Fig. 1A). Each subunit of CaMKII is comprised of a kinase domain, regulatory segment, variable linker region, and hub domain (Fig. 1B). The hub domain is responsible for oligomerization, which organizes into two stacked hexameric (or heptameric) rings to form a dodecameric (or tetradecameric) holoenzyme (Bhattacharyya et al., 2016; Chao et al., 2011; Rosenberg et al., 2006). In the absence of Ca2+, the regulatory segment binds to and blocks the substrate-binding pocket. Ca2+/calmodulin (Ca2+/CaM) turns CaMKII on by competitively binding the regulatory segment and exposing the substrate-binding pocket (Fig. 1C).
It has been demonstrated that CaMKII has a threshold frequency for activation (Chao et al., 2011; De Koninck & Schulman, 1998). There are four human CaMKII genes; CaMKIIα and β are predominantly expressed in neurons, CaMKIIδ is predominantly expressed in the heart and CaMKIIγ is found in multiple organ systems, including the reproductive organs. The kinase and hub domains of all four genes are highly conserved (~90% on average), however, the linker domain connecting the kinase and hub domains is variable in length and composition. Details elucidating the importance of the variable linker region remain to be uncovered, but there are >30 different splice variants of each of the four genes, which mostly vary in the linker region only. It has been shown that CaMKII activity is tuned by the length of the variable linker in vitro (Bayer, De Koninck, & Schulman, 2002; Chao et al., 2011). Specifically, as the variable linker is lengthened, less Ca2+ is needed for activation (i.e., activation of CaMKII is easier). Thus, it is important for us to consider the complexity of endogenous CaMKII expressed in various cell types.
Camui is currently the only biosensor for CaMKII activity (Takao et al., 2005). Camui is a Förster resonance energy transfer (FRET)-based biosensor for CaMKII activity, which exploits the conformational change that CaMKII undergoes when it binds to Ca2+/CaM (Fig. 2A). To date, Camui has been a very useful tool to study and understand CaMKII activity in various cell types (mainly neurons and cardiomyocytes) and under various conditions (Erickson, Patel, Ferguson, Bossuyt, & Bers, 2011; Kwok et al., 2008; Takao et al., 2005). However, a major limitation is that the Camui sensor is constructed of a CaMKII variant itself, and thus will only report on this particular variant. To enhance our understanding of this complex protein, we need a way to measure endogenous CaMKII activity. One option is to re-engineer Camui with the appropriate CaMKII isoform to be studied, however this becomes limiting when there are multiple isoforms expressed in a single cell type, such as during the development of the female gamete, the egg. We now report the development of a novel biosensor that detects endogenous CaMKII activity. Herein, we show the efficacy of this new sensor in mouse eggs.
RESULTS AND DISCUSSION
Development of a novel biosensor for endogenous CaMKII activity
We developed a novel substrate-based sensor for CaMKII activity, FRESCA (FRET based Sensor for CaMKII Activity, Fig. 2A). We monitored CaMKII activity using FRESCA in real-time following the induction of Ca2+ responses to several agonists that are capable of initiating egg activation and embryogenesis. Building on a previous design for an Aurora kinase biosensor (Liu, Vader, Vromans, Lampson, & Lens, 2009), we replaced the sequence encoding the Aurora kinase substrate for the CaMKII substrate (syntide). The design also employs FHA2, a phosphate-binding domain, to facilitate a conformational change once the adjacent CaMKII substrate is phosphorylated (Durocher et al., 2000). FHA2 will bind to this phosphorylated Thr residue and produce a decrease in FRET between the terminal CFP/YFP pair.
Measuring the FRESCA response in HEK293T cells
We first tested the selectivity of FRESCA in HEK293T cells, which express negligible levels of CaMKII. We transfected HEK293T cells with either (i) CaMKII, calmodulin and FRESCA, or (ii) calmodulin and FRESCA. Ionomycin was added to the HEK293T cells to induce Ca2+ release and simultaneously monitored FRET (CFP/YFP ratio). We observed that with CaMKII present, the addition of ionomycin causes a reduction in FRET, indicating that CaMKII is active and phosphorylating FRESCA (Fig. 2B, red lines). Importantly, we did not observe a FRET change when CaMKII was not co-transfected, demonstrating that FRESCA is selective for CaMKII and not being phosphorylated by other HEK cell kinases (Fig. 2B, blue lines).
Using FRESCA to monitor CaMKII activity in mouse eggs
HEK293T cells provided a good model for a highly controlled evaluation of CaMKII activity and the ability of FRESCA to specifically report on CaMKII in the presence of other cellular kinases. However, we wanted to test FRESCA in a more complex and native system, importantly, with endogenous CaMKII and where the Ca2+ response has a clear physiological function. To accomplish this, we expressed FRESCA in mouse eggs to measure endogenous CaMKIIγ activity following increases in intracellular Ca2+ induced by a variety of agonists.
In all mammals, Ca2+ oscillations are required for initiation of embryogenesis (Deguchi, Shirakawa, Oda, Mohri, & Miyazaki, 2000; Fissore, Dobrinsky, Balise, Duby, & Robl, 1992). Mammalian eggs are arrested at metaphase II of meiosis; both Ca2+ oscillations and consequent CaMKII activity are required for release from this arrest (Fig. 1D) (Backs et al., 2010; Chang, Minahan, Merriman, & Jones, 2009; Miao, Stein, Jefferson, Padilla-Banks, & Williams, 2012; Miyazaki et al., 1992; Presler et al., 2017). Ca2+ oscillations are induced following gamete fusion when the sperm releases into the egg a sperm specific protein (PLCzeta; ζ), which triggers the Ca2+ responses (Ducibella et al., 2002; Saunders et al., 2002). On average, there is one Ca2+ rise every 20 minutes and oscillations in mouse zygotes last for ~4 hours, which coincides with the formation of the pronuclei (PN) (Jones, Carroll, Merriman, Whittingham, & Kono, 1995). The source of this Ca2+ is from internal stores, which are replenished by Ca2+ influx from the extracellular media. CaMKII is activated simultaneously with the initiation of Ca2+ oscillations and female mice that are CaMKIIγ null are sterile (Backs et al., 2009). Despite the role of CaMKIIγ in the initiation of development, the complete profile of CaMKII activity during fertilization in mammals is not known. Further, CaMKII activity also seems to play a role in preventing apoptosis in Xenopus and mouse eggs, although the pattern and degree of activation for this activity are even less studied (Nutt et al., 2005).
To date, CaMKII activity has only been assessed based on a few Ca2+ rises using in vitro kinase assays and during only the first hour of oscillations, which is considerably shorter than the time scale for normal oscillations in the mouse. Therefore, there is a need to monitor CaMKII activity in live cells and for an extended time, which is what we address here.
FRESCA and ionomycin-induced Ca2+ oscillations in mouse eggs
FRESCA expression and distribution in germinal vesicle (GV) oocytes and MII stage oocytes, henceforth referred to as eggs, was widespread and cytoplasmic (Fig. 2C). However, a small amount of FRESCA appeared to enter the nucleus of GV oocytes (Fig. 2, supplement 1).
Given the immediate and large Ca2+ rise caused by the addition of ionomycin, we first tested FRESCA responses in eggs using this ionophore. We analyzed the effect of 3 concentrations of ionomycin: 0.5 μM, 2.5 μM and 5 μM. Upon addition of ionomycin to eggs expressing FRESCA, we observed a FRET decrease, indicating CaMKII activity (Fig. 2D-I). At the lowest ionomycin concentration (0.5 μM), CaMKII activity appears to perfectly track the Ca2+ pulse (Fig. 2D, E). Conversely, at higher ionomycin concentrations, CaMKII activity is unstable during the duration of the Ca2+ pulse, although higher concentrations appeared to prolong and increase the FRET response of FRESCA (Fig. 2, supplement 2). The time to FRET peak was faster with addition of higher ionomycin concentrations (Fig. 2, supplement 2).
We tested the specificity of FRESCA for CaMKII in mouse eggs. We first used CaMKII inhibitors, which should eliminate the FRET response if CaMKII is the only kinase phosphorylating FRESCA in eggs (Fig. 2, supplement 3). We show that the addition of KN93, a commonly used allosteric inhibitor for CaMKII, significantly reduces FRET (Madgwick, Levasseur, & Jones, 2005; Smyth et al., 2002). Addition of AS105, an ATP competitive CaMKII specific inhibitor, also significantly reduces FRET, while its inactive analog (AS461) does not affect FRET (Neef et al., 2018). We also tested inhibition and activation of protein kinase C (PKC), since PKC is the other major Ca2+ sensitive kinase in mouse eggs (Medvedev, Stein, & Schultz, 2014; Wang et al., 2010). Addition of PKC inhibitors Bim1 (Halet, 2004) and GO6983 (Gou, Wang, Zou, Qi, & Xu, 2018) do not affect the FRESCA signal, indicating that PKC is not phosphorylating FRESCA. Eggs do not express conventional PKC isoforms, so the results of the broad-spectrum PKC inhibitor (GO6983) reinforced the results of Bim1. Finally, addition of PMA, a PKC activator shown to stimulate this enzyme in mouse eggs (Halet, 2004), also does not induce FRET (Fig. 2, supplement 3). Taken together, we report that FRESCA is a specific reporter of CaMKII activity in mouse eggs.
FRESCA and Sr2+-induced Ca2+ oscillations
Addition of 10 mM Sr2+ to the extracellular media in place of external Ca2+ is a common method of parthenogenetic activation in mouse eggs, and it induces highly consistent oscillations in these cells (Fig. 3, black lines); these oscillations initiate all events of egg activation (Bosmikich & Whittingham, 1995; Carvacho, Lee, Fissore, & Clapham, 2013; Kline & Kline, 1992). Further, the TRPV3 channel has recently been identified as the channel responsible for Sr2+ influx in mouse eggs (Carvacho et al., 2013). We therefore examined the response of endogenous CaMKII to Sr2+-induced oscillations. We observed endogenous CaMKIIγ activity (monitored by FRESCA) almost simultaneously with the initiation of oscillations. Indeed, nearly all eggs (11/13) showed CaMKII activity within the first two rises. Importantly, CaMKII activity is reproduced over time, as FRESCA continues to track each Ca2+ rise for >2 hours (Fig. 3B).
We propose a molecular model to describe this data. The Ca2+ rises progressively decrease in duration/amplitude over time (Deguchi et al., 2000), however, the FRESCA responses are not diminished and the peak kinase activity seems to outlast the peak elevation of Ca2+/Sr2+. This suggests autophosphorylation of CaMKII at Thr 286, which facilitates activation at subsequent Ca2+ pulses by increasing the affinity for Ca2+/CaM (see cartoons in Fig. 3A) (Meyer, Hanson, Stryer, & Schulman, 1992). Additionally, a prolonged time course of FRESCA response to Sr2+ indicates that FRESCA continues to faithfully track endogenous CaMKII up to 6 hours (Fig. 3, supplement 2).
Using Camui to measure CaMKII activity in mouse eggs
As described, the highly used Camui biosensor is comprised of CaMKII itself (see Fig. 4A), specifically CaMKIIα, which has a 30-residue variable linker region (Fig. 4C). Despite its widespread use, Camui has not yet been used to monitor CaMKII activity in mouse eggs. Given that it has been demonstrated that CaMKII activity is tuned by the length of the variable linker (Bayer et al., 2002; Chao et al., 2011), specifically, as the variable linker is lengthened, less Ca2+ is needed for activation, we hypothesized that FRESCA may report CaMKII activity in mouse eggs more faithfully than Camui. This assumption is based on the knowledge that mouse eggs express equimolar concentrations of the two versions of CaMKIIγ (γ3 and γJ), which have 69 and 90 residue variable linkers, respectively (Fig. 4C) (Hatch & Capco, 2001; Suzuki, Hara, Takagi, Yamamoto, & Ueno, 2011), considerably longer than the 30-residue linker of CaMKIIα. We therefore tested the response of Camui compared to FRESCA.
We expressed the Camui reporter in mouse eggs using mRNA injection, and similar to FRESCA, expression was robust within ~30 minutes and we began FRET measurements ~4 hours post injection to attain stable Camui levels. As shown by confocal microscopy, Camui attained a widespread cytoplasmic expression in eggs, although in GVs it was excluded from the nucleus, which is consistent with the reported expression of CaMKII in the cytosol of mouse eggs (Fig. 4B and Fig 4., supplement 1) (Hatch & Capco, 2001).
Camui and ionomycin-induced Ca2+ oscillations
We first induced Ca2+ release in eggs by adding ionomycin as previously described and simultaneously monitored changes in FRET values (YFP/CFP) (Fig. 4C-H). As with FRESCA, a decrease in FRET is indicative of an increase in CaMKII activity. It is clear that CaMKII activity increases (red line) coincident with the increase in Ca2+ (black line) in all conditions. The Ca2+ and Camui responses increased dose-dependently and approximately synchronously, as the large increase in the amount of Ca2+ release caused by increasing ionomycin from 0.5 μM to 2.5 μM, results in a 1.9-fold increase in CaMKII activity (mean amplitude of FRET change) (Fig. 4, supplement 2). Further increasing ionomycin from 2.5 μM to 5 μM produces nearly no change in total Ca2+ release, although it is very likely that the reporting range of Rhod-2 is saturated at these levels of Ca2+ release. The CaMKII activity also appears to remain constant, although this may also represent saturation of the FRET signal (Fig. 4, supplement 2). Notably, addition of 5 μM ionomycin results in a prolonged duration of activity compared to lower concentrations, but it is unclear whether this reflects the extended activation of the enzyme or cellular stress.
The absolute amplitude of the change in the FRET ratio for Camui after the addition of 0.5 μM ionomycin is ~10-fold greater than what is observed for FRESCA (0.014 for FRESCA compared to 0.14 for Camui), however, this measurable signal change in FRESCA is sufficient to monitor endogenous CaMKIIγ activity, which we cannot detect with Camui, which reports CaMKIIα activity. In addition, it is worth noting that the shape of the FRESCA traces is slightly different from those of Camui for the same stimulus. The peak activity of FRESCA is shorter than the corresponding Ca2+ peak, although the return to basal activity is more protracted, whereas the Camui response more perfectly tracks the shape of the Ca2+ peak.
Camui and Sr2+ induced Ca2+ oscillations
We next examined the Camui response to Sr2+-induced oscillations (Fig. 5). Remarkably, despite the presence of robust changes in intracellular Ca2+ levels, Camui (Fig. 5, red line) did not report any CaMKII activity until the ~6th significant Ca2+ rise (Fig. 5B, arrow and inset). In total, only 33% of the eggs expressing Camui showed activity in the first two rises, whereas nearly all FRESCA expressing eggs showed activity within the first two rises. These data suggest that the endogenous CaMKIIγ in eggs is more sensitive to Ca2+/CaM than CaMKIIα. This finding is in line with previous data showing that longer linker CaMKII splice variants (CaMKIIγ3 and CaMKIIγJ) are activated at lower concentrations of Ca2+/CaM than shorter linker variants (CaMKIIα) (see Fig. 4C) (Chao et al., 2011). Additionally, the delayed response seen in the Camui eggs could also be a result of endogenous CaMKII being activated first (lower EC50 for Ca2+/CaM) thereby competing with Camui for the available activating ligand.
Another distinctive feature of the Camui response caused by Sr2+ oscillations is that whereas the initial Camui responses were delayed, once they commenced, they displayed an integrated activation with each subsequent pulse. For example, we analyzed the mean amplitude for the first three observable FRET changes. From the first to the second FRET change, there was a 1.6-fold increase in CaMKII activity. From the second to the third FRET change, there was a negligible change, and these changed occurred while the amplitude of the Ca2+ peaks progressively decreased and/or remained unchanged (Fig. 5, supplement 1). These data indicate that CaMKII activity, once stimulated, is cooperative with each additional Ca2+ pulse. This result is consistent with previous data showing that CaMKII activity is highly cooperative in vitro (Chao et al., 2010; Chao et al., 2011). As depicted in Figure 5A, a potential explanation for this is phosphorylation at Thr286 which may persist even in the absence of elevated Ca2+. It has been clearly shown that CaMKII with Thr286 phosphorylated has a significantly higher affinity for Ca2+/CaM (Meyer et al., 1992). This would also explain why the FRET level does not return to baseline in between later Ca2+ oscillations (Fig. 5B, blue lines).
Measuring CaMKII activity under native fertilization conditions
In mammals, fertilization-associated Ca2+ oscillations are induced by the release of sperm’s PLCζ into the ooplasm (Saunders et al., 2002). We tested the response of both FRESCA and Camui in response to the expression of PLCζ.
FRESCA and PLCζ-induced Ca2+ oscillations
We accomplished PLCζ expression by injection of its mRNA into FRESCA expressing eggs, and thereafter began monitoring changes in FRESCA responses (Fig. 6A, B). The initiation of oscillations stimulated the early activity of the endogenous CaMKIIγ, and this activity was detected with each additional rise. Similar to Sr2+ induced oscillations, we observed a relative decrease in the amplitude of the Ca2+ pulses over time, yet the FRESCA response was largely maintained (Fig. 6, supplement 1). These observations are the longest evaluation of CaMKII activity following natural oscillations ever reported following fertilization, as previous studies only reported up to 60 min post-initiation of oscillations (Markoulaki, Matson, & Ducibella, 2004; Ozil et al., 2005).
Camui and PLCζ-induced Ca2+ oscillations
We next assessed how Camui would report CaMKII activity induced by Ca2+ oscillations, and compare the response to those induced by Sr2+. To do this, eggs expressing Camui were injected with PLCζ mRNA and Ca2+ and FRET responses were monitored. Similar to FRESCA, we observed that Ca2+ oscillations nearly immediately induced CaMKII activity as monitored by Camui (Fig. 6D, arrow and bottom inset). However, this initial activity was not detected in subsequent rises, and only the first and second (and to a less extent, third) Ca2+ rises induced Camui responses despite the presence of robust and frequent Ca2+oscillations (Fig. 6C, D, supplement 2). Additionally, it is worth pointing out that the area under the curve for the third Ca2+ rise in these experiments was significantly reduced. This may be due to the fact that Camui itself is contributing significantly to the existing CaMKII in the egg, and potentially altering Ca2+ dynamics. These results raised the possibility that Camui is not well suited to detect CaMKII activity initiated by sporadic and low magnitude Ca2+ rises, which are characteristic of mammalian fertilization. Regardless, it remains to be elucidated why Sr2+ induced oscillations are able to protractedly promote robust and persistent Camui responses whereas the Camui response to PLCζ-induced oscillations fades rapidly.
CONCLUDING REMARKS
It has been appreciated for decades that both Ca2+ oscillations and CaMKII activation in mouse eggs is crucial to fertilization and initiation of embryo development. Here, we provide an analysis of CaMKII activation in real-time in eggs using FRET-based CaMKII biosensors. Importantly, our new biosensor, FRESCA, allowed us to monitor endogenous CaMKII (CaMKIIγ3 and γJ) activation in real-time as a consequence of different activation stimuli (ionomycin, Sr2+, and PLCζ). The FRESCA response was noticeably different from the Camui sensor, which reports on CaMKIIα. When different Ca2+ oscillation patterns are induced, we observe subsequent differences in CaMKII activity. From our data, it is clear that both the (i) pattern of Ca2+ oscillations as well as the (ii) specific CaMKII isoform responding play a role in CaMKII activation.
The pivotal role of CaMKII activation in causing release of the meiotic arrest and activation of the embryonic developmental program in vertebrates was recently and more specifically evidenced by careful mass spectrometry experiments (Presler et al., 2017). This study showed that soon after fertilization, and temporally coinciding with the Ca2+ wave, there is a strong increase in protein phosphorylation that far outweighs the biochemical changes caused by protein degradation that accompanies fertilization. Remarkably, the study also found that 25% of the phosphorylated sites matched the minimal phosphorylation motif of CaMKII. It is therefore important to determine how Ca2+ rises turn on CaMKII activity, and what parameter(s) of individual rises within an oscillatory pattern are necessary for periodic and consistent stimulation of its activity. We propose that the magnitude of the initial activation of CaMKII depends on the magnitude of the stimulus and on internal regulation of CaMKII, which is largely based on the variable linker region. Knowing the minimal Ca2+ signal that increases the activity of CaMKIIγ is important as we seek to develop more physiological methods of parthenogenetic activation to treat some cases of infertility.
More broadly, now that we have demonstrated the utility of FRESCA in mouse eggs, this opens the door to measuring endogenous CaMKII activity in other cell types, such as neurons and cardiomyocytes. CaMKII activation has been heavily studied in vitro (Chao et al., 2010; Chao et al., 2011; Rosenberg, Deindl, Sung, Nairn, & Kuriyan, 2005), and it is intriguing to also consider the potential effects of subunit exchange in cellular conditions (Bhattacharyya et al., 2016; Stratton et al., 2013). It will be necessary to increase the signal to noise ratio of the FRESCA sensor in order to achieve a more robust signal for accurate quantification of kinetics and amplitudes. Once this is accomplished, we believe that FRESCA will provide new insights into CaMKII activity in cells and allow us to unravel the complexity of this unique protein kinase.
MATERIALS AND METHODS
Plasmid design
In order to accommodate the requirements for FHA2 binding (Durocher et al., 2000), syntide was modified from PLARTLSVAGLPGKK to PLARALTVAGLPGKK to create syntide-2. Syntide-2 was generated by annealing GATCCGGCGGCGCCGGCGGCGGCccgctggcgcgcgccctgaccgtggcgggcctgccgggcaaaaa aGGC and GGCCGCCttttttgcccggcaggcccgccacggtcagggcgcgcgccagcggGCCGCCGCCGGCGCCG CCG (IDT), which produced BamHI site at the 5’ end and a NotI site on the 3’ end. This product was phosphorylated (Ambion Pnk), purified (Thermo Fisher) and then ligated using T4 DNA ligase (Invitrogen) into a plasmid encoding the Aurora kinase FRET sensor (kind gift from Thomas Maresca). The final FRESCA sensor (with syntide-2 in place of the Aurora substrate) was cloned into pCDNA3.1.
HEK 293T Cell culture
All HEK293T cell cultures were grown in Dulbecco’s Modified Eagle’s Medium (Sigma) supplemented with 10% fetal bovine serum (Sigma) and maintained at 37°C and 5% carbon dioxide levels. The identity of these cells was authenticated by ATCC (CRL-3216 ATC 293T, Lot #63226319). Cells were transfected using Lipofectamine® 2000 Reagent (Invitrogen) and 150 ng of DNA constructs.
Collection of mouse eggs
Metaphase II (MII) eggs were collected from the oviducts of 6- to 10-week-old CD-1 female mice 12–14 h after administration of 5 IU of human chorionic gonadotropin (hCG), which was administered 46–48h after the injection of 5 IU of pregnant mare serum gonadotropin (PMSG; Sigma; Saint Louis, MO). Cumulus cells were removed with 0.1% bovine testes hyaluronidase (Sigma). MII eggs were placed in KSOM with amino acids (Millipore Sigma) under mineral oil at 37°C in a humidified atmosphere of 5% CO2 until the time of monitoring. All animal procedures were performed according to research animal protocols approved by the University of Massachusetts Institutional Animal Care and Use Committee.
Preparation of cRNAs and Microinjections
The sequences encoding Camui and FRESCA were subcloned into a pcDNA6 vector (pcDNA6/Myc-His B; Invitrogen, Carlsbad, CA) between the XhoI and PmeI restriction sites. Mouse PLCζ was a kind gift from Dr K. Fukami (Tokyo University of Pharmacy and Life Science, Japan) and subcloned into a PCS2+ vector, as previously described by us (Kurokawa et al., 2007). Plasmids were linearized with a restriction enzyme downstream of the insert to be transcribed and cDNAs were in vitro transcribed using the T7 or SP6 mMESSAGE mMACHINE Kit (Ambion, Austin, TX) according to the promoter present in the construct. A Poly (A)-tail was added to the mRNAs using a Tailing Kit (Ambion) and poly(A)-tailed RNAs were eluted with RNAase-free water and stored in aliquots at −80 °C. Microinjections were performed as described previously (Lee et al., 2016). cRNAs were centrifuged, and the top 1–2 μl was used to prepare micro drops from which glass micropipettes were loaded by aspiration. cRNA were delivered into eggs by pneumatic pressure (PLI-100 picoinjector, Harvard Apparatus, Cambridge, MA). Each egg received 5–10 pl, which is approximately 1–3% of the total volume of the egg. Injected MII eggs were allowed for translation up to 4h in KSOM. Group of eggs were injected with mouse PLCζ after 4h of FRET construct injection.
FRET and Calcium imaging
To estimate relative changes in the cytoplasmic activity of Camui and/or FRESCA, emission ratio imaging of the Camui and FRESCA (YFP/CFP) was performed using a CFP excitation filter, dichroic beam splitter, CFP and YFP emission filters (Chroma technology, Rockingham, VT; ET436/20X, 89007bs, ET480/40m and ET535/30m). To measure Camui and/or FRESCA activity and [Ca2+]i simultaneously, eggs that had been injected with Camui and/or FRESCA cRNAs were loaded ~ 4 hours post-injection with 1 μM Rhod-2AM supplemented with 0.02% pluronic acid for 20 minutes at RT. Eggs were then attached on glass-bottom dishes (MatTek Corp., Ashland, MA) and placed on the stage of an inverted microscope. CFP, YFP and Rhod-2 intensities were collected every 20 second by a cooled Photometrics SenSys CCD camera (Roper Scientific, Tucson, AZ). The rotation of excitation and emission filter wheels was controlled using the MAC5000 filter wheel/shutter control box (Ludl) and NIS-elements software (Nikon). Imaging was performed on an inverted epifluorescence microscope (Nikon Eclipse TE 300, Analis Ghent, Belgium) using a 20x objective. For studies where ionomycin was used to induce Ca2+ responses, eggs were transferred into a 360 μl Ca2+-free TL-Hepes drop on a glass bottom dish, after which and following a brief monitoring period to determine baseline [Ca2+]i values, different concentrations of ionomycin were added and Ca2+ responses monitored. For Sr2+ studies, eggs were transferred into a Ca2+-free TL-Hepes, containing 10mM Sr2+. In cases where [Ca2+]i oscillations were induced by injection of mPLCζ cRNA, eggs were placed in TL-Hepes media containing 2 mM Ca2+ within 20 minutes of the injection of mPLCζ which occurred 4 hours post-injection of the FRET constructs (Camui or FRESCA).
Pharmacological tests in mouse eggs
Mouse eggs were transferred to Ca2+ free TL-Hepes containing desired concetnrations of pharmacological compounds 5 min prior to Ca2+ imaging. FRET (YFP/CFP) was monitored simultaneously with Ca2+ (rhodamine signal). First, we determined how much inhibitor could be added without affecting Ca2+ entry. Concentrations of inhibitors were chosen based on this information as well as what was used in previous studies, see text for references. All media was Ca2+ free with the exception of PMA. The following concentrations were used: KN93 (0.5 μM), GO6983 (3 μM), Bim1 (5 μM), PMA (1 μM), AS105 (5 μM), AS461 (5 μM). AS105 has not been used in mouse eggs, so we adjusted the concentration to a level where the Ca2+ entry was not affected. Eggs with the first 4 compounds added were stimulated with 0.5 μM ionomycin 5 min after monitoring started, while the eggs with AS compounds were stimulated with 2.5 μM ionomycin. Side by side controls were performed under the same conditions (0.5 μM vs. 2.5 μM ionomycin).
Data processing & statistical analyses
Graphs reporting FRET changes and Ca2+ responses were prepared using the values of the YFP (436×535)/CFP (436×480) ratios on the left axis, whereas Rhod-2 values were calculated using the following formula (F)/F0 (actual value at x time/average baseline values for the first 2 minutes of monitoring) and the scale placed on the right axis. Values from three or more experiments performed on different batches of eggs are presented as means ± s.e.m and were analyzed by the Student’s t-test. Differences were considered significant at P <0.05.
Acknowledgements
We thank Changli He for assistance on RNA purification. We thank Allosteros therapeutics for providing AS105, AS461 and Howard Schulman for helpful discussion. We also thank Peter Chien, Eric Strieter, Scott Garman for discussions and John Kuriyan for helpful comments on the manuscript.