Ratnesh K Srivastav

Susan Schwede

Malte Klaus

Jessica Schwermann

Matthias Gaestel

Rainer Niedenthal
email: niedenthal.rainer@mh-hannover.de

Monitoring protein-protein-interactions in mammalian cells by Trans-SUMOylation

Keywords: Trans-SUMOylation, in vivo labelling, protein interaction, in vivo analysis, Ubc9 fusion-directed SUMOylation, binding domains

Abbreviations: TRS (Trans-SUMOylation), SUMO (small ubiquitin-like modifier), Ubc9 (SUMO conjugating enzyme), HEK293 (human embryo kidney cells), p38α (mitogen activated kinase p38α), MK2 (mitogen activated kinase-activated protein kinase 2), MK5 (mitogen activated kinase-activated protein kinase 5), p53 (cellular tumor antigen p53), ERK3 (extracellular signal-regulated kinase 3), Y2H (yeast two hybrid), FPs (fluorescent proteins), FRET (fluorescence resonance energy transfer), PCA (protein fragment complementation assays), GFP (green fluorescent protein), UFDS (Ubc9 fusion-directed SUMOylation), HA (haemaglutinin), STAT1 (signal transducer and activator of transcription), CDK4 (cell division protein kinase 4), CRSP9 (cofactor required for Sp1 transcriptional activation, subunit 9), CSNK2B (casein kinase II subunit beta), FOS (proto-oncogene c-Fos), CDC37 ( Hsp90 co-chaperone Cdc37), DRG1 (developmentally-regulated GTP-binding protein 1), HSF2BP (heat shock factor 2-binding protein), TAF10 (transcription initiation factor TFIID subunit 10), PSMC3 (Homologous-pairing protein 2 homolog), MEKK1ca (mitogen-activated protein kinase kinase kinase 1 constitutive active), TBP (TATAbox-binding protein), FKBP (peptidyl-prolyl cis-trans isomerase FKBP1A), FLAG (N-DYKDDDDK-C octapeptid), Myc (myc protooncogene), HSP27 (heat shock protein beta-1), MEK2 (dual specificity mitogen-activated protein kinase kinase 2), MEK1 (dual specificity mitogen-activated protein kinase kinase 1), ERK2 (extracellular

INTRODUCTION

Specific and regulated protein-protein-interaction is essential for the existence of productive multi protein complexes that maintain and regulate almost all processes in the cell -from sugar, fat and amino acid metabolism to transcription, translation, replication, chromosome segregation and cytokinesis. Our understanding of these processes mainly depends on the identification and characterization of the specific proteins involved and on the properties of their interactions. Therefore, various methods have been developed in the past, which qualitatively and/or quantitatively analyse protein-protein interactions [reviewed in [START_REF] Charbonnier | The social network of a cell: recent advances in interactome mapping[END_REF][START_REF] Figeys | Mapping the human protein interactome[END_REF]. The most prominent methods, which have also been adapted for proteome wide analysis, are the yeast two hybrid (Y2H) system [START_REF] Fields | A novel genetic system to detect protein-protein interactions[END_REF] and affinity purification combined with mass spectrometry [reviewed in 4]. There are several versions of both methods and each has different advantages and limitations. The isolation of endogenous protein complexes using specific antibodies for co-immunopurification could give insights into protein complexes close to the physiological reality. However, appropriate antibodies are rare and proteins weakly expressed or weak transient interactions are hardly detectable. These problems are partially solved by the use of transiently or stably overexpressed tagged proteins [START_REF] Junttila | Single-step Strep-tag purification for the isolation and identification of protein complexes from mammalian cells[END_REF][START_REF] Korf | Large-scale protein expression for proteome research[END_REF][START_REF] Lichty | Comparison of affinity tags for protein purification[END_REF][START_REF] Terpe | Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems[END_REF]. However, these may also increase the number of false positive interactors, as some protein interactions only take place during the protein extract preparation and therefore do not correspond to the actual conditions in the living cell.

Alternatively, Y2H represents a genetic approach that also identifies weak binary protein interactions and directly provides the DNA coding for the interacting proteins. It was developed for the yeast Saccharomyces cerevisiae, but was also adapted to Schizosaccharomyces pombe and to Escherichia coli. Y2H has been used for the identification of thousands of protein-protein-interactions of a variety of organisms [summarized in 1]. However, in these approaches proteins are expressed in a heterologous organism (yeast or E. coli), most of the native posttranslational modifications and chaperone protein interactions are missing and, in yeast, the system also depends on the transport of the interacting proteins to the nucleus. Accordingly, after nearly two decades of extensive use of Y2H, this method is now estimated to produce between 25 and 45% of false positive results [START_REF] Huang | Where have all the interactions gone? Estimating the coverage of two-hybrid protein interaction maps[END_REF]. Further in vitro methods for monitoring protein-protein interactions, such as pull down assays, peptide microarrays or protein cocrystallization are relatively far from physiological reality. Other methods, such as fluorescence spectroscopy and imaging technologies, attempt to analyse the interaction of proteins in their native environment. New imaging technologies coupled with genetically constructed fluorescent proteins (FPs) are used for direct co-localization of proteins by fluorescence resonance energy transfer (FRET) [START_REF] Jares-Erijman | FRET imaging[END_REF][START_REF] Jares-Erijman | Imaging molecular interactions in living cells by FRET microscopy[END_REF] or by protein fragment complementation assays (PCA) with split-GFP [START_REF] Ghosh | Antiparallel leucine zipper-directed protein reassembly: application to the green fluorescent protein[END_REF]. These elegant methods open new opportunities to analyse single protein-protein interactions but, so far, they seem not to be suited for general and a proteome wide analysis [summarized in 13].

Here we describe Trans-SUMOylation (TRS), a new method that monitors protein-protein-interactions in vivo and operates under protein expression levels similar to that of the endogenous proteins. In TRS the fusion of the SUMOylating enzyme Ubc9 to the protein of interest (protein 1) results in covalent modification of an interacting protein (protein 2) by SUMOylation, a ubiquitin-like posttranslational protein modification of eukaryotic cells where a mature SUMO protein of 101 amino acids (SUMO1) is reversibly

RESULTS

Trans-SUMOylation monitors protein-protein-interactions. SUMOylation substrate proteins are strongly SUMOylated when they are fused to the SUMO conjugating enzyme Ubc9 (Ubc9 fusion-directed SUMOylation, UFDS; Figure 1A) [START_REF] Jakobs | Ubc9 fusion-directed SUMOylation (UFDS): a method to analyse function of protein SUMOylation[END_REF][START_REF] Jakobs | Ubc9 fusiondirected SUMOylation identifies constitutive and inducible SUMOylation[END_REF] and this protein fusion functionally can be replaced by a rapamycin-induced FRB/FKBP12 heterodimerization [START_REF] Zimnik | Mutually exclusive STAT1 modifications identified by Ubc9/substrate dimerization-dependent SUMOylation[END_REF]. Here we have analysed whether an Ubc9 fusion protein can also SUMOylate its binding proteins (Trans-SUMOylation, TRS; Figure 1B), thus making SUMOylation a monitor for protein-protein interactions in vivo. Therefore, we first co-expressed a protein of interest (protein 1) fused to Ubc9 together with an interacting protein (protein 2) in HEK293 cells, which also express EGFP-SUMO1. As a result of interactions between protein 1 and 2 and subject to the condition that a SUMOylation site is available in protein 2, EGFP-SUMOylation will normally result in altered electrophoretic mobility of protein 2 and can be detected by Western blot. To analyse TRS for its ability to identify protein interactions in mammalian cells in vivo, we tested protein binding pairs known from the literature [summarized in the BioGRID database (www.thebiogrid.org)] and Entrez Gene [www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene]; for references also see supplementary Tables I-IV)). We always co-expressed protein 2 and a protein 1-Ubc9 fusion, or, as a negative control, non-fused and non-binding protein-fused Ubc9 in the absence and presence of EGFP-tagged SUMO1. Proteins were analysed for SUMOylation by Western blot using protein 2 specific antibodies. Western blots demonstrating protein interactions detected by TRS are shown in Figure 2A-2D and in Supplementary Figure 1. In many cases intense SUMOylation of the interacting proteins can be detected indicating that TRS occurs as a general phenomenon (summarized in supplementary Tables I andII). Here it should be noted for all shown Western blots that the shift of a protein band by a SUMOylation is not simply as it is estimated by the addition of the mass of the conjugated SUMO or EGFP-SUMO. Additionally the localisation of the conjugation site at the C(N)-Terminus or in the centre of the conjugated polypeptide determines how strong the protein band is shifted. While conjugation to the ends of the polypeptide chain does not shift significantly more than due the molecular mass added, conjugation to the centre of the polypeptide chain leads often to a significant extra shift. By TRS, we confirmed sixteen established protein interactions (supplementary Table I), such as interactions between the protein kinases MK2 and p38 (Figure 2A and 2B), between the tumor-suppressor p53 and p38 (Figure 2C), the homodimerisation of proteasome regulatory subunit PSMC3/PSMC3 (Figure 2D), between MK2 and the polycomb member Edr2 (Supplementary Figure 1A), between the AP-1 transcription factors c-JUN and c-FOS (Supplementary Figure 1B and1C) and also between p53 and the regulatory kinase subunit CSNK2B (Supplementary Figure 1D), (see also supplementary Table I). Furthermore, we identified seven new protein interactions (supplementary Table II): the homodimerisation of heat shock transcription factor-binding protein HSF2BP (Figure 3A), of the regulatory kinase subunit CSNK2B (Figure 3B), of the Hsp90 cochaperone CDC37 and of the protein kinase MK2 (Supplementary Figure 1E and1F) as well as the interactions between MK2/CSNK2B (Supplementary Figure 1G), MK2/MK5 (Supplementary Figure 1G) and MK2/ERK1 (Supplementary Figure 1H). Four of the identified new protein interactions, the homodimerisation of HSF2BP (Figure 3C) and of CSNK2B (Figure 3D) as well as the interactions between MK2/MK5 (Figure 3E) and MK2/CSNK2B (Figure 3F) were confirmed by GST pull down.

We recently have shown that MK2 associates with the polycomb group complex (PcG) and regulates via its interaction with Edr2 the hematopoietic stem cell homeostasis [START_REF] Schwermann | MAPKAP kinase MK2 maintains self-renewal capacity of hematopoietic stem cells[END_REF]. When we verified MK2/Edr2 interactions by TRS and used p38α and Edr2 as control, we also become aware of a positive interaction signal for p38α and Edr2 in TRS (Supplementary Figure 1A). This novel interaction between p38α and Edr2 could be confirmed by nuclear colocalization of both protein, when fused to GFP or RFP, respectively, and visualized in confocal microscopy (Figure 3G). Since Edr2 is part of the multi-protein PcG, we then analysed whether proteins in this complex are substrates of MK2 or p38α. Interestingly, p38α but not MK2 phosphorylates the Edr2-interacting PcG protein Bmi1 at the proline-directed threonine 275 (Figure 3H and3I). Binding of p38α to Edr2 and most likely also to MK2 leads to its recruitment to the PcG complex where it can phosphorylate the PcG protein Bmi1. The Edr2-dependent docking of p38α to PcG and the subsequent phosphorylation of the complex component Bmi1 could indicate functional consequences and biological relevance of this novel interaction detected by TRS.

TRS using a SUMOylation-site-tag.

There are several possible explanations why some protein interactions could not be confirmed by TRS: Firstly, TRS is not sensitive enough to detect very weak protein interactions, like those found between enzymes and their substrates. Secondly, the interactions identified under artificial conditions does not exist in vivo, thirdly, because of sterical restrictions and structural constrains in reaching the SUMOylation site by Ubc9 and, lastly, because there is no SUMOylation site present in the protein 2. To apply TRS for the latter two cases, we introduced a SUMOylation-site-tag (ST), which can introduce the ideal SUMOylation site of p53 into any protein of interest (Figure 4A). The ST(377-393)-peptide TSRHKKLMFKTEGPDSD was added to the C-terminus of the non-SUMOylated mutant p53-K386R (Figure 4A and4B) with or without three and six glycine residues as linker, respectively (Figure 4A). As expected, the ST-fusion leads to significant increased TRS of p53K386R-ST(377-393) by Ubc9-p38 (Figure 4C, Supplementary Figure 2A and2D) and by CSNK2B-Ubc9 (Supplementary Figure 2D) and of MK2-ST(377-393) and MK2-ST(377-391) by Ubc9-p38 (Supplementary Figure 2B and2C). Furthermore, we tested other SUMOylation-site-tags (ST(377-389), ST(377-391), ST(RAGP1), ST8; Figure 4C and Supplementary Figure 2E) and demonstrated that even a peptide of 8 amino acids (ST8) functions in TRS (Figure 4C). These data indicate that STs can be utilized to extend TRS analysis to proteins which do not contain endogenous SUMOylation sites.

TRS identifies interaction domains. Next we asked whether TRS can be used to characterize protein interaction domains. To that end, we analyzed the specific interactions between the protein kinase MK5 and the atypical MAPK ERK3 (Figure 5A and 5B), which was already characterized by us in pull down experiments [START_REF] Schumacher | Scaffolding by ERK3 regulates MK5 in development[END_REF], also in TRS. First, we coexpressed Ubc9-MK5 and, as negative controls, also Ubc9-MK2 and Ubc9-p38 together with full length GST-ERK3 and analysed the protein extracts by Western blotting (Figure 5B). The known specificity of interactions between ERK3 and MK5 [START_REF] Schumacher | Scaffolding by ERK3 regulates MK5 in development[END_REF][START_REF] Seternes | Activation of MK5/PRAK by the atypical MAP kinase ERK3 defines a novel signal transduction pathway[END_REF] was confirmed by TRS of GST-ERK3 detected for co-expression of Ubc9-MK5 while co-expression of Ubc9-MK2 and the Ubc9-p38 does not lead to TRS of GST-ERK3 in EGFP-SUMO1 expressing HEK293 cells. By pull down experiments of MK5 together with C-terminal ERK3-deletion mutants it has been previously shown that the overlapping ERK3-regions between amino acids 301 and 357 [START_REF] Schumacher | Scaffolding by ERK3 regulates MK5 in development[END_REF] and amino acids 330 and 340 [START_REF] Seternes | Activation of MK5/PRAK by the atypical MAP kinase ERK3 defines a novel signal transduction pathway[END_REF] are essential for MK5 binding. Here, we analysed TRS of the C-terminal ERK3 deletion mutants as shown in Figure 5A and obtained confirmatory results with this new method: While Ubc9-MK5 leads to TRS of the ERK3(1-471) and ERK3(1-357) fragments (Figure 5B), no TRS of GST-ERK3(1-301) is observed (Figure 5B). In addition, we were able to show that there is no TRS of the entire C-terminus of ERK3(317-720) and of ERK3(101-720) (Figure 5B) by Ubc9-MK5, indicating that binding of MK5 to ERK3 requires both parts of the kinase domain of ERK3 and parts of its C-terminal extension. The analyses of MK2/p38α, and p53/p38α interactions by TRS also confirmed the known molecular facts (Supplementary Figure 3A-3D) TRS of proteins expressed at endogenous levels. Protein-protein interaction analyses are prone to false positive results, particularly at the time when proteins are mixed in high concentrations or when they are co-overexpressed. To show that TRS can determine protein interactions under native in vivo conditions at expression levels which are similar to the endogenous ones, we analysed three different TRS pairs for protein-protein interactions at protein levels similar to endogenous ones. Therefore, the indicated amounts (330 pg -10 ng) of specific fusion protein expression vectors were cotransfected with the indicated amounts of co-expression plasmids into HEK293 cells (Figure 6). Under these conditions and using uncleaveable (UC)SUMO3(Q89P) that reduces deconjugation we reinvestigated the TRS of GST-MK2 by p38α-Ubc9 (Figure 6A), of GST-p38α by MK2-Ubc9 (Figure 6B) and of GST-ERK3 by MK5-Ubc9 (Figure 6C). When the GST and Ubc9 fused proteins were expressed at levels similar to the nonfused endogenous proteins, we detected a weak TRS band (marked by empty grey arrow heads) in all lanes where the GST fusion protein was coexpressed with the Ubc9 fusion protein and SUMO (EGFP-SUMO1 and UCSUMO3). No SUMOylation was detected with the massive overexpression of Ubc9 alone (controls of Figure 6A and6B), demonstrating the specificity of TRS. This proves that TRS can determine real in vivo interactions of proteins at expression levels of endogenous proteins.

DISCUSSION

The identification of protein binding partners is an essential step towards the functional characterization of a protein. Here we describe TRS, a new method to identify and study in vivo protein-protein interactions by coexpression in mammalian cells. TRS is based on the finding that Ubc9 fusion proteins can trans-SUMOylate their binding partners. For 16 known and 7 new protein interactions we have demonstrated that TRS can be used to monitor the binding of the proteins in vivo and that TRS can also contribute to the characterization of in vivo interacting regions of protein pairs, such as MK5/ERK3, MK2/p38 and p53/p38. While the analyses of the MK5/ERK3 complex completely confirmed the known interacting domains, we cannot exclude that the used deletions in ERK3 possibly removed the SUMOylation site(s) in that protein. This possibility was excluded in the analyses of the MK2/p38 and p53/p38 complex, where the deletions of protein 1 have been fused to Ubc9 and the SUMOylation substrate protein 2 remains untouched. For future analyses of interaction domains by TRS, we would always suggest to tag protein 2 with a SUMOylation-site to preserve its SUMOylatability.

Of note, using the protein pairs MK2/p38 and MK5/ERK3, which were moderately expressed in HEK293 cells, we have demonstrated that TRS can detect the interactions of proteins at levels similar to their endogenous expression. Hence, TRS could be regarded as an in vivo-labelling mechanism for coexpressed proteins that interact with a specific Ubc9 fusion protein under most native conditions within the cell. The analysis we have shown here for moderately expressed proteins p38α, MK2, MK5 and Erk3 could also be adapted to lower expressed proteins, if the protein 2 that is analysed for SUMOylation, is enriched before the Western analysis using a suitable tag.

What are the advantages and disadvantages of TRS compared to other methods for studying proteinprotein interactions in vivo in the living cell? Often, it is suggested that co-immunoprecipitation of endogenous interacting proteins is the most suited system to confirm protein-protein interactions. Although this method starts with the native non-manipulated cellular system, immunoprecipitation of endogenous interacting proteins is performed after cell lysis in protein extracts, where proteins, which are normally separated by their distinct cellular localization or by local variations of physicochemical conditions, can artificially interact. Similar limitations exist for the methods that show protein-protein interactions by purification of protein complexes under native conditions, such as pull down, Tandemaffinity-purification (TAP) [START_REF] Rigaut | A generic protein purification method for protein complex characterization and proteome exploration[END_REF], the Cross-and-Capture method [START_REF] Suter | Examining protein-protein interactions using endogenously tagged yeast arrays: the Cross-and-Capture system[END_REF] and the Lumier-system [START_REF] Barrios-Rodiles | High-throughput mapping of a dynamic signaling network in mammalian cells[END_REF] and also the QUICK-system [START_REF] Selbach | Protein interaction screening by quantitative immunoprecipitation combined with knockdown (QUICK)[END_REF], a method that can minimize unspecific contaminants, cannot reduce falsepositive interactions that exclusively take place in the protein extract. TRS minimizes the probability of such false-positive results, because SUMO-labelling takes place in an interaction-dependent manner only in living cell and because the conjugating enzyme Ubc9 is directly inactivated during cell lysis by boiling in SDS loading buffer. As further control we replaced the Ubc9 fusion protein by an overexpressed unfused Ubc9. This enabled us to clearly differentiate between Ubc9-dependent and Ubc9-fusion proteindependent SUMOylation of the target protein.

Of the genetic methods for analysis of protein-protein interactions, the most relevant are the yeast two hybrid approaches. These analyse protein-protein interactions in the living yeast cell, where various modifications of mammalian proteins necessary for the interactions cannot be generated and where yeast proteins can possibly interfere with the mammalian protein-protein interactions analysed. Therefore, mammalian two hybrid systems have been developed, which are based on the reconstitution of a transcription factor [START_REF] Shioda | A green fluorescent protein-reporter mammalian two-hybrid system with extrachromosomal maintenance of a prey expression plasmid: application to interaction screening[END_REF], of the signalling of a membrane spanning receptor signalling cascade (MAPPIT) [START_REF] Eyckerman | Design and application of a cytokine-receptor-based interaction trap[END_REF], by the protein fragment complementation of an enzymatic activity (Split-TEV) [START_REF] Wehr | Monitoring regulated protein-protein interactions using split TEV[END_REF], by the bimolecular complementation of a fluorescence signal (BIFC) [START_REF] Hu | Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation[END_REF] or also by fluorescence/bioluminescence resonance energy transfer (FRET/BRET) [START_REF] Siegel | Measurement of molecular interactions in living cells by fluorescence resonance energy transfer between variants of the green fluorescent protein[END_REF][START_REF] Xu | A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins[END_REF]. These methods may all better reflect the real situation in the cell and have been successfully used to identify interacting proteins. However, their limitations arise from the facts that interactions have to take place in a specific cellular localization (mammalian Two hybrid, MAPPIT, Split-TEV and Tango) and that there are structural constraints to reach the protein fragment complementation (Split-TEV, BIFC and FRET/BRET). Furthermore, most of these methods are necessarily combined with an indirect reporter system that can also cause false-positive results. TRS is not restricted to a special cellular localization for the protein-protein interactions and it does not need a special reporter system, because the resulting modification is detected directly at the interacting protein.

Eleven protein interactions previously described could not be confirmed by TRS using EGFP-SUMO1. Possible reasons for this could be the existence of structural constraints between the binding proteins, a problem that concerns all systems based on fusion proteins. Our observation that TRS is not efficient with all tested constructs for the p53/p38α or the p53/ERK2 binding pairs (cf. supplementary material, Table I) supports the notion of sterical constraints of TRS. We tried to minimize these sterical constraints for TRS by the development of special linkers that will give the fused Ubc9 more of the flexibility necessary to reach the SUMOylation site of the associating proteins but so far without success (data not shown). However, we could demonstrate that both sterical constraints and missing SUMOylation sites in the protein 2 can be compensated by the introduction of highly flexible SUMOylation-site-tags (STs) into protein 2. STs enable TRS of proteins without natural SUMOylation site(s), as shown for p53K386R, and strongly enhance the TRS of other proteins 2, such as MK2. In this context it also should be noted that the Ubc9-fusion leads to a forced SUMOylation of the fusion partner in UFDS [START_REF] Jakobs | Ubc9 fusion-directed SUMOylation (UFDS): a method to analyse function of protein SUMOylation[END_REF][START_REF] Jakobs | Ubc9 fusiondirected SUMOylation identifies constitutive and inducible SUMOylation[END_REF] and also to the interaction partner in TRS (this study). Hence, even weak SUMOylation sites can display Trans-SUMOylation. However, a further explanation for missing protein 2 SUMOylation could be that some transient interactions between enzymes and substrates, such as JNKs/JUN, MK2/Hsp27 or ERK1/p53 (supplementary material, Table II), are too weak to be identified by TRS using EGFP-SUMO1. Here, further developments of TRS are under way, using SUMO with a reduced deconjugation, SUMOylationsite-tags with an increased SUMOylation or a linker-fused Ubc9 with higher conjugation activity.

TRS in its present form depends on the coexpression of the interacting proteins, where the prey protein should contain a SUMO-site or tag, and is surely suited to confirm protein interactions in vivo that have been identified with systems like the Y2H. Furthermore it can be used for a systematic coexpression of protein pairs to identify new interacting protein pairs in vivo. To ensure that as much as possible interactions will be identified, we suggest to use N-and C-terminal fusions of Ubc9 to a certain protein 1 and a protein 2 that carries a SUMOylation-site-Tag. TRS determines the protein-protein interactions of interest in the homologue organism and rules out misleading results caused by the heterologous organism used.

TRS is a completely new concept that uses a mammalian posttranslational modification system to covalently label coexpressed interacting proteins in vivo and is capable of studying protein-protein interactions at levels similar to endogenous expression. Therefore, TRS has the potential to contribute significantly to the physiologically validated protein-protein interactome. TRS will also be suited to monitor the action of small molecule inhibitors on protein-protein interactions in the living cell and, hence, could help to validate pharmacologically important targets. Furthermore, we have first promising indications that TRS can be the basis for the future development of a screening method for endogenous interacting proteins in mammalian cells in vivo, where endogenous proteins trans-SUMOylated by an expressed Ubc9 fusion protein could be purified and analysed by mass spectrometry.

AUTHOR CONTRIBUTION

Ratnesh Srivastav designed and performed experiments and analysed data. Malte Klaus, Susan Schwede and Jessica Schwermann performed experiments. Rainer Niedenthal directed and supervised the research, designed the experiments, analysed data and wrote the paper. Matthias Gaestel cosupervised the research, analysed the data and wrote the paper. A c c e p t e d M a n u s c r i p t

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. Trans-SUMOylation identifies protein-protein interactions.

(A) GST-MK2 and EGFP-SUMO1 or (B) GST-p38 and EGFP-SUMO1 were cotransfected into HEK293 cells either alone (-) or together with HA-Ubc9, p53-Ubc9, Ubc9-MK5, Ubc9-MK2, MK2-Ubc9, Ubc9-p38, JUN-Ubc9 or Ubc9-CSNK2B (+). (C) p53 and EGFP-SUMO1 were cotransfected into HEK293 cells either alone (-) or together with HA-Ubc9, STAT1-Ubc9, ERK2-Ubc9, Ubc9-ERK2, Ubc9-TBP, Ubc9-CDK4 or Ubc9-p38 (+). (D) GST-PSMC3 and EGFP-SUMO1 were cotransfected into HEK293 cells either alone (-) or together with HA-Ubc9, Ubc9-PSMC3 or MK2-Ubc9 (+). 24 h after transfection the Ubc9 fusion protein binding proteins in the protein extracts of the transfectants were detected by immunoblotting using protein or tag specific antibodies (GST, p53, Ubc9). After stripping, the Ubc9 fusion proteins were detected with Ubc9 antibody (Ubc9). Binding proteins, binding proteins conjugated with coexpressed EGFP-SUMO1 (E-S1), Ubc9 fusion proteins and Ubc9 fusion proteins conjugated with coexpressed EGFP-SUMO1 (E-S1) are indicated by black arrow heads or black lines, weak Trans-SUMOylation bands are indicated by grey empty arrow heads. For all the Western blots it should be noted, that the shift of a protein band by a SUMOylation is not simply as it is estimated by the addition of the mass of the conjugated SUMO or EGFP-SUMO. Additionally the localisation of the conjugation site at the C(N)-Terminus or in the centre of the conjugated polypeptide determines strongly how much the protein band is shifted. The conjugation to the ends of the polypeptide chain does not shift strongly additional to the added mass, but the conjugation to the centre of the polypeptide chain.

Figure 3 Confirmation of protein-protein interactions identified by Trans-SUMOylation.

(A) GST-HSF2BP and EGFP-SUMO1 were cotransfected into HEK293 cells either alone (-) or together with HA-Ubc9, Ubc9-HDGF or Ubc9-HSF2BP (+). (B) GST-CSNK2B with (+) or without (-) EGFP-SUMO1 was cotransfected into HEK293 cells either alone (-) or together with CSNK2B-Ubc9 (+) and with (+) or without (-) the constitutive active (ca) MAP3-kinase MEKK1. 24 h after transfection the Ubc9 fusion protein binding proteins in the protein extracts of the transfectants were detected by immunoblotting using a GST specific antibody (GST). Binding proteins and binding proteins conjugated with coexpressed EGFP-SUMO1 (E-S1) are indicated by black arrow heads. (C) Ubc9-HSF2BP was cotransfected into HEK293 cells either with GST or together with GST-HSF2BP. (D) CSNK2B-FKBP was cotransfected into HEK293 cells either with GST or together with GST-CSNK2B. (E) GFP-MK5 was cotransfected into HEK293 cells either with GST or together with GST-MK2. (F) Ubc9-CSNK2B was cotransfected into HEK293 cells either with GST or together with GST-MK2. 24 h after transfection the GST and GSTfusion proteins bound to glutathione sepharose were pulled down from the protein extracts of the transfectants. Co-purification of Ubc9-, FKBP-and GFP-fusion proteins were detected by immunoblotting using tag specific antibodies (Ubc9, FKBP, GFP). After stripping the GST fusion proteins were detected with GST antibody (GST). The detected coexpressed proteins are indicated by black arrow heads. An unspecific band in B) was indicated by a grey star. (G) The colocalisation of p38α with Edr2. HEK293 cells were transfected with expression vectors for GFP-Edr2 and RFP-p38α. Fusion proteins were visualised by fluorescence microscopy. Nuclei were stained using TO-PRO. RFP-p38α-AGF is the fusion protein of the catalytically inactive mutant of p38α which lacks the phosphorylatable threonine and tyrosine in the activation loop. p38α phosphorylates Bmi1 at T275. (H) In vitro phosphorylation assay for Bmi1 and Hsp25 using His-MK2 alone and together with its activating MAPK GST-p38α. Kinase assay was performed in the presence of γ-33P-ATP and phosphorylated proteins were visualised by phosphorimaging. (I) Different potential phosphorylation site mutants corresponding to alanine replacement of the proline-directed S/T residues shown in bold in the C-terminal sequence part of Bmi1 were subjected to in vitro phosphorylation using GST-p38α. Phosphorylation was detected as in H .

Figure 4

Trans-SUMOylation of SUMOylation site tags.

(A) Structure of p53 and the p53K386R mutant fused to the indicated SUMOylation-site-tags. (B) p53 and EGFP-SUMO1 or p53K386R and EGFP-SUMO1 were cotransfected into HEK293 cells either alone (-) or together with Ubc9-p38, Ubc9-BACH1 or MK2-Ubc9 (+). (C) Ubc9-p38 and EGFP-SUMO1 were cotransfected into HEK293 cells together with p53wt, p53K386R, p53K386R-ST(377-393), p53K386R-3G-ST(377-393), p53K386R-6G-ST(377-393), p53K386R-ST(377-391), p53K386R-ST(377-389), p53K386R-ST(RAGP1) or p53K386R-ST8 (+).24 h after transfection the Ubc9 fusion protein binding proteins in the protein extracts of the transfectants were detected by immunoblotting using a p53 specific antibody). After stripping the Ubc9 fusion proteins were detected with an Ubc9 antibody (Ubc9). Binding proteins, binding proteins conjugated with coexpressed EGFP-SUMO1 (E-S1), Ubc9 fusion proteins and Ubc9 fusion proteins conjugated with coexpressed EGFP-SUMO (E-S1) are indicated by black arrow heads or black lines. An unspecific band in C) was indicated by a grey star.

Figure 5

Protein-protein interaction domains characterized by Trans-SUMOylation.

(A) Schematic representation of ERK3 deletion mutants fused to GST. (B) Trans-SUMOylation analysis of ERK3 deletion mutants fused to GST. GST-ERK3 and EGFP-SUMO1 were cotransfected into HEK293 cells either alone (-) or together with HA-Ubc9, Ubc9-p38, Ubc9-MK2 or Ubc9-MK5 (+). GST-ERK3(101-720), GST-ERK3(1-301), GST-ERK3(316-720), GST-ERK3(1-471) or GST-ERK3(1-357) were cotransfected into HEK293 cells together with HA-Ubc9 and EGFP-SUMO1 or Ubc9-MK5 and EGFP-SUMO1 (+). 24 h after transfection the Ubc9 fusion protein binding proteins in protein extracts of the transfectants were detected by immunoblotting using a GST specific antibody (GST). After stripping the Ubc9 fusion proteins were detected with an Ubc9 antibody (Ubc9). Binding proteins, binding proteins conjugated with coexpressed EGFP-SUMO1 (E-S1), Ubc9 fusion proteins and Ubc9 fusion proteins conjugated with coexpressed EGFP-SUMO (E-S1) are indicated by black arrow heads or black lines.

Figure 6

Trans-SUMOylation of proteins expressed similar to endogenous levels. (A) GST-MK2 and UCSUMO3, (B) GST-p38 and EGFP-SUMO1 or UCSUMO3 and (C) GST-ERK3 and EGFP-SUMO1 or UCSUMO3 were cotransfected into HEK293 cells either alone (-) or together with the indicated amounts of expression plasmids of HA-Ubc9, Ubc9-p38, MK2-Ubc9 or Ubc9-MK5 (+) to reach expression levels similar to that of endogenous proteins. 24 h after transfection the Ubc9 fusion protein binding proteins and the endogenous proteins in the protein extracts of the transfectants were separated on a 7.5% SDS-PAGE and detected by immunoblotting using protein specific antibodies (MK2, p38, Ubc9, ERK3, MK5). After stripping the Ubc9 fusion proteins and the endogenous proteins were detected with specific antibodies (MK2, MK5, p38, Ubc9). The Ubc9 and the Ubc9 fusion proteins were also detected after a separation on a 13% SDS-PAGE by immunoblotting using the Ubc9 antibody (Ubc9). The binding proteins, the binding proteins conjugated with coexpressed EGFP-SUMO1 (E-S1) or UCSUMO3 (S3), the Ubc9 fusion proteins, the HA-Ubc9, the endogenous Ubc9, MK2, MK5, ERK3

Figure 1

 1 Figure 1Schematic representation of the Trans-SUMOylation (TRS) system.(A) In the Ubc9 fusion-directed SUMOylation (UFDS), a protein directly fused to Ubc9 is SUMOylated by the SUMO loaded Ubc9 in cis. (B) A protein 2 that binds to protein 1 can be SUMOylated in trans by the SUMO loaded Ubc9 fused to protein 1.

Figure 2

 2 Figure 2

  Figure 6Trans-SUMOylation of proteins expressed similar to endogenous levels. (A) GST-MK2 and UCSUMO3, (B) GST-p38 and EGFP-SUMO1 or UCSUMO3 and (C) GST-ERK3 and EGFP-SUMO1 or UCSUMO3 were cotransfected into HEK293 cells either alone (-) or together with the indicated amounts of expression plasmids of HA-Ubc9, Ubc9-p38, MK2-Ubc9 or Ubc9-MK5 (+) to reach expression levels similar to that of endogenous proteins. 24 h after transfection the Ubc9 fusion protein binding proteins and the endogenous proteins in the protein extracts of the transfectants were separated on a 7.5% SDS-PAGE and detected by immunoblotting using protein specific antibodies (MK2, p38, Ubc9, ERK3, MK5). After stripping the Ubc9 fusion proteins and the endogenous proteins were detected with specific antibodies (MK2, MK5, p38, Ubc9). The Ubc9 and the Ubc9 fusion proteins were also detected after a separation on a 13% SDS-PAGE by immunoblotting using the Ubc9 antibody (Ubc9). The binding proteins, the binding proteins conjugated with coexpressed EGFP-SUMO1 (E-S1) or UCSUMO3 (S3), the Ubc9 fusion proteins, the HA-Ubc9, the endogenous Ubc9, MK2, MK5, ERK3 and p38 are indicated by black arrow heads. UCSUMO3 represents the human SUMO3Q89P mutant. Weak Trans-SUMOylation bands are indicated by grey empty arrow heads. Unspecific bands in A) and B) were indicated by grey stars.

  Figure 1 (Niedenthal)

Figure 2

 2 Figure 2 (Niedenthal)

Figure 3

 3 Figure 3 (Niedenthal)

Figure 3 Figure 4

 34 Figure 3 (Niedenthal)

Figure 5 Figure 6

 56 Figure 5(Niedenthal)

ACKNOWLEDGEMENT

We thank Astrid Jakobs for help in cell culture and Western blot experiments. We also thank Dr. Alexey Kotlyarov (Hannover Medical School) for helpful discussions and Dr. Thomas Binz (Hannover Medical School) for critical reading of the manuscript.

FUNDING

This work publication was supported by the Institute for Physiological Chemistry/Biochemistry and the HiLF program of the Hannover Medical School (to R. N.).


Sally V Phillips

Yuansong Yu

Andreas Rossbach

Michail Nomikos

Vyronia Vassilakopoulou

Evangelia Livaniou

Bevan Cumbes

F Anthony Lai

Christopher H George

Karl Swann
email: swannk1@cardiff.ac.uk

Divergent effect of mammalian PLC-zeta in generating Ca2+ oscillations in somatic cells versus eggs

Keywords: phospholipase C (PLC), calcium, oscillations, CHO cells, eggs, sperm, Biochemical

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Phospholipase C-zeta (PLC]) is an isoform of phosphoinositide-specific PLC that is specifically expressed in mammalian sperm cells [START_REF] Saunders | PLC]: a sperm-specific trigger of Ca 2+ oscillations in eggs and embryo development[END_REF]. Microinjection of mouse PLC] cRNA into mouse, human, or bovine eggs triggers a prolonged series of Ca 2+ oscillations similar to those observed at fertilization [START_REF] Saunders | PLC]: a sperm-specific trigger of Ca 2+ oscillations in eggs and embryo development[END_REF][START_REF] Swann | PLC](zeta): A sperm protein that triggers Ca 2+ oscillations and egg activation in mammals[END_REF][START_REF] Kouchi | Recombinant phospholipase Czeta has high Ca 2+ sensitivity and induces Ca 2+ oscillations in mouse eggs[END_REF][START_REF] Miyazaki | Calcium signals for egg activation in mammals[END_REF][START_REF] Kurakawa | Mammalian fertilization: from sperm factor to phospholilpase Czeta[END_REF]. PLC] has been identified as the protein responsible for causing Ca 2+ oscillations in mouse eggs when either cytosolic sperm extracts, or whole intact sperm, are microinjected into the egg cytoplasm [START_REF] Saunders | PLC]: a sperm-specific trigger of Ca 2+ oscillations in eggs and embryo development[END_REF][START_REF] Swann | PLC](zeta): A sperm protein that triggers Ca 2+ oscillations and egg activation in mammals[END_REF][START_REF] Kouchi | Recombinant phospholipase Czeta has high Ca 2+ sensitivity and induces Ca 2+ oscillations in mouse eggs[END_REF][START_REF] Miyazaki | Calcium signals for egg activation in mammals[END_REF][START_REF] Kurakawa | Mammalian fertilization: from sperm factor to phospholilpase Czeta[END_REF][START_REF] Fujimoto | Mammalian phospholipase Czeta induces oocyte activation from the sperm perinuclear matrix[END_REF]. The critical level of PLC] protein required to trigger Ca 2+ release in mouse eggs is around 10-50fg, which is similar to the estimated levels in a single mouse sperm [START_REF] Saunders | PLC]: a sperm-specific trigger of Ca 2+ oscillations in eggs and embryo development[END_REF]. Furthermore, reduced expression of PLC] during spermatogenesis by transgenic RNA interference approaches has been shown to decrease the number of Ca 2+ oscillations at fertilization in mouse eggs [START_REF] Knott | Transgenic RNAi reveals role for mouse sperm phospholipase C] in triggering Ca 2+ oscillations during fertilization[END_REF]. These data have led to the proposal that sperm PLC] is the physiological activator of mammalian development that is delivered to the egg after sperm-egg membrane fusion [START_REF] Saunders | PLC]: a sperm-specific trigger of Ca 2+ oscillations in eggs and embryo development[END_REF][START_REF] Swann | PLC](zeta): A sperm protein that triggers Ca 2+ oscillations and egg activation in mammals[END_REF][START_REF] Kouchi | Recombinant phospholipase Czeta has high Ca 2+ sensitivity and induces Ca 2+ oscillations in mouse eggs[END_REF][START_REF] Miyazaki | Calcium signals for egg activation in mammals[END_REF][START_REF] Kurakawa | Mammalian fertilization: from sperm factor to phospholilpase Czeta[END_REF]. PLC] mediates its effects in eggs by virtue of its ability to hydrolyse PIP 2 and generate InsP 3 since mutation of aspartate-210 to an arginine (D210R), which is a critical residue in the PLC] catalytic domain, abolishes its ability to cause Ca 2+ oscillations or activate eggs [START_REF] Saunders | PLC]: a sperm-specific trigger of Ca 2+ oscillations in eggs and embryo development[END_REF]. Hence, PLC] can account for all of the Ca 2+ oscillations in eggs at fertilization since they are entirely dependent upon the stimulation of Ca 2+ release via the type 1 InsP 3 receptor [START_REF] Miyazaki | Calcium signals for egg activation in mammals[END_REF][START_REF] Kurakawa | Mammalian fertilization: from sperm factor to phospholilpase Czeta[END_REF][START_REF] Brind | Inositol 1,4,5-trisphosphate receptors are downregulated in mouse oocytes in response to sperm or adenophostin A but not to increases in intracellular Ca 2+ or egg activation[END_REF][START_REF] Jellerette | Down-regulation of the inositol 1,4,5-trisphosphate receptor in mouse eggs following fertilization or parthenogenetic activation[END_REF].

It is not known whether PLC] activity is regulated either in sperm or eggs. One significant feature of PLC] that distinguishes it from many other mammalian PLCs is that its activity in vitro is retained in very low concentrations of Ca 2+ without the requirement for other protein factors [START_REF] Kouchi | Recombinant phospholipase Czeta has high Ca 2+ sensitivity and induces Ca 2+ oscillations in mouse eggs[END_REF][START_REF] Nomikos | Role of phospholipase C-{zeta} domains in Ca 2+ -dependent phosphatidylinositol 4,5-bisphosphate hydrolysis and cytoplasmic Ca 2+ oscillations[END_REF]. PLC] is up to 50% active at the resting cytoplasmic Ca 2+ concentrations [START_REF] Kouchi | Recombinant phospholipase Czeta has high Ca 2+ sensitivity and induces Ca 2+ oscillations in mouse eggs[END_REF][START_REF] Nomikos | Role of phospholipase C-{zeta} domains in Ca 2+ -dependent phosphatidylinositol 4,5-bisphosphate hydrolysis and cytoplasmic Ca 2+ oscillations[END_REF]. This implies that PLC] would be autonomously active in generating Ca 2+ oscillations when it is injected into the egg cytoplasm at low concentrations. In this case it is remarkable that the sperm contains more than a thousand fold higher PLC] concentration than the egg, yet sperm have a normal resting Ca 2+ level and undergo controlled Ca 2+ oscillations in response to stimuli such as progesterone [START_REF] Publicover | Ca 2+ signalling in the control of motility and guidance in mammalian sperm[END_REF]. This implies that PLC] might be inactive in sperm and that its activity is regulated differently in sperm compared to eggs. However, the nature of this differential regulation remains unclear.

Some previous studies have characterized recombinant PLC] expressed in cell lines or somatic tissues [START_REF] Coward | Expression of a fluorescent recombinant form of sperm protein phospholipase C zeta in mouse epididymal sperm by in vivo gene transfer into the testis[END_REF][START_REF] Kuroda | The role of X/Y linker region and N-terminal EFhand domain in nuclear translocation and Ca 2+ oscillation-inducing activities of phospholipase Czeta, a mammalian egg-activating factor[END_REF]. It was reported that mouse PLC] expression in COS cells is associated with the generation of Ca 2+ oscillations [START_REF] Kuroda | The role of X/Y linker region and N-terminal EFhand domain in nuclear translocation and Ca 2+ oscillation-inducing activities of phospholipase Czeta, a mammalian egg-activating factor[END_REF]. Injection of boar sperm extracts (containing PLC]) into hepatocytes has also been shown to trigger Ca 2+ oscillations [START_REF] Berrie | A cytosolic sperm factor triggers calcium oscillations in rat hepatocytes[END_REF]. These data suggest that PLC] can be autonomously active in somatic cells. However, another report showed that low levels of PLC] expressed in all tissues of transgenic mice has no discernable affect upon somatic cells, despite the fact that it activated oocytes within the ovaries of females [START_REF] Yoshida | Borad, extopic expression of the sperm protein PLCZ1 induces parthenogenesis and ovarian tumours in mice[END_REF]. Ca 2+ changes were not examined in that study but it implies that low levels of PLC] may be ineffective in A c c e p t e d M a n u s c r i p t somatic tissues. The only known mechanism for inactivating PLC] is nuclear sequestration which can account for the cessation of Ca 2+ oscillations after fertilization in the mouse [START_REF] Larman | Cell cycle-dependent Ca 2+ oscillations in mouse embryos are regulated by nuclear targeting of PLC[END_REF][START_REF] Yoda | Ca 2+ oscillation-inducing phospholipase C zeta expressed in mouse eggs is accumulated to the pronucleus during egg activation[END_REF]. However, it is unclear whether PLC] localization accounts for the different reports on the effect of PLC] expression in somatic cells. PLC] has been reported to be present in either the cytoplasm or the nucleus of somatic cells in a manner that may depend upon time, the cell line used, or species source of PLC] [START_REF] Coward | Expression of a fluorescent recombinant form of sperm protein phospholipase C zeta in mouse epididymal sperm by in vivo gene transfer into the testis[END_REF][START_REF] Ito | Difference in Ca 2+ oscillation-inducing activity and nuclear translocation ability of PLCZ1, an egg-activating sperm factor candidate, between mouse, rat, human, and medaka fish[END_REF].

Here, we have expressed mouse PLC] in CHO cells that have been widely used to study the mechanism of Ca 2+ oscillations [START_REF] Nash | Reassessment of the Ca 2+ sensing property of a type I metabotropic glutamate receptor by simultaneous measurements of inositol 1,4,5trisphophate and Ca 2+ in single cells[END_REF] We find that very high levels of PLC] expression in CHO cells has little affect upon Ca 2+ homeostasis, suggesting it is inactive in these cells. Nuclear localization of PLC] depended upon time after transfection, but is unlikely to account for the lack of Ca 2+ oscillation in these cells. Remarkably we found that, despite the absence of an effect on CHO cell Ca 2+ level, the PLC]-expressing CHO cells are able to cause Ca 2+ oscillations when introduced into mouse eggs, and can be extracted and shown to have a high intrinsic PLC activity.

EXPERIMENTAL Cell culture and transfection

Chinese hamster ovary (CHO) cells were cultured in complete F-12 media (Invitrogen Ltd) in 5% CO 2 at 37 o C and seeded in 6 well plates or in polylysine coated glass-bottomed culture dishes (World Precision Instruments Inc) 24 hours prior to transfection at ~80-90% confluency. For transient expression, the nuclei of cells maintained in Leibovitz L-15 media were microinjected with plasmid DNA (~10ng/ul) in KCl Hepes buffer [START_REF] Swann | Use of luciferase chimera to monitor PLC] expression in mouse eggs[END_REF] with 2.5mg/ml Alexa 594-dextran (10,000 MW) or 0.5mM Oregon Green BAPTA dextran (Invitrogen) using a Femtojet and InjectMan NI2 (Eppendorf) with an injection pressure of 90hPa. The injection system was mounted on an Axiovert 200 fluorescence microscope (Zeiss Ltd). After injecting 50-100 cells the medium was exchanged back to F-12 and cells were returned to the incubator prior to imaging. For stable expression, CHO cells were transfected with DNA Lipofectamine2000 (Invitrogen) and then incubated for 16-18 hours prior to testing for heterologous PLC] expression. PLC]expressing cells were selected on the basis of resistance to G418 by using an increasing concentration of G418 (0 to 1 mg/ml in 100Pg increments) and cells exhibiting appreciable YFP fluorescence were enriched using FACS (DakoCytomation, MoFlo). The PLC]-luciferase fusion protein construct was made in a pCR3 vector (Invitrogen) as described previously [START_REF] Nomikos | Role of phospholipase C-{zeta} domains in Ca 2+ -dependent phosphatidylinositol 4,5-bisphosphate hydrolysis and cytoplasmic Ca 2+ oscillations[END_REF]. The YFP-PLC] construct was made in pcDNA3.1 with YFP placed at the N-terminus of PLC]. The eYFP-D210R PLC] construct was derived from YFP-PLC] by site directed mutagenesis of aspartate 210 to arginine using the Stratagene Quikchange XL SDM kit.

Injection of eggs with extracts or cells

Metaphase II mouse eggs were collected and microinjected with ~10pl of extracts (about 5% of the egg volume) as described previously [START_REF] Nomikos | Role of phospholipase C-{zeta} domains in Ca 2+ -dependent phosphatidylinositol 4,5-bisphosphate hydrolysis and cytoplasmic Ca 2+ oscillations[END_REF][START_REF] Swann | Use of luciferase chimera to monitor PLC] expression in mouse eggs[END_REF]. To make cell extracts, CHO cells (~ 2 x 10 7 ) stably transfected with YFP fusion constructs were harvested by trypsinisation then pelleted and resuspended A c c e p t e d M a n u s c r i p t several times in KCl Hepes buffer [START_REF] Nomikos | Role of phospholipase C-{zeta} domains in Ca 2+ -dependent phosphatidylinositol 4,5-bisphosphate hydrolysis and cytoplasmic Ca 2+ oscillations[END_REF]. Cells were lysed with 3 cycles of freeze-thaw using liquid nitrogen, before being subjected to ultracentrifugation at 100,000 x g for 1 hour at 4 o C. The resulting supernatant was concentrated using centrifugal filters and stored at -80 o C. Microinjection of cells was performed using a Piezo micro-manipulator (PrimeTech Ltd) that mimics the process of somatic cell nuclear transfer [START_REF] Kishigami | Somatic cell nuclear transfer in the mouse[END_REF]. Single CHO cells were drawn into the end of a pipette narrow enough to rupture their cell membrane then immediately injected into a mouse egg by application of low pressure. Before injection, eggs were loaded with either fura red or fura PE3 by incubation for 30 mins in 2-4PM of the AM forms for the dyes. Small groups of eggs were microinjected with cell or sperm extracts, or cells and then rapidly transferred to the imaging microscope. Ca 2+ was measured in eggs by monitoring fluorescence with a Nikon TiU epifluorescence microscope equipped with a Coolsnap HQ 2 CCD camera (Photometrics), with excitation light of 430nm/490nm (emission measured with a 615nm long pass filter) for fura red and excitation of 350nm/380nm (emission measured at 535nm) for fura PE3, or excitation of 550nm and an emission of 600nm for rhod dextran.

Microinjecting CHO cells with sperm extracts.

Boar sperm extracts (~5mg/ml) were prepared as the cytosolic protein fraction of lysed pig sperm as described previously [START_REF] Berrie | A cytosolic sperm factor triggers calcium oscillations in rat hepatocytes[END_REF]. Sperm extracts were mixed with an equal volume of 10000MW dextran-conjugated Alexa647 (Invitrogen) and microinjected into confluent fluo3-loaded CHO cells [24] using injection pressures of 120-140hPa. The successful transduction of exogenous material into cells was confirmed by visualising Alexa 647 fluorescence using a 633nm He:Ne laser fitted to an SP2 confocal laser scanning microscope (Leica Microsystems). Following microinjection, Ca 2+ -dependent fluo3 signals were acquired at 1Hz for 20mins as described [START_REF] Berrie | A cytosolic sperm factor triggers calcium oscillations in rat hepatocytes[END_REF]. Post-Ca 2+ imaging, data was only analysed from those cells that retained >75% of their initial Alexa647 fluorescence.

PLC assays and Western blots.

PIP 2 hydrolytic activity of PLC present in YFP-or YFP-PLC]transfected CHO cell extracts was assayed as described previously [START_REF] Nomikos | Role of phospholipase C-{zeta} domains in Ca 2+ -dependent phosphatidylinositol 4,5-bisphosphate hydrolysis and cytoplasmic Ca 2+ oscillations[END_REF]. The assay mixture final volume was 50 μl containing 100 mM NaCl, 0.4% sodium cholate (w/v), 2 mM CaCl 2 , 4 mM EGTA, 20 μg of bovine serum albumin, 5 mM 2-mercaptoethanol, and 20 mM Tris-HCl buffer, pH 6.8. The PIP 2 concentration in the reaction mixture was 220 μM, containing 0.05 μCi of [ 3 H]PIP 2 . Assay conditions were optimized for linearity, requiring a 10 min, 25 °C incubation with 20 pmol protein. Transfected CHO cell line proteins were separated by SDS-PAGE and immunoblot analysis performed as described previously [START_REF] Nomikos | Role of phospholipase C-{zeta} domains in Ca 2+ -dependent phosphatidylinositol 4,5-bisphosphate hydrolysis and cytoplasmic Ca 2+ oscillations[END_REF]. Proteins were probed with a peptide affinity-purified rabbit anti-PLC] polyclonal antibody (1:10,000 dilution) generated to the EF-hand domain amino acid residues 16-31.

CHO cell imaging

Luciferase expression was monitored from cells incubated in 100PM luciferin using an Axiovert 100 microscope (Carl Zeiss Ltd) with a 40x, 1.4NA oil immersion objective lens and a cooled intensified charge-coupled device (ICCD) camera (Photek Ltd) [START_REF] Swann | Use of luciferase chimera to monitor PLC] expression in mouse eggs[END_REF]. Luminescence was collected for 20 minutes and then Ca 2+ was monitored in the same cells for ~1 hour using low level fluorescence excitation light from a halogen lamp with a 490nm excitation filter. Fluorescence from these cells was integrated every 10 sec with sensitivity of the ICCD at 10% [START_REF] Swann | Use of luciferase chimera to monitor PLC] expression in mouse eggs[END_REF]. Resting Ca 2+ concentrations were estimated using the equation Ca 2+ =K d (F max -F)/(F-F min ) with F min taken as zero and the K d for Oregon Green BAPTA in cytoplasm taken as 430nM [START_REF] Thomas | A comparison of fluorescent Ca 2+ indicator properties and their use in measuring elementary and global Ca 2+ signals[END_REF]. The F max was obtained by adding ionomycin to saturate the fluorescence of OGBD or fluo3. Cells expressing YFP fusion proteins were imaged with either a confocal or wide field microscope. For confocal imaging we used a TCS SP5 and a DMI 6000 inverted microscope (Leica). Cells were imaged with a 63x 1.23NA oil immersion objective lens and 512x512 pixel resolution. The YFP, fluo3 and fura red fluorescence was excited with the 488nm laser line with emission at 510-540nm for YFP or fluo3, and at 580-610nm for fura red fluorescence. Alexa-594 fluorescence was visualised using the 561nm He:Ne laser and emission at 600-630nm. For wide field imaging we used a Nikon TiU epifluorescence microscope with a Photometrics Coolsnap HQ 2 CCD camera equipped with excitation and emission filter wheels. Excitation was with a 490nm and 420nm band-pass filter, and emission was monitored with a 530nm band-pass filter (for YFP) or a 620 long-pass filter (for fura red). Where specified, cells were loaded with the Ca 2+ indicator dyes by incubation in 10μM of the AM form in un-supplemented F-12 medium for 60-90mins before being washed and transferred to Krebs-Ringer-Hepes buffer [24,25]. For the confocal analysis of intracellular Ca 2+ transients, cells were imaged every 100ms for 100 frames before the addition of ATP (100µM) and for a further 500 frames (1 min total). Signal variability (SV) is an index of the magnitude of amplitude and temporal characteristics of Ca 2+ fluxes during steady-state Ca 2+ cycling processes [24,25]. For signal variability analysis, data was acquired every 100ms for 30 seconds. Following an experimental series, 15-20 cells were selected per area (typically 4-6 areas per dish) and data analysed from regions of interest (ROIs) of ~40μm 2 (>150 pixels) using Leica software. Further analysis was carried out using Microsoft Excel and GraphPad Prism. Derivation of mathematical operations used to calculate signal variability is given in a previous report [START_REF] Thomas | A comparison of fluorescent Ca 2+ indicator properties and their use in measuring elementary and global Ca 2+ signals[END_REF]. For wide field imaging of YFP-transfected cells an initial YFP image was collected, and the fura red images were collected every 10 seconds with a 420nm/490nm excitation and data were analysed using Image-Pro Plus and Sigma Plot. Data was analysed for significance using ANOVA or a student's t test.

RESULTS

Expression of PLC] ]-luc in CHO cells

The PLC]-luciferase fusion protein (PLC]-luc) has previously been shown to trigger Ca 2+ oscillations when expressed in mouse eggs [START_REF] Nomikos | Role of phospholipase C-{zeta} domains in Ca 2+ -dependent phosphatidylinositol 4,5-bisphosphate hydrolysis and cytoplasmic Ca 2+ oscillations[END_REF]. CHO cells were microinjected with cDNA encoding either PLC]-luc or luciferase, together with the Ca 2+ indicator Oregon Green BAPTA dextran (OGBD). Figure 1a shows a luminescence image from a typical group of PLC]-luc expression plasmidinjected cells indicating successful transient expression of the fusion protein, as reported by the corresponding fluorescence image of OGBD specifically in injected cells (Fig. 1b). Intracellular Ca 2+ was measured in PLC]-luc expressing cells 3, 5 and 24 hours following microinjection and the mean It can be seen that there were small low frequency Ca 2+ oscillations in all three type of cells but there was no clear difference between PLC]-transfected cells and other cells transfected or not (see Table 1). Since CHO cells express the endogenous purinergic P2Y1, P2Y2 and P2X7 receptors, they were tested for responsiveness to extracellular ATP [26]. All the cells underwent a single large rise in Ca 2+ in response to ATP (Fig. 1c). Table 1 summarizes data from transfected cells analysed at 3, 5 and 24 hours post-microinjection. At all three time points there were no differences in the proportion of cells showing low frequency Ca 2+ oscillations, or in those showing a Ca 2+ increase in response to addition of ATP, between PLC]-luc and luc control CHO cells and control OGBD injected cells (see Table 1). The resting Ca 2+ levels in CHO cells were estimated by the addition of Ca 2+ ionophore . Fig. 1d shows that the resting Ca 2+ levels were not significantly different between PLC]-luc or luc control cells, or in comparison to OGBD injected cells, at either 3, 5 or 24 hours after microinjection. These data suggest that transient PLCζ-luc expression does not have a significant effect upon Ca 2+ homeostasis in CHO cells. The presence of low frequency, small amplitude Ca 2+ oscillations in some CHO cells evidently occurs in these cells under certain conditions.

Localization of YFP-PLC] ] in CHO cells

Nuclear sequestration of PLC] appears to cause the cessation of Ca 2+ oscillations in mouse eggs and could account for the lack of CHO cell Ca 2+ oscillations in the above experiments [START_REF] Larman | Cell cycle-dependent Ca 2+ oscillations in mouse embryos are regulated by nuclear targeting of PLC[END_REF][START_REF] Yoda | Ca 2+ oscillation-inducing phospholipase C zeta expressed in mouse eggs is accumulated to the pronucleus during egg activation[END_REF]. We examined the distribution of PLC] using a YFP-tagged PLC] which, similar to VenusGFP-PLC], is also effective in causing Ca 2+ oscillations in mouse eggs (see Fig. S1a). Figure 2a shows confocal images of CHO cells at 3, 5, 24 and 48 hours after being injected with the YFP-PLC] expression plasmid. Distinct levels of recombinant YFP expression were detected as early as 3 hours after DNA microinjection and persisted for at least 48 hours for all of the DNA constructs examined. Expression of YFP alone was homogeneous throughout the nucleus and cytoplasm in all cells. In contrast, YFP-PLC] showed two distinct patterns of localisation. At 3 and 5 hours post-injection all cells showed a cytoplasmic distribution, whereas after 24 and 48 hours the majority of cells showed nuclear translocation with enhanced staining in the nucleoli. Some cells that did not show obvious nuclear translocation at 24 and 48 hours maintained a clear cytoplasmic distribution identical to that observed at 3 and 5 hours. Unlike YFP-PLC], YFP-D210R PLC] did not show any obvious signs of nuclear accumulation at any time points and the majority of cells (>90%) maintained the same cytoplasmic localisation.

As well as studying transiently transfected CHO cells, we also examined the distribution of PLC] in stable cell lines. We prepared CHO cell lines that stably expressed YFP-PLC] or YFP alone after two rounds of cell sorting. The distribution of YFP-PLC] inCHO cells was variable and again could be seen in either the cytoplasm or the nucleus (Fig. 2b). Along with the transient transfection studies, these data support the previous observations that PLC] can localize to the nucleus of cells [START_REF] Coward | Expression of a fluorescent recombinant form of sperm protein phospholipase C zeta in mouse epididymal sperm by in vivo gene transfer into the testis[END_REF]. However, since localization does not occur in all cells or takes more than 5 hours, this suggests that nuclear translocation cannot account for its apparent inactivity in CHO cells.

Ca 2+ homeostasis in YFP-PLC] transfected CHO cells

Following microinjection of CHO cells with YFP-PLC] we measured cytoplasmic Ca 2+ changes using confocal microscopy. We studied the response of transduced cells to the addition of ATP, as described above with PLC]-luc expressing cells, but the faster rate of image acquisition (10Hz) enabled by confocal microscopy permits a more detailed kinetic analysis of basal Ca 2+ handling and ATP-evoked Ca 2+ release in these cells. Cells were loaded with fluo3 and after exposure to ATP the amplitude and relative change in fluorescence was taken as a measure of response for YFP, YFP-PLC] and YFP-D210R PLC] transfected cells, and for control injected cells. The initial increase in Ca 2+ was unaffected by removal of extracellular Ca 2+ indicating that P2Y and InsP 3 -dependent intracellular Ca 2+ store release was involved (unpublished observations). Fig. 3a shows examples of the Ca 2+ response to a maximal concentration of ATP (at the arrow) for all four cell types, which consisted of a large and rapid response followed by a gradual decline as shown in Fig. 1c. The responses in Fig. 3a are at 5 and 24 hours but similar results were seen at 3 and 48 hours after DNA injection. Fig. 3b summarizes the data from for all four types of cells, at all time points. There was no difference in either the proportion of cells responding to ATP, the relative amplitude of the Ca 2+ rise, nor in the time to peak of the Ca 2+ rise evoked by ATP, at any of the different times after microinjection of DNA. These data again suggest that P2Y receptor driven InsP 3 production and Ca 2+ store release is unaltered by PLC] expression.

The use of YFP-PLC] and confocal microscopy allowed us to examine more subtle changes in Ca 2+ homeostasis than may be revealed by the above experiments. Previous studies of cardiac ryanodine receptor (RyR2)transfected CHO cells have shown that there is an increased variability in resting cytoplasmic Ca 2+ fluxes associated with heterologous expression of RyR2 [24,25]. Such signal variability (SV) is an index that robustly describes the signal noise in the basal Ca 2+ -dependent fluorescence from Ca 2+ dyes in the cell cytoplasm and has previously been used to characterize subtle perturbations of steady-state Ca 2+ cycling and their functional consequences (e.g. induction of apoptosis) [24,25]. To examine whether recombinant expression of YFP-PLC] resulted in subtle changes to CHO cell Ca 2+ handling, we monitored Ca 2+ with fura red since the emission peaks of YFP and fura red are separated by >100nm. As well as examining Ca 2+ dynamics in CHO cells transiently expressing recombinant protein we analysed Ca 2+ in the YFP-PLC] stable cell lines and studied the Ca 2+ response to a full range of ATP concentrations. Fig. 4a shows the Ca 2+ transients generated by different concentrations of ATP in control CHO cells and in YFP-PLC] transfected cells. We categorised the cell populations into those that expressed high or low levels of recombinant protein, based on the FACS YFP fluorescence signals and analysed the populations on this basis. As with the transient transfections, there were no differences in the Ca 2+ response to high concentrations of ATP. Nevertheless, the data in Fig. 4 show that, for low concentrations of ATP, a smaller proportion of both types of YFP-PLC] expressing cells responded with a Ca 2+ transient. The response in YFP-PLC] cells was also significantly delayed compared to control CHO cells (Fig. 4b). However, this small difference in response was independent of the expression level of YFP-PLC] since it was equivalent in those cells expressing either high or low levels of YFP-PLC] recombinant protein.

The lack of any dramatic change in the Ca 2+ homeostasis in YFP-PLC] expressing cells (Fig. 3b) did not appear to be due to any degradation of PLC portion of the YFP-PLC] fusion protein, nor in any loss of its intrinsic PLC activity. We made cytosolic extracts from YFP-PLC] cells and tested then for PLC] activity and expression. We specifically assayed PLC activity of cells in a mixed micelle assay with a free Ca 2+ concentration at 200nM which is near maximal for stimulating PLC], but below the level required for stimulating most somatic PLC isoforms [START_REF] Kouchi | Recombinant phospholipase Czeta has high Ca 2+ sensitivity and induces Ca 2+ oscillations in mouse eggs[END_REF][START_REF] Nomikos | Role of phospholipase C-{zeta} domains in Ca 2+ -dependent phosphatidylinositol 4,5-bisphosphate hydrolysis and cytoplasmic Ca 2+ oscillations[END_REF]. Fig. 5a shows that cytosolic extracts made from YFP-PLC] expressing cells had a much higher PLC activity in vitro compared with control YFP-transfected cells. This is consistent with the robust expression of the full length YFP-PLC] fusion protein (103kDa), as illustrated by the clear immunoreactive protein of ~100kDa recognised by an affinitypurified anti-PLC] antibody on the western blot (Fig. 5b).

CHO cell expressed YFP-PLC] causes Ca 2+ oscillations in mouse eggs.

One reason why PLC] may not be active in somatic cells could be a requirement for a specific factor(s) present only within the egg. To test this idea we sought to introduce recombinant PLC] synthesised in CHO cells into mouse eggs. Cell extracts were prepared from untransfected CHO cells (negative control), or cells exhibiting stable expression of YFP (~73.4 ± 0.6% positively-labelled), or else YFP-PLC] (97.6 ± 0.1% positively labelled). These extracts were prepared in the same way as previously described for making cytosolic sperm extracts [START_REF] Saunders | PLC]: a sperm-specific trigger of Ca 2+ oscillations in eggs and embryo development[END_REF]27]. Fig. 6a shows that injecting ~5% volume of YFP-PLC] CHO cell extract into mouse eggs triggered a series of Ca 2+ oscillations in all injected eggs (n=14). The frequency of the Ca 2+ oscillations observed was 11.4 ± 1.4 spikes/hour, which persisted for the duration of the recording (typically 1-1.5 hours). These Ca 2+ oscillations are similar in form to We also tested the activity of PLC] expressed in CHO cells by directly injecting these cells into the egg cytoplasm in the manner used in the somatic cell transfer procedure [START_REF] Kishigami | Somatic cell nuclear transfer in the mouse[END_REF]. Intracellular Ca 2+ was monitored immediately following microinjection of either untransfected CHO cells (negative control), or cells expressing YFP-PLC] (from an enriched stable cell population). Single CHO cells were injected into single mouse eggs then transferred to the fluorescence imaging system within 1-2 minutes. Fig. 6c shows that the injection of cells expressing YFP-PLC] induced a series of repetitive Ca 2+ oscillations in the majority (15/23) of eggs. As was observed with the CHO cell extract injections, the egg Ca 2+ response to single CHO cell injection displayed rapid spikes with the initial transient taking place within minutes of the cell injection. The frequency of the Ca 2+ oscillations observed was 4.3 ± 0.8 per hour which persisted for the duration of the recording and is similar to that seen during mouse egg fertilization [START_REF] Swann | PLC](zeta): A sperm protein that triggers Ca 2+ oscillations and egg activation in mammals[END_REF][START_REF] Miyazaki | Calcium signals for egg activation in mammals[END_REF]. In contrast, Fig. 6d shows that no change in intracellular Ca 2+ occurred following microinjection of negative control cells. These data suggest that the YFP-PLC] transfected CHO cells are expressing YFP-PLC] at levels capable of inducing Ca 2+ oscillations in mouse eggs. Furthermore, the rapid onset of Ca 2+ oscillations following injection into egg cytoplasm suggests that the PLC] expressed in CHO cells exhibits full functional activity.

Since PLC] appears to be inactive in CHO cells we considered whether this might be due to an intrinsic inability of these cells to respond to PLC]. Previously sperm extracts were used as a source of an endogenously active Ca 2+ releasing factor and they contain high levels of PLC] [START_REF] Saunders | PLC]: a sperm-specific trigger of Ca 2+ oscillations in eggs and embryo development[END_REF]. We made cytosolic sperm boar extracts and microinjected them into CHO cells. Fig. 7ad shows experiments in which boar sperm extracts were injected into cells along with a fluorescent tracer dye. In all cases, the injection of such sperm extracts failed to cause any changes in resting Ca 2+ , and their response to sperm extracts was indistinguishable from microinjection of fluorescent tracer dye alone (Fig. 7c andd). The same extracts caused high frequency Ca 2+ oscillations when injected into mouse eggs (Fig. 7e), so these data suggests that CHO cells are not able to generate respond to native sperm PLC] even when it is introduced directly by microinjection into their cytoplasm.

DISCUSSION

Consistent with previous expression studies using Venus-or YFP-tagged PLC] in COS cells we found that either YFP-or luciferase-PLC] could be expressed in CHO cells [START_REF] Coward | Expression of a fluorescent recombinant form of sperm protein phospholipase C zeta in mouse epididymal sperm by in vivo gene transfer into the testis[END_REF][START_REF] Kuroda | The role of X/Y linker region and N-terminal EFhand domain in nuclear translocation and Ca 2+ oscillation-inducing activities of phospholipase Czeta, a mammalian egg-activating factor[END_REF]. The previous reports of PLC] expression in non-gametes gave apparently contradictory results in that a study using cell lines showed that PLC] expression triggered Ca 2+ oscillations, whereas transgenic expression of PLC] throughout the mouse soma did not lead to any clear phenotypic change [START_REF] Yoshida | Borad, extopic expression of the sperm protein PLCZ1 induces parthenogenesis and ovarian tumours in mice[END_REF]. The PLC] transgenic whole soma expression levels were probably much lower than our studies with mammalian Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

cell lines since we could readily detect expression and yet expression in the transgenic mice was not detectable using a Venus-PLC] fusion protein [START_REF] Yoshida | Borad, extopic expression of the sperm protein PLCZ1 induces parthenogenesis and ovarian tumours in mice[END_REF]. Notably, our data indicates that readily detectable expression of two different PLC] fusion proteins can be achieved in a cell line with no apparent perturbation of resting Ca 2+ levels, no major change in Ca 2+ homeostasis and no altered response to agonists that increase cytoplasmic Ca 2+ . The absence of any effect of expressed PLC] on cytoplasmic Ca 2+ is surprising since previous work implied that PLC] is autonomously activated inside cells. Moreover, our data also demonstrates that the PLC] expressed within CHO cells is either already active, or can subsequently become activated, when it is assayed in vitro or introduced into eggs, thus implying that PLC] may be held in an inactive state in CHO cells. This is also consistent with our findings that cytosolic sperm extracts, that contain an endogenous active PLC] >@, were also incapable of causing any Ca 2+ release when directly microinjected into normal CHO cells.

Previous studies showing expression of mouse PLC] in somatic cells lines have reported either nuclear or cytoplasmic localization [START_REF] Coward | Expression of a fluorescent recombinant form of sperm protein phospholipase C zeta in mouse epididymal sperm by in vivo gene transfer into the testis[END_REF]. These observations could provide an explanation for some of the differences in the reported functional effects of PLC] expression. The current data shows that the specific localization of PLC] within cells may be dependent upon the elapsed time after transfection. We observed this time-dependence because in our studies we microinjected the plasmid DNA into cells, hence the timing of cDNA transduction was more precisely determined than with other methods. The localization of YFP-PLC] is primarily retained within the cytoplasm for the first 3-5 hours after injection and is subsequently found in the nucleus after 24 hours. This time period should be sufficient for some cells to complete a cell cycle and hence may imply that differential localization occurs after a round of mitosis. It was of interest that YFP-D210R PLC] did not undergo any nuclear localization in CHO cells, which is similar to what we have seen in mouse eggs (Yu and Swann, unpublished data). Since this mutant form of PLC] lacks the ability to bind its substrate this may suggest that PIP 2 binding plays some role in nuclear translocation. Previous studies with Venus-PLC] shows that a rapid (<1 hour) localization occurred into the nuclei of mouse zygotes and embryos [START_REF] Larman | Cell cycle-dependent Ca 2+ oscillations in mouse embryos are regulated by nuclear targeting of PLC[END_REF][START_REF] Yoda | Ca 2+ oscillation-inducing phospholipase C zeta expressed in mouse eggs is accumulated to the pronucleus during egg activation[END_REF], suggesting that the mouse PLC] expressed in CHO cells is either very slowly transported into the nucleus or alternatively it may require a mitotic transit to become localized. In either case, the time-dependent localization we observe may explain the variation in previous reports of localization of PLC] that may be due to the uncertain time at which transcription starts using liposome-based methods. Nevertheless, the observed nuclear localization is unlikely to account for the lack of PLC] activity in CHO cells since it occurs many hours after transfection and with high levels of YFP-PLC] protein expression some cytoplasmic localization will invariably overlap with nuclear localization. In stably transfected YFP-PLC] cells there was also some variability in nuclear localization, but the homogeneity of Ca 2+ handling in all cells confirmed that nuclear sequestration cannot readily account for an apparent lack of activity of PLC] in cells Semi-quantitative estimates suggest we have expressed relatively high levels of recombinant PLC] protein in CHO cells. From our previous calibration of luciferase expressed in mouse eggs [START_REF] Nomikos | Role of phospholipase C-{zeta} domains in Ca 2+ -dependent phosphatidylinositol 4,5-bisphosphate hydrolysis and cytoplasmic Ca 2+ oscillations[END_REF] we can determine that the luminescence from single CHO cells expressing PLC]luc (~10cps) corresponds to >100 fg of the fusion protein. We can also calculate the amount of YFP-PLC] protein present in our experiments. The amount of YFP-PLC] in the selected high-expression cells is at least ten times the detection threshold level, which is ~1PM [28]. So our levels should correspond to around 10PM. Given a CHO cell volume of ~2pl, our transfected and selected cells should contain ~2pg of YFP-PLC] protein (relative molecular mass ~100 kD). The estimated ~2pg/CHO cell is significantly greater than the 20-50fg of PLC] that was previously shown to trigger a physiological pattern of Ca 2+ oscillations in mouse eggs [START_REF] Saunders | PLC]: a sperm-specific trigger of Ca 2+ oscillations in eggs and embryo development[END_REF]. Given that CHO cells are only ~1% of the volume of a mouse egg, it appears that CHO cells can tolerate expression levels of ~200-fold PLC]-luc and ~2000-fold YFP-PLC] higher than is required to cause InsP 3 -dependent Ca 2+ oscillations in mouse eggs.

It is not clear why PLC] is apparently inactive in CHO cells. One possibility is that our transfected CHO cells have adapted to high levels of PLC] by downregulating the number of InsP 3 receptors. Given the high levels of expressed PLC] this would amount to the effective ablation of InsP 3 receptors. However, this seems unlikely to be occurring in our experiments because such downregulation only occurs after generation of considerable amounts of InsP 3 that are sufficient to cause high frequency Ca 2+ oscillations [START_REF] Brind | Inositol 1,4,5-trisphosphate receptors are downregulated in mouse oocytes in response to sperm or adenophostin A but not to increases in intracellular Ca 2+ or egg activation[END_REF][START_REF] Jellerette | Down-regulation of the inositol 1,4,5-trisphosphate receptor in mouse eggs following fertilization or parthenogenetic activation[END_REF]. Notably, we failed to detect any Ca 2+ changes, even at early times after transfection with PLC]. There is also insufficient time for InsP 3 receptor downrgulation to explain the lack of response to the sperm extracts. Furthermore, any InsP 3 receptor down-regulation leads to a loss of agonist-induced Ca 2+ increase [29]. We did find that there was a reduction in the Ca 2+ response to low doses of ATP for stably transfected of YFP-PLC] cells. However this effect was not related to the expression level of YFP-PLC] It is also noteworthy that previous work on ATP-induced Ca 2+ response in CHO cells showed that PKC stimulation causes a marked feedback inhibition of Ca 2+ transients [ 30 ]. Therefore, if PLC] was causing sustained PIP 2 hydrolysis and hence DAG production, we would anticipate a substantial block of the ATP Ca 2+ transients. Since we consistently failed to detect any change in Ca 2+ response at high ATP concentrations, the simplest interpretation of our data is that the recombinant PLC] is inactivated when expressed in CHO cells.

The apparent lack of PLC] activity in transfected CHO cells, coupled with clear Ca 2+ releasing activity when these cells are introduced into eggs, resembles the physiological situation with regards to fertilization. Given the size difference between the sperm and egg, the mammalian sperm should contain levels of PLC] that must be at least three orders of magnitude higher than is effective in causing Ca 2+ release in eggs. The mammalian sperm has a resting Ca 2+ level similar to that of other cells and can exhibit Ca 2+ increase in response to agonists, some of which appear to work via InsP 3 generation [START_REF] Publicover | Ca 2+ signalling in the control of motility and guidance in mammalian sperm[END_REF]. This implies that the PLC] present in sperm is also inactive. However, PLC] is capable of causing the first Ca 2+ increase at fertilization within . Therefore, the experiments where we inject transfected CHO cells into the egg mimics the fertilization of the egg by a sperm in that an inactive PLC] rapidly becomes active upon exposure to the egg cytoplasm. The high intrinsic PLC activity of sperm extracts also resembles the high in vitro PLC activity we find in of YFP-PLC] transfected CHO cell cytosolic extracts. There may be two explanations for such effects. One is that PLC] may be held in an inactive state in sperm, and that whatever the mechanism the inhibitory components are also present in CHO cells. Otherwise the activity of PLC] observed upon entry into eggs could be because it interacts specifically with a protein(s) that is only expressed in the egg cytoplasm. This possibility would be consistent with the fact that other known mammalian PI-specific PLCs are also stimulated by interaction with accessory proteins [32].

Biochemical

  Journal Immediate Publication. Published on 22 Jun 2011 as manuscript BJ20101581 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101581

Biochemical

  Journal Immediate Publication. Published on 22 Jun 2011 as manuscript BJ20101581 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101581

Biochemical

  Journal Immediate Publication. Published on 22 Jun 2011 as manuscript BJ20101581 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101581 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 22 Jun 2011 as manuscript BJ20101581 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101581 A c c e p t e d M a n u s c r i p t luminescence in single CHO cells expressing PLC]-luc was in the range of 1-10cps, compared with a range of 1-15cps from cells injected with control Luc vector. Fig 1c shows typical recordings of Ca 2+ levels in PLC]-luc and luc control cells as well as OGBD control injected cells 5 hours after microinjection.

  Fig.2b shows that after such sorting there was a clear difference in YFP Biochemical Journal Immediate Publication. Published on 22 Jun 2011 as manuscript BJ20101581 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101581 A c c e p t e d M a n u s c r i p t channel fluorescence between selected cells and the wild-type CHO cells.

  Fig 3b bottom right panel shows the analysis of fura red fluorescence noise as measured by signal variability for YFP, YFP-PLC] and YFP-D210 PLC] transfected cells, and for control injected cells at 5 hours and 24 hours after microinjection. There was no difference Biochemical Journal Immediate Publication. Published on 22 Jun 2011 as manuscript BJ20101581 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101581A c c e p t e d M a n u s c r i p t observed in the signal variability from all four groups of cells. These experiments suggest that there are no subtle alterations in Ca 2+ homeostasis in CHO cells expressing YFP-PLC], supporting the conclusion that the YFP-PLC] expressed is functionally inactive in CHO cells.

Biochemical

  Journal Immediate Publication. Published on 22 Jun 2011 as manuscript BJ20101581 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101581 A c c e p t e d M a n u s c r i p t those previously described after injection of mouse eggs with cytosolic sperm extracts [27]. Injection of extracts from untransfected CHO cells, or YFPtransfected CHO cells did not cause any changes in Ca 2+ in mouse eggs (Fig. 6b).

Biochemical

  Journal Immediate Publication. Published on 22 Jun 2011 as manuscript BJ20101581 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101581 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 22 Jun 2011 as manuscript BJ20101581 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101581 A c c e p t e d M a n u s c r i p t

Biochemical

  Journal Immediate Publication. Published on 22 Jun 2011 as manuscript BJ20101581 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101581 A c c e p t e d M a n u s c r i p t minutes of sperm-egg membrane fusion [31]

  s c r i p t 24. George CH, Higgs GV, Mackrill JJ, Lai FA. (2003) Dysregulated ryanodine receptors mediate cellular toxicity: restoration of normal phenotype by FKBP12.6. J Biol. Chem. 278, 28856-28864. 25. George,C.H., Jundi,H., Walters,N., Thomas,N.L., West,R.R. and Lai,F.A. (2006). Arrhythmogenic mutation-linked defects in ryanodine receptor autoregulation reveal a novel mechanism of Ca 2+ release channel dysfunction. Circ. Res. 98, 88-97. 26. Iredale,P.A. and Hill,S.J. (1993). Increases in intracellular calcium via activation of an endogenous P2-purinoceptor in cultured CHO-K1 cells. Br J Pharmacol, 110, 1305-10. 27. Parrington,J. Jones,K.T., Lai,F.A. and Swann,K. (1999) The soluble sperm factor that causes Ca 2+ release from sea urchin egg homogenates also triggers Ca 2+ oscillations after injection into mouse eggs. Biochem. J. 341, 1-4. 28. Niswender,K.D., Blackman, S.M., Rohde, L., Magnuson, M.A. and Piston D.W. (1995). Quantitative imaging of green fluorescent protein in cultured cells: comparison of microscopic techniques, use in fusion proteins and detection limits. J. Microsc, 180, 109-16. 29. Wojcikiewicz,R.J. Furuichi,T., Nakade,S., Mikoshiba,K. and Nahorski,S.R. (1994) Muscarinic receptor activation down-regulates the type I inositol 1,4,5trisphosphate receptor by accelerating its degredation. J. Biol. Chem. 269, 7963-7969 30. Strassheim,D. and Williams,C.L. (2000) P2Y 2 purinergic and M 3 muscarinic acetylcholine receptors activate different phospholipase CE isoforms that are uniquely susceptible to protein kinase C dependent phosphorylation and inactivation. J. Biol. Chem. 275, 39767-39772 31. Lawrence,Y., Whitaker,M. and Swann,K. (1997) Sperm-egg fusion is the prelude to the initial Ca 2+ increase at fertilization in the mouse. Development 124, 223-241 32. Suh,PG., Park,J.I., Manzoli,L., Cocco,L., Peak,J.C., Katan,M., Fukami,K., Kataoka,T., Yun,S. and Ryu,S.H. (2008) BMB Reports 41, 415-433 Biochemical Journal Immediate Publication. Published on 22 Jun 2011 as manuscript BJ20101581 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101581 A c c e p t e d M a n u s c r i p t

Figure 1 .

 1 Figure 1. Ca 2+ dynamics in CHO cells expressing PLC] ]-luc Luminescence (a) and fluorescence (b) images are shown of a representative group of CHO cells injected with plasmid DNA encoding PLC]-luc and OGBD. Note that not all the injected cells (shown by fluorescence) are expressing luciferase (shown by the luminescence). In c) are shown representative recordings of fluorescence (from OGBD in arbitrary units (a.u.) indicating Ca 2+ changes observed in cells within each experimental group. Cells were imaged for fluorescence for at least 30 minutes prior to the addition of ATP (100μM, at the arrow). The histogram in d) shows the mean and SEM for the resting Ca 2+ levels, estimated after addition of ionomycin, for CHO cells injected with luc, or PLC]-luc DNA, as well as control cells injected with OGBD only. Bars are coded as indicated on the right of graph. There was no significant difference in the resting Ca2+ levels between the different groups of cells at any one time point (ANOVA).

Figure 2 .

 2 Figure 2. Localization of YFP-PLC] expressed in CHO cells In a) is shown the localisation of YFP, the enzymatically inactive YFP-D210R PLC] mutant and YFP-PLC], in transiently transfected CHO cells. Confocal images acquired at different times after microinjection are indicated by the times above the panel. Each image is representative of the pattern of localisation observed in the majority of cells at each time point. The number of cells displaying the indicated localisation pattern is shown in white followed by the total number of cells analysed in each instance. The scale bar represents 10μm. In b) is shown the FACS analysis of cells sorted for expression of YFP-PLC] with expressing cells above in grey and control CHO cells below in black. The fluorescence (F) is plotted for cells against the forward scatter (FSC). On the right are two confocal fluorescence images of cells with either nuclear or overall staining of YFP-PLC], with again the proportion of cells with the pattern shown indicated by the numbers on each image.

Figure 3 .

 3 Figure 3. Expression of PLC] has no effect on the ATP-induced Ca 2+ transient in transiently transfected CHO cells. In a) are shown groups of sample traces of fluo3 fluorescence increases in response (F in arbitrary units) to 100PM ATP addition. The responses are shown for control CHO cells and for cell expressing YFP alone, YFP-PLC] , or YFP-D210R PLC], at 5 hours and 24 hours after injection of DNA. Data obtained from six discrete cells randomly distributed across the field of view is shown (grey) with with the average of that group plotted in black. In b) is shown the analysis of parameters of the ATP-induced Ca 2+ transient at time points between 3 and 24 hours in control CHO cells (open circles), and for cells expressing YFP (open squares),YFP-D210R PLC] (black circles) or YFP-PLC] (black squares). Data are given as mean ± SEM (n=4 separate experiments each and the total number of cells analysed is shown in parentheses). Each time point analysis involved 43-480 individual cells, and there were no significant differences between any of the groups of cells at the time points of experiments (ANOVA).

Figure 4 .

 4 Figure 4. ATP-induced Ca 2+ transients responses of YFP-PLC] ] stably transfected CHO cells. In a) stably YFP-PLC] transfected and wild type CHO cells were stimulated by the addition of ATP over a range of concentrations and the Ca 2+ response monitored with fura red fluorescence ratio (in arbitrary units). The concentration of ATP added (at the arrow head), is shown down the left hand side and proportion of cells showing the displayed response indicated above each trace. The YFP-PLC] cells were divided into 'high' and 'low' level expression depending upon the level of YFP fluorescence (see Fig. S1b), and the data is plotted for each class separately. In b) the dose response curves are plotted for cells in terms of the percentage of cells showing a Ca 2+ increase as well as the latency between addition of ATP and the start of the Ca 2+ transient. Data is plotted for control CHO cells (black circles), and again for cells expressing high (open circles) versus low levels (black triangles) of YFP-PLC]. Data with an * is significantly different from control CHO cells (students t test, p<0.05).

Figure 5 .

 5 Figure 5. PLC enzyme activity and expression of YFP-PLC] in stably transfected CHO cells. Cells that had been sorted for stable expression of YFP-PLC] were used to make cytosolic extracts. In a) the PLC enzymatic activity in cell extracts is shown for YFP-PLC] transfected cells (open bar) and control YFP-transfected CHO cells (closed bar). The specific activity was 165 ± 9 nmol/min/mg for YFP-PLC] transfected cells, and 23 ± 10 nmol/min/mg for YFP control-transfected cells. Panel b) shows a Coomassie blue-stained SDS gel (left) and a Western blot (right) for the same batches of extracts. The Western blot was developed following incubation with an affinitypurified rabbit polyclonal antibody to PLC].

Figure 6 .

 6 Figure 6. Ca 2+ dynamics in mouse eggs after injection of CHO cell or CHO cell extracts Intracellular Ca 2+ responses in mouse eggs were monitored following the microinjection of extracts prepared from CHO cells. In a) a representative trace after injection of extract prepared from the YFP-PLC] cell line is shown. Ca 2+ oscillations were seen in 14/14 eggs (11.4 ± 1.4 spikes/hour). In b) extracts from YFP-transfected or non-transfected CHO cells produced no Ca 2+ response in any of the 6 or 4 eggs injected, respectively. The recording in c) is from an egg injected with a whole CHO cell that was expressing YFP-PLC]. Ca 2+ oscillations were seen in 15/23 such whole CHO cell-injected eggs (4.3 ± 0.8 spikes/hour). In d) is shown an example of an egg injected with a whole CHO cell expressing YFP alone. No Ca 2+ response was observed in any of the 14 injected eggs. Each trace is a recording from a single egg and is representative of the observations made in each experimental group. Traces a) and b) were recorded with the use of fura-red, while c) and d) used fura-PE3.

Figure 7 .

 7 Figure 7. Microinjection of YFP-PLC] ] transfected cell extracts into control CHO cells. Boar sperm extracts were microinjected into control CHO cells and then imaged for 20 mins. The protocol for the experiments are shown in a) and images in b) where taken before (Pre) during and after (Post) the microinjection (MI) of extracts. The sample images in b) show a brightfield image of cells (BF), the Ca 2+ -dependent fluorescence of fluo3 (Ca 2+ ), and the fluorescence of the Alexa647 tracer (Alexa 645) that was used to visualise successful injection. In c) the black traces show typical fluo3 fluorescence recordings from the 'Ca 2+ imaging' phase of the experiment (time 10-30, panel (a)) following microinjection (MI) with boar sperm extract (BSE) mixed with Alexa647 (BSE:dye), or injection of Alexa647 alone (dye only).Representative traces obtained from those cells that had not been microinjected (non-MI), but were present in the same field of view, are given (grey). In d) are shown the average resting Ca 2+ levels and the Ca 2+ noise after microinjection of boar sperm extract or dye alone, compared with the same parameters from CHO cells in the same dish that were not injected (non-MI). >40 injections were performed per coverslip (n=4), and the efficacy of injection, determined by the visualisation and retention of Alexa647 over the 20minute imaging period, exceeded 75%. All data in c) and d) are taken from cells that retained Alexa647 to levels of fluorescence >75% of initial values following the 20minute Ca2+ imaging window. In e) a sample of the boar sperm extract mixed with an equal volume of Alexa647 that was microinjected into CHO cells was microinjected into unfertilized mouse eggs. The illustrated pattern of Ca 2+ oscillations measured with Rhod dextran fluorescence (F) was seen in 6/6 injected eggs.

  Fig.1

F

  Fig. 2

Fig. 4 Fura

 4 Fig. 4

  Fig. 5

Fig. 7 F

 7 Fig. 6

Table 1 . Analysis of Ca 2+ dynamics in CHO cells.

 1

		Biochemical Journal Immediate Publication. Published on 22 Jun 2011 as manuscript BJ20101581
			3 hours
		% of cells	Frequency	% of cells	n number
		oscillating	(spikes/hr)	responding to	(number of
	THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101581	ATP 99.8 +/-0.3 99.3 +/-0.5 100 % of cells responding to ATP 100 99.8 +/-0.3 100 % of cells responding to ATP 99.0 +/-0.4 99.0 +/ -0.6 99.3 +/ -0.3 CHO cells were injected with OGBD + plasmid DNA encoding luciferase or cells) Luc 35.1 +/-3.6 6.9 +/-0.9 4 (47) PLC]-luc 36.0 +/-3.0 7.9 +/-1.0 4 (45) OGBD 31.9 +/-3.7 7.0 +/-0.7 3 (18) 5 hours % of cells oscillating Frequency (spikes/hr) n number (number of cells) Luc 25.1 +/-8.6 6.0 +/-0.9 4 (29) PLC]-luc 31.0 +/-3.2 5.7 +/-0.6 4 (50) OGBD 31.1 +/-5.9 6.0 +/-0.9 3 (13) 24 hours % of cells oscillating Frequency (spikes/hr) n number (number of cells) Luc 31.0 +/-4.8 5.9 +/-0.8 4 (22) PLC]-luc 33.4 +/-3.9 6.2 +/-1.0 4(21) OGBD 29.0 +/-2.4 6.5 +/-0.8 4 (14) PLC]-luc or were injected with OGBD alone. Cells were imaged for OGBD fluorescence at 3, 5 and 24 hours following microinjection. Data is given as mean ± S.E.M. n= the number of experimental runs. The total number of cells analysed is shown in parentheses. A c c e p t e d M a n u s c r i p t

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.© 2011 The Authors Journal compilation © 2011 Portland Press Limited

© 2011 The Authors Journal compilation © 2011 Portland Press Limited

ACKNOWLEDGMENTS.

We thank Nazar Amso for his support of SVP on the early part of this project.

FUNDING

This work was supported by grants from the Biotechnology and Biological Sciences Research Council (BB/C513142/1) , Medical Research Council (G0801676) and the Wellcome Trust (080701/Z/06/Z). Biochemical Journal Immediate Publication. Published on 22 Jun 2011 as manuscript BJ20101581 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20101581

AUTHOR CONTRIBUTION

Sally Phillips carried out most of the experiments on CHO cells, Yuansong Yu carried out experiments on eggs and some of studies on CHO cells. Andreas Rossbach prepared the DNA constructs. Chris George contributed to the analysis of confocal studies of CHO cells and microinjected extracts for Fig 7 .  Michail Nomikos conducted biochemical analysis of PLC protein, Vyronia Vassilakopoulou, Evangelia Livaniou and Bevan Cumbes prepared the rabbit anti-PLC antibody. F. Anthony Lai contributed towards the conception of the project and drafting of the manuscript. Karl Swann supervised the project and prepared the final version of the manuscript.