Joanna Fombonne

Pierre-Antoine Bissey

Chantal Thibert

Catherine Guix

Remy Sadoul

Patrick Mehlen
email: patrick.mehlen@lyon.unicancer.fr

The Patched dependence receptor triggers apoptosis through ubiquitination of caspase-9

Keywords: Ptc, Patched; Ub, Ubiquitin -

Patched (Ptc), the main receptor for Sonic Hedghog, is a tumor suppressor.

Ptc has been shown to be a dependence receptor, and as such triggers apoptosis in the absence of its ligand. This apoptosis induction occurs through the recruitment by the Ptc intracellular domain of a caspase-activating complex, which includes the adaptor proteins DRAL and TUCAN, and the apical caspase-9. We show here that this caspase-activating complex also includes the E3 ubiquitin ligase NEDD4. We demonstrate that Ptc-mediated apoptosis and Ptc-induced caspase-9 activation require NEDD4. We show that Ptc, but not Bax, the prototypical inducer of the intrinsic cell death pathway, triggers polyubiquitination of caspase-9. Moreover, a caspase-9 mutant that could not be ubiquitinated failed to mediate Ptc-induced apoptosis. Taken together, these data support the view that the Ptc dependence receptor specifically allows the activation of caspase-9 via its ubiquitination, which occurs via the recruitment by Ptc of NEDD4.

Introduction

Apoptosis is a central mechanism during embryonic development and adult tissue maintenance. Its dysregulation is causally implicated in numerous pathologies such as cancer, neurodegenerative disorders, and autoimmune diseases [START_REF] Zornig | Apoptosis regulators and their role in tumorigenesis[END_REF]. In higher organisms, apoptosis typically occurs through the activation of caspases, cysteinyl aspartate proteases that are the central executioners of the cell death program [START_REF] Green | The central executioners of apoptosis: caspases or mitochondria?[END_REF][START_REF] Boatright | Mechanisms of caspase activation[END_REF]. As caspases display the property to cleave and activate other caspases in an amplification loop, apoptosis induction is often seen as the result of the initial activation of the so-called apical caspases, -i.e. caspase-8, 10 or caspase-9 [START_REF] Boatright | A unified model for apical caspase activation[END_REF].

Such apical caspase activation appears to occur in specific and dedicated caspase-activating complexes [START_REF] Mace | Molecular cell death platforms and assemblies[END_REF]. While the DISC complex allows the recruitment and activation of caspase-8 by death receptors, the apoptosome complex allows the activation of caspase-9 (5-7). Caspase-9 activation typically occurs via the apoptosome, whose composition and mechanism of action have been elucidated [START_REF] Pop | The apoptosome activates caspase-9 by dimerization[END_REF][START_REF] Riedl | The apoptosome: signalling platform of cell death[END_REF]. However, caspase-9 has also been shown to be activated directly by a specific type of receptor called dependence receptors [START_REF] Forcet | The dependence receptor DCC (deleted in colorectal cancer) defines an alternative mechanism for caspase activation[END_REF][START_REF] Mille | The Patched dependence receptor triggers apoptosis through a DRAL-caspase-9 complex[END_REF]. Dependence receptors display dual signaling that is dependent on ligand availability: in the presence of their trophic ligands, they transduce various signals, whereas in the absence of their ligands, they are not inactive but rather actively trigger apoptosis [START_REF] Bredesen | Receptors that mediate cellular dependence[END_REF][START_REF] Goldschneider | Dependence receptors: a new paradigm in cell signaling and cancer therapy[END_REF][START_REF] Mehlen | Dependence receptors: from basic research to drug development[END_REF]. We recently showed that the dependence receptor Patched triggers apoptosis in the absence of its ligand Sonic Hedgehog through the formation of a caspase-activating complex -i.e., the dependosome-that includes DRAL, TUCAN, and caspase-9 [START_REF] Mille | The Patched dependence receptor triggers apoptosis through a DRAL-caspase-9 complex[END_REF]. We investigated here the mechanism by which this Ptccomplex triggers caspase-9 activation and we observed the recruitment of Nedd4 in this complex.

Nedd4 plays a major role in protein ubiquitination, which usually requires coordinated action of a Ub-activating enzyme (E1), a Ub-conjugating enzyme (E2), and a Ub ligase (E3) such as Nedd4. Ubiquitination was initially described to play a major role in protein degradation by providing a signal for proteasome-mediated degradation. However, it has been shown recently that ubiquitination is important not only for protein degradation but also for the regulation of both pro-survival and proapoptotic signals [START_REF] Wertz | Signaling to NF-kappaB: regulation by ubiquitination Cold Spring[END_REF][START_REF] Sun | Bcr-Abl ubiquitination and Usp9x inhibition block kinase signaling and promote CML cell apoptosis[END_REF][START_REF] Vucic | Ubiquitylation in apoptosis: a posttranslational modification at the edge of life and death[END_REF]. Considering recent data describing the ubiquitination of caspase-8 as a mechanism for caspase-8 activation [START_REF] Jin | Cullin3-based polyubiquitination and p62-dependent aggregation of caspase-8 mediate extrinsic apoptosis signaling[END_REF], we investigated whether caspase-9 activation in the Ptc-complex is associated with caspase-9 ubiquitination.

We show here that while caspase-9 is not ubiquitinated upon activation of the intrinsic pathway for apoptosis, caspase-9 is ubiquitinated in the Ptc complex upon SHH withdrawal and we present evidence that caspase-9 ubiquitination in Ptc complex is required for Ptc-mediated apoptosis.

Results and Discussion

Following a two-hybrid screen, using as bait the pro-apoptotic domain of Ptc (Ptc 1165-1392, shown to be required for apoptosis and to interact with caspase-9 [START_REF] Mille | The Patched dependence receptor triggers apoptosis through a DRAL-caspase-9 complex[END_REF][START_REF] Thibert | Inhibition of neuroepithelial patched-induced apoptosis by sonic hedgehog[END_REF]), Nedd4, an E3 ubiquitin ligase, was identified as a putative partner of Ptc (Fig. 1A). Nedd4 was previously shown to interact with Ptc in Drosophila [START_REF] Lu | The C-terminal tail of the Hedgehog receptor Patched regulates both localization and turnover[END_REF]. Nedd4 was further demonstrated to interact with Ptc by co-immunoprecipitation in HEK293T cells (Fig. 1B). This interaction appeared specific since, whereas full-length Ptc and Ptc 1-1392 clearly interacted with Nedd4, Ptc deleted of the seventh intracellular domain (Ptc 1-1165) failed to pull down Nedd4 (Fig. 1B). The Ptc/Nedd4 interaction was not affected by SHH presence, suggesting that Ptc constitutively interacts with Nedd4 (Fig. 1C).

Nedd4 as Ub ligase (E3) plays a major role in protein ubiquitination. Because ubiquitination was initially described to play a major role in protein degradation by providing a signal for proteasome-mediated degradation, the Nedd4 interaction with Ptc might at first appear to be a mechanism to increase proteasome-dependent degradation of Ptc and thus to negatively regulate Ptc-induced apoptosis. However, as shown in Fig. 1D, forced expression of Nedd4 in HEK293T failed to reduce the level of Ptc expression, and more importantly, dramatically potentiated Ptc-induced apoptosis as measured by caspase-3 activation. This effect was not related to protein degradation, since treatment with the proteasome inhibitor MG132, while blocking degradation of proteins such as P21, had no effect on the Nedd4-mediated potentiation of Ptc-induced caspase-3 activation (Fig. 1E and Suppl. Fig. 1A). A similar potentiating effect was observed when cell death per se rather than caspase activation was analysed (Fig. 1F). This effect appeared to be specific for Nedd4 since another E3 ubiquitin ligase-mdm2-did not potentiate Ptc-induced apoptosis (Suppl. Fig. 1B). Potentiation of Ptc-induced apoptosis was due to the ligase activity of Nedd4, since forced expression of Nedd4 C1286S, a Nedd4 catalytic dead mutant, failed to promote Ptc-induced caspase-3 activation (Fig. 1G). We next performed a loss-of-function experiment to determine whether Nedd4 is required for Ptc-mediated apoptosis. As shown in Fig. 1H, Ptc-induced apoptosis was completely inhibited when Nedd4 was silenced by a siRNA approach (Suppl. Fig. 1C). Thus Nedd4 promotes Ptc-induced apoptosis via its E3 ligase activity and is required for Ptc pro-apoptotic signaling.

Ptc triggers apoptosis through the recruitment of a caspase-activating complex -the dependosome-that includes DRAL and caspase-9, and leads to direct caspase-9 activation. Nedd4 was shown to be a component of this caspaseactivating complex since, in HEK293T co-immunoprecipitation, Nedd4 was pulled down with both DRAL and caspase-9 (Fig. 2AB). The presence of SHH, which has been shown to disrupt this caspase-activating complex [START_REF] Mille | The Patched dependence receptor triggers apoptosis through a DRAL-caspase-9 complex[END_REF], inhibited the interaction of Nedd4 with caspase-9 (Fig. 2B). Moreover, while Ptc triggers an increased caspase-9 activation when Nedd4 was overexpressed, such Ptc-mediated caspase-9 activation was not detected upon forced expression of the Nedd4 catalytic dead mutant (Fig. 2C). Conversely Nedd4 silencing by siRNA transfection inhibited caspase-9 activation (Fig. 2D), supporting the view that Nedd4 is recruited to the dependosome in the absence of SHH and participates in caspase-9 activation.

We therefore investigated the mechanism by which Nedd4 E3 ligase activity within the dependosome complex promotes caspase-9 activation, and consequently cell death. Considering recent data describing the ubiquitination of caspase-8 as a mechanism for caspase-8 activation (16), we investigated whether caspase-9 activation in the dependosome is associated with caspase-9 ubiquitination. In HEK293T cells forced to express a caspase-9 mutated at its catalytic cysteine (this mutant was used instead of wild-type caspase-9 to avoid caspase amplification), a caspase-9 immunoblot performed after cell extraction with sodium dodecyl sulfate (but not the milder detergent Triton X-100) revealed putative ubiquitination of caspase-9 upon Ptc expression, while Bax, the prototypical inducer of the intrinsic pathway for apoptosis, had no effect (Fig. 3A). As expected from the dependence receptor paradigm, the addition of SHH inhibited Ptc-associated caspase-9 ubiquitination. Similarly, enforced expression of Ptc deleted of the pro-apoptotic domain (Ptc 1-1165) was not associated with caspase-9 ubiquitination (Fig. 3A).

Caspase-9 immunoprecipitation, performed under denaturing conditions to disrupt binding to other proteins, followed by Ub immunoblot, confirmed the ubiquitination of caspase-9 (Fig. 3B). Ubiquitination of endogenous caspase-9 was also observed upon Ptc expression after immunoprecipitation of HA-tagged Ub (Fig. 3C). To further analyse whether the caspase-9 recruited to the dependosome is ubiquitinated, we performed Ptc immunoprecipitation and assayed for caspase-9 ubiquitination. As shown in Fig. 3D, the Ptc-associated caspase-9 was ubiquitinated. Thus, in the absence of SHH, Ptc triggers ubiquitination of caspase-9 within the dependosome.

Ubiquitination involves covalent attachment of Ub to proteins, and this occurs either through addition of monoubiquitin or polyubiquitin chains linked via internal lysines. While K48-linked polyUb frequently provides a signal for proteasomemediated degradation, K63-linked chains are more frequently associated with a functional effect on the targeted protein [START_REF] Martinez-Forero | Lysine 63 polyubiquitination in immunotherapy and in cancer-promoting inflammation[END_REF]. The pattern of ubiquination of Ptcmediated caspase-9 ubiquitination supports a poly-ubiquitination of caspase-9 (Fig. 3ABD), and we therefore investigated the nature of this polyubiquitination. Using specific antibodies recognizing either K48 or K63-linked chain, we observed that Ptc did not trigger any K48-Ub modification of caspase-9, while caspase-9 was clearly covalently linked to K63-Ub (Fig. 3E).

To determine whether this caspase-9 K63-ubiquitination could affect caspase-9 activity, we attempted to silence caspase-9 ubiquitination. Caspase-9 contains in its P10 fragment 5 putative lysines that represent potential ubiquitination sites (Fig. 4A).

We therefore mutated these 5 lysines in caspase-9: K394/398/409/410/414R. Enforced expression of Ptc was associated with a strong reduction in caspase-9 ubiquitination when the caspase-9 mutant-i.e., caspase-9 5KR-was used instead of wild-type caspase-9 (Fig. 4B). We therefore assessed caspase activation in response to Bax or Ptc expression in the presence of either wild-type caspase-9 or the caspase-9 5KR mutant. A first set of experiments was performed in HEK293T cells.

As shown in Fig. 4C, while Ptc triggered caspase-3 activation in wild-type caspase-9 settings, Ptc-induced caspase-3 activation was markedly reduced when caspase-9 5KR was used. The suggested ubiquitination-dependent activation of caspase is specific for Ptc, since Bax triggered caspase activation whether caspase-9 wild-type or 5KR was used (Fig. 4D). Since, in the HEK293T cells, endogenous caspase-9 is expressed and may interfere with the ectopically expressed caspase-9, we performed the same experiment in MEF cells deficient for caspase-9 (Suppl. Fig. 1D) [START_REF] Marsden | Apoptosis initiated by Bcl-2-regulated caspase activation independently of the cytochrome c/Apaf-1/caspase-9 apoptosome[END_REF]. As shown in Fig. 4E, while expression of wild-type caspase-9 allowed Ptc and Baxinduced caspase activation, expression of caspase-9 5KR was associated with apoptosis induction via Bax but not with Ptc. Thus, Ptc triggers caspase activation via ubiquination of caspase-9. Because caspase-9 cleavage has been shown to play a role in caspase-9 activation and amplification(6), HEK293T cells were forced to express either a non-cleavable form of caspase-9 or a non-cleavable caspase-9 mutated at the 5 lysines that represent potential ubiquitination sites. As shown in Fig. 4F, Ptc triggered caspase-3 activation in uncleavable caspase-9 settings, while it did not when uncleavable caspase-9 5KR was used. Thus, caspase-9 activation mediated by Ptc requires Nedd4 and ubiquitination of caspase-9, but no caspase-9 cleavage.

Together these data support the view that Ptc triggers direct activation of caspase-9 within the dependosome by a mechanism that requires Nedd4 recruitment and caspase-9 ubiquitination. Interestingly, there is a strong analogy with the elegant observation made by Ashkenazi and colleagues, who first described the contribution of cullin3-based polyubiquitination of caspase-8 in caspase-8 activation [START_REF] Jin | Cullin3-based polyubiquitination and p62-dependent aggregation of caspase-8 mediate extrinsic apoptosis signaling[END_REF].

Interestingly, both type of receptors-the death receptors DR4 and DR5, and the dependence receptor Ptc-recruit at the membrane caspase-activating complexes: the DISC in the former case, and the dependosome in the latter. These then trigger initiator caspase activation, caspase-8 by the death receptors DR4 and DR5, and caspase-9 by the dependence receptor Ptc. It is even more intriguing to note that, while caspase-9 ubiquitination appears to be a prerequisite for caspase-9 activation in the Ptc dependosome, we failed to detect any caspase-9 ubiquitination upon apoptosome activation, and we failed to observe, upon Bax overexpression, any loss of caspase-9 activity using a Ub-dead mutant of caspase-9. Thus, depending on whether an extrinsic or intrinsic pathway is used, two different mechanisms of initiator caspase activation are employed: one requires initiator caspase ubiquitination, be it caspase-8 or caspase-9, and occurs at the membrane; whereas the other is independent of initiator caspase ubiquitination, and occurs in the cytosol. One may wonder what is the role of adding K63 polyubiquitination to promote caspase-9 activation, specifically for the dependosome and not the apoptosome. In the case of caspase-8, ubiquitination was shown to allow caspase-8 translocation from receptor-associated DISC to Ub-rich foci, although to date there has been no evidence that these Ub-rich foci actively participate in caspase-8 activity. In the case of caspase-9, future biochemical work will need to be performed to define the role of polyubiquitination in caspase-9 catalytic activation.

Experimental Procedures

Site directed mutagenesis and plasmid constructs.

The pRK5-Ptc, pRK5-Ptc-HA, constructs encoding Ptc deleted of its last intracellular domain (pRK5-PtcD7IC) or after caspase cleavage site (D1392) (pRK5-Ptc1-1392) prK5-SHH and pcDNA3-DRAL-3xflagM2 were described in [START_REF] Mille | The Patched dependence receptor triggers apoptosis through a DRAL-caspase-9 complex[END_REF]. The pcDNA3-Bax-HA, the pcDNA3-Nedd4-myc and the pCMV-Mdm2 were respectively a kind gift from Dr. A. Arrigo Dr. G. Melino and Dr L. Corbo. For direct two-hybrid, the coding sequence of the 4 WW domains of Nedd4 was amplified from pcDNA3-Nedd4-myc by PCR using the primers Nedd4WW-F and Nedd4WW-R (sequences given in Suppl.

Table1) and then inserted into pGADT7 plasmid by digestion through EcoRI-XhoI.

The pGBKT7-Ptc1165-1392 has been described previously [START_REF] Mille | The Patched dependence receptor triggers apoptosis through a DRAL-caspase-9 complex[END_REF]. For immunoblot experiments, in order to detect ubiquitination, either pRK5-Ubiquitin-HA or pcDNA3-Ubiquitin-3xflagM2 was used. The pcDNA3-Nedd4C1286S-myc was obtained by directed mutagenesis via Quickchange strategy (Stratagene) on pcDNA3-Nedd4-myc with the primers indicated in Suppl. Table1 (Nedd4C1286S-F and Nedd4C1286S-R).

For co-immunoprecipitations, and immunoblots, the pcDNA3-caspase-9 dominant negative (pcDNA3-C9DN) was used for transfection [START_REF] Forcet | The dependence receptor DCC (deleted in colorectal cancer) defines an alternative mechanism for caspase activation[END_REF]. For caspase activity assay pcDNA3-active caspase-9 was used (pcDNA3-C9) [START_REF] Forcet | The dependence receptor DCC (deleted in colorectal cancer) defines an alternative mechanism for caspase activation[END_REF]. pcDNA3-C9DN or pcDNA3-C9 were used as templates to generate the pcDNA3-non cleavable caspase-9 dominant negative (pcDNA3-NCC9-DN) or pcDNA3-non cleavable caspase-9 (pcDNA3-NCC9) constructs by inserting two mutations (D315/330A) via Quickchange strategy (Primers are indicated in Suppl. Table1). The pcDNA3-C9DN, pcDNA3-NCC9-DN, pcDNA3-C9 and pcDNA3-NCC9 were then used as templates to generate the mutation of 5 lysines into arginine located at the C-terminus part of the caspase-9 (K394/398/409/410/414R).

Cell cultures, transfection procedures, reagents

Transient transfection of Human Embryonic Kidney 293T cells (HEK293T) was performed with calcium phosphate for co-immunoprecipitation or with Jetprime (Polyplus) for cell death assay and immunoblot according to the manufacturer's instructions. Caspase-9 null Mouse Embryonic Fibroblasts (MEF C9-/-) were a kind gift from Dr. D.Green. MEF cells were cultured in DMEM media supplemented with 10% calf fetal serum and 2µl of β-mercaptoethanol. MEF cells were transfected with Jetprime (Polyplus) for cell death assay. Recombinant SHH-N was from R&D systems and were added at the time of transfection at 600ng/ml. MG132 was used at 0.5 µg/ml for 2h and were purchased from Sigma. For siRNA experiments, cells were transfected with 60 pmols siRNA using Jetprime reagent. Nedd4 and control siRNAs were from Sigma.

Two-hybrid analysis

Matchmaker two-hybrid system III (Clontech) was used according to the manufacturer's instructions using AH109 yeast co-transformed with pGBKT7-DNA binding domain GAL4 fused to Ptc 1165-1392 (pGBKT7-Ptc7IC) and the pGADT7-GAL4 transcriptional activation domain AD fused to 4 WW domains of Nedd4 (pGADT7-WWNedd4)(Clontech). Cells were then allowed to grow in the absence of leucine, tryptophane, adenine, and histidine, and in the presence of X-alpha Gal (5bromo-4-chloro-3-indolyl-D-galactopyranoside (10mg/ml) and 3-amino-1,2,4-triazole (55mM). As a negative control, yeast cells were co-transformed with pGBKT7-Ptc7IC and empty pGADT7 vector, or with pGADT7-WWNedd4 and empty pGBKT7 vector.

Co-immunoprecipitation and immunoblotting analysis

In cellulo co-immunoprecipitation were carried out on HEK293T cells with various constructs as described previously [START_REF] Mille | The Patched dependence receptor triggers apoptosis through a DRAL-caspase-9 complex[END_REF]. For experiments requiring SHH, in addition to transfection with SHH encoding vector, recombinant SHH was added at a final concentration of 600 ng/ml 24 hours before harvesting the cells. HEK293T cells were then lysed in 50 mM HEPES pH 7.6, 150 mM NaCl, 5 mM EDTA and 1% NP-40 in the presence of protease inhibitor cocktail (Roche), and further incubated with anti-Ptc-1 (Santa Cruz Biotechnology, 0.8 μg/ml), anti-FlagM2 (Sigma, 2.4 μg/ml), anti-Myc (Sigma, 2.4 μg/ml) or anti-caspase-9 (Santa Cruz, Biotechnology, 0.8 μg/ml) antibodies and protein-A (Sigma) or protein-G Sepharose (GE Healthcare). Washes were done in 50 mM HEPES pH 7.6, 150 mM NaCl, 5 mM EDTA (4 washes). For western blot and co-immunoprecipitation performed in order to detect ubiquitinated Caspase-9, HEK293T cells were harvested 48h after transfection. For western blot, cells were lysed in 30 mM Tris pH 7.5, 150 mM NaCl, 10% glycerol and 1% SDS in the presence of protease inhibitor cocktail (Roche). For co-immunoprecipitation, cells were heated in SDS 1% at 95ºC for 5 min and then diluted in 30 mM Tris pH 7.5, 150 mM NaCl, 10% glycerol. Lysates were further incubated with anti-Caspase-9 (Santa Cruz Biotechnology, 0.8 μg/ml) or anti-HA (Sigma, 2.4 μg/ml) antibodies and protein-A Sepharose (Sigma). Washes were performed in 30 mM Tris pH 7.5, 150 mM NaCl, 10% glycerol (4 washes). Immunoblots were performed as previously described ( 9) using anti-Myc (Sigma, 1/1000), anti-FlagM2 (Sigma, 1/5000), anti-HA (Sigma, 1/10000), anti-Ptc-1 (Santa Cruz Biotechnology, 1/1000), anti-Caspase-9 (Cell Signaling, 1/2000), anti-Caspase-9 (Immunotech, 1/2000), anti-ßactin (Millipore, 1/5000), anti-polyUbiquitin (EnzoLife, 1/2000), anti-Ubiquitin, Lys63-specific (Millipore, 1/2000), anti-Ubiquitin, Lys48-specific (Millipore, 1/2000) and anti-p21 (Dako, 1/1000) antibodies.

Cell death analysis and caspase assays.

Cell death was analyzed 24h after transfection using trypan blue staining procedures.

Caspase-3 activity assay was performed 24h after transfection using the caspase-3 fluorometric assay kit (BioVision) as described previously [START_REF] Mille | The Patched dependence receptor triggers apoptosis through a DRAL-caspase-9 complex[END_REF]. Caspase-9 activity was measured 18h after transfection using the luminescent Caspase-Glo® 9 Assay according to the manufacturer's instructions (Promega).

Quantitative Reverse Transcription -Polymerase Chain Reaction.

To assay Nedd4 mRNA expression in HEK293T cells transfected with siRNA against Nedd4, total RNA was extracted from cells with the Nucleospin RNAII kit (Macherey-Nagel, Duren, Germany) and 1μg of RNA was reverse transcribed using the iScript cDNA Synthesis kit (Bio-Rad, Hercules, CA). Real-time Q-RT-PCR was performed on a LightCycler 2.0 apparatus using the LightCycler® TaqMan® Master kit (Roche Applied Science, Basel, Switzerland) according to the manufacter's instructions. The ubiquitously expressed ß-actin gene was used as internal control. Primers and probe were given by Universal Probe Library Assay Design Center website (Roche Applied Science). Sequences are shown in Supplementary Table 1.

Statistics.

The statistical significance of differences between groups was evaluated by the Mann -Whitney U test. Mean values for all outcome variables are presented with standard errors of the mean. Data presented are representative of at least four independent experiments. All statistical tests were two-sided, and P values less than 0.05 were considered to be statistically significant.

Figure 1 :

 1 Figure 1: Nedd4 is required for Ptc-induced apoptosis. A. Schematic representation of a two-hybrid screen with the pro-apoptotic domain of Ptc-1. The Ptc-Nedd4 interaction was confirmed by direct two-hybrid: AH109 yeast transformed either with a Gal4AD plasmid fused with 4 WW domains of Nedd4 and a Gal4BD plasmid fused with the Ptc pro-apoptotic domain (Nedd4+Ptc), with a mock Gal4BD plasmid and a Gal4AD plasmid fused with 4 WW domains of Nedd4 (Nedd4), or with a mock Gal4AD plasmid and a Gal4BD plasmid fused with the Ptc pro-apoptotic domain (Ptc). B, C. Co-immunoprecipitations were performed on HEK293T cells transiently expressing (B) Nedd4-myc alone or with Ptc (Ptc), Ptc-∆7IC (P∆7IC) or Ptc1-1392 (P1-1392) (C) Ptc-HA alone or with Nedd4-myc in the absence (Nedd4-myc) or in the presence of SHH (Nedd4-myc+SHH). Pull-down with anti-Ptc (B) or anti-myc (C) antibodies was used to immunoprecipitate Ptc and Nedd4, respectively. Nedd4 and Ptc were then revealed by Western blot by using anti-myc and anti-HA antibodies, respectively. Western blot on lysates before pulldown are shown (Tot. prot.). D, E, G, H. Caspase-3 activity assay was performed on HEK293T cells 24h after transfection (D) with empty vector (Ctrl), Ptc encoding vector (Ptc), Nedd4 encoding vector (Nedd4) or Ptc and Nedd4 encoding vectors (Ptc+Nedd4) (E) with Ptc encoding vector (Ptc) or Ptc and Nedd4 encoding vectors (Ptc+Nedd4) in the absence (-) or in the presence (+) of MG132 (G) with empty vector (Ctrl), Ptc encoding vector (Ptc), Nedd4 encoding vector (Nedd4), Ptc and Nedd4 encoding vectors (Ptc+Nedd4), Nedd4C1286S encoding vector (Nedd4 C1286S) or Ptc and Nedd4C1286S encoding vectors (Ptc+Nedd4C1286S) (H) with empty vector (Ctrl) or Ptc encoding vector (Ptc), in the presence of siRNA control (siCtrl) or siRNA Nedd4 (siNedd4). (D, G, H) Anti-HA (αHA-Ptc) and anti-myc (αmyc-Nedd4) immunoblot are shown as a control of specificity and loading. F. Cell death was analyzed by trypan blue exclusion assay in HEK293T cells 24h after transfection of an empty vector (Ctrl), a Ptc encoding vector (Ptc), a Nedd4 encoding vector (Nedd4) or Ptc and Nedd4 encoding vectors (Ptc+Nedd4). For all caspase-3 activity and cell death assays, folds over control of each experiment are represented and error bars are standard errors of the mean. *; p<0.05, **; p<0.01 and ***; p<0.001

Figure 2 :

 2 Figure 2: Nedd4 is part of the dependosome and regulates caspase-9 activation. A. DRAL immunoprecipitation (IP:αflag) was performed on HEK293T cells transfected with either Nedd4 alone or Nedd4 and DRAL together (DRAL-flag). DRAL interaction with Nedd4 was revealed by Western blot using an anti-myc (Nedd4-myc) antibody. Western blot on lysates before pull-down are shown (Tot. prot.). B. Coimmunoprecipitations were performed on HEK293T cells transiently expressing DN-Caspase-9 alone or with Nedd4-myc in the absence (Nedd4-myc) or in the presence of SHH (Nedd4-myc+SHH). Western blot on lysates before pull-down are shown (Tot. prot.). C, D. Caspase-9 activity assay was performed using a proluminogenic caspase-9 substrate 18h after transfection of HEK293T cells with wild-type caspase-9 together (C) with Nedd4 expressing construct or Nedd4C1286S encoding vector or (D) with siRNA control (C9+siCtrl) or siRNA Nedd4 (C9+siNedd4), in the absence (Ctrl) or in the presence of Ptc (Ptc). Anti-HA (αHA-Ptc and αHA-Bax), anti-Caspase-9 (αC9) and anti-myc (αmyc-Nedd4) immunoblots are shown as a control of specificity and loading. Folds over control are represented and error bars are standard error of the mean. **; p<0.01

Figure 3 :

 3 Figure 3: Caspase-9 is polyubiquitinated during Ptc-induced apoptosis. A. Western blot using caspase-9 antibody was performed on HEK293T cells lysed in lysis buffer containing 1% SDS, 48h after transient transfection of DN-caspase-9 (DN-C9) together with an empty vector, with Bax encoding vector (Bax-HA), with Ptc encoding vector in the absence (Ptc-HA) or in the presence (Ptc-HA+SHH) of SHH, or with Ptc-∆7IC encoding vector (P∆7IC). ß actin (αß actin), Ptc (αPtc) and Bax (αHA) immunoblot are shown as a control of loading. B, E. Immunoprecipitation of caspase-9 (IP: αCasp9) was performed on SDS-lysed HEK293T cells expressing DN-caspase-9 (DN-C9) and Ubiquitin proteins in the absence or in the presence of Ptc (Ptc). Ubiquitinated Caspase-9 (Ub) was revealed by Western blot using an antiubiquitin (B) or anti-K48-ubiquitin and anti-K63-ubiquitin (E) antibodies. Western blots on lysates before pull-down are shown (Tot. prot.). C. Immunoprecipitation of HAubiquitinated proteins (IP αHA-Ub) was performed on HEK293T cells lysed in the

Figure 4 :Figure 1 :

 41 Figure 4: Ubiquitination of caspase-9 is required for Ptc-induced caspase activation. A. Schematic representation of Caspase-9. The position of 5 lysines mutated in C9-5KR constructs is indicated. B. Anti-caspase-9 immunoblot was performed on HEK293T cells transfected with wild-type caspase-9 (C9) or caspase-9 mutated on 5 lysines (C9-5KR) in the absence (Ctrl) or in the presence of Ptc (Ptc). Anti-HA (αHA-Ptc) immunoblot is shown as a control of loading. C-F. Caspase-3 activity assay was performed in HEK293T cells (C,D,F) or in Mouse embryonic Fibroblast deficient for caspase-9 (MEF C9-/-) (E), 24h after transfection with wild-type caspase-9 (C9), caspase-9 mutated on 5 lysines (C9-5KR) or non cleavable caspase-9 (NCC9) together with either an empty vector (Ctrl), a Ptc encoding vector (Ptc) or a Bax encoding vector (Bax). Anti-HA (αHA-Ptc and αHA-Bax) and anti-caspase-9 (αC9) immunoblots are shown as controls of specificity and loading. Folds over control are represented and error bars are standard error of the mean. **; p<0.01

  Figure 1

Acknowledgments:

We wish to thank DE Bredesen for critical reading of the manuscript, AP. Arrigo, D.

Green and G. Melino for sharing reagents. This work was supported by institutional grants from the Ligue Contre le Cancer, INCA, ANR, and IP ApoSys.

Correspondence and requests for materials should be addressed to P.M. (e-mail: patrick.mehlen@lyon.unicancer.fr).


Olivier Chesneau
email: olivier.chesneau@oca.eu

Eric Lagadec

M Otulakowska-Hypka

D P K Banerjee

C E Woodward

E Harvey

A Spang

P Kervella

F Millour

N Nardetto

N M Ashok

M J Barlow

M Bode

A Evans

D K Lynch

T J O'brien

R J Rudy

R W Russel

The expanding dusty bipolar nebula around the nova V1280 Sco

Keywords: high angular resolution, (Stars:) novae, cataclysmic variables, individual: V1280 Sco, Stars: circumstellar matter, Stars: mass-loss

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Introduction

White dwarfs (WDs) are the end products of the evolution of low-and intermediate-mass stars, with initial masses between 0.8 and 8 M ⊙ . If a white dwarf is in a close binary system, it can accrete hydrogen from its close companion's atmosphere, which then triggers the ignition of runaway nuclear reaction. This leads to a bright outburst, called a classical nova.

The classical nova V1280 Sco was discovered in outburst by Yamaoka et al. (2007) light (m V ∼4) and formed dust only two weeks after reaching maximum in visible (Das et al. 2008). This nova experienced one of the slowest evolutions historically reported and the nebular phase was entered about 50 months (t∼1600d) after outburst [START_REF] Naito | The Astronomer's Telegram[END_REF]. This is a clear indication that the mass of the erupting WD is low (i.e. ∼0.6M ⊙ or even smaller).

Extensive monitoring of the event was performed using the Very Large Telescope Interferometer (VLTI) during the first four months (until t=145d) of the event [START_REF] Chesneau | Asymmetric Planetary Nebulae 5 Conference[END_REF]. Owing to the sparse uv coverage, an interpretation involving a spherical dusty shell was developed, providing estimates of the dust formation rate. Based on an estimate of the total dust mass formed during the 250 days after outburst, [START_REF] Chesneau | Asymmetric Planetary Nebulae 5 Conference[END_REF] suggested that a mass as large as 10 -4 M ⊙ was ejected. [START_REF] Naito | The Astronomer's Telegram[END_REF] provided an independent estimate from neutral oxy-gen lines that confirmed the high mass ejected, an additional argument for a low-mass WD progenitor, known to statistically eject more mass that fast, massive ones. The interferometric observations also provided a consistent picture of the expansion rate in the plane of the sky estimated at 0.35 mas/d. Radial velocities were published by Das et al. (2008) in the near-IR domain, and by Sadakane et al. (2010) in the visible. Sadakane et al. (2010) reported multiple high-velocity narrow components in the NaI D line from 650 to 900 km.s -1 . They interpreted this as being due to the formation of clumps, as a consequence of a strong shock between the slow, dust-forming ejecta, and a fast wind (∼ 2000 km.s -1 ) generated during the second brightening of the source (Chesneau et al. 2008, t=110d). P-Cygni lines were systematically observed even four years after the outburst, which is indicative of a sustained and significant mass loss (Schwarz et al. 2011). The typical P-Cygni type velocities reached 500 km.s -1 , from which [START_REF] Chesneau | Asymmetric Planetary Nebulae 5 Conference[END_REF] inferred a distance of 1.6±0.4kpc. However, Hounsell et al. (2010) recently derived a distance of 630±100 pc from space-based visual light curves that span the first 20 days after discovery. This much smaller distance estimate demands that the VLTI-derived distance be scrutinized. Using the spatial expansion rate from the VLTI but a revised velocity range of the dust-forming ejecta of 350±160km • s -1 based on the mean velocity measured from blue-shifted absorption lines of OI and SiII, [START_REF] Naito | The Astronomer's Telegram[END_REF] derived a revised distance of 1.1±0.5kpc.

The dust event and the large amount of circumstellar material prevented any X-ray detection of V1280 Sco until May 2009, t=834d (Ness et al. 2009). After this date the source was detected several times from t=834d to t=939d after outburst (Schwarz et al. 2011), although the dense ejecta considerably absorbed the potentially important X-ray flux.

We report on observations of the Very Large Telescope performed between 2009 (t=857d) and 2011 (t=1664d) that reveal a striking bipolar nebula in expansion. The very slow ejecta of V1280 Sco likely expanded while being significantly influenced by the companion, although an intrinsically polar-oriented ejection scenario must also be investigated. This is a unique opportunity to observe a newly formed bipolar nebula. Our study has a broader relevance in the context of the shaping of asymmetrical bipolar nebulae and the formation of disks, such as that seen in some planetary or pre-planetary nebulae (Chesneau 2011;De Marco & Soker 2011;Lykou et al. 2011;Chesneau et al. 2007), born-again sources (Lau et al. 2011;[START_REF] Chesneau | [END_REF] or in massive stars (Millour et al. 2011;Meilland et al. 2010;Kastner et al. 2010;[START_REF] Kraus | Binaries -Key to Comprehension of the Universe[END_REF]) and for a better understanding of the still poorly known common-envelope phenomenon (de Marco et al. 2011).

The observations are presented in Sect. 2. In Sect. 3 we derive some physical parameters of the nebula, and we discuss our results in Sect. 4.

Observations

The source was observed with the NACO, VISIR and SINFONI instruments of the Very Large Telescope (VLT). The log of the observation is shown in Table 1 and the visible light curve is reproduced in Fig. 1.

NACO observations

We observed V1280 Sco in J, H, Ks with the S13 camera, which provides a field of view (FOV) of 14"x14" and a pixel scale of 13.26±0.03 mas. The AutoJitter mode was used in which the telescope moves according to a random pattern in a 5" box at each exposure. The nearby star GSC 07364-01316 (Lasker et al. 2008) was selected as the reference star for the visible wavefront sensor of the adaptive optics (AO) for determining the point spread function (PSF) of the instrument and to serve as a rough flux calibrator. The PSF obtained in 2009 with NACO in standard mode was not good enough to allow a meaningful deconvolution of the compact source. In 2010, the NACO observations benefited from the newly implemented 'cube mode', which provides a large set of short exposures (30ms) over part of the detector. 8000 frames were recorded per filter, representing an exposure time of 480s each. This new capability is essential to reach truly diffraction-limited imaging at the shortest wavelengths (Kervella et al. 2009). The cube mode provides a large cube of short exposures from which a sub-set with the highest Strehl ratio is selected (principle of 'lucky imaging').

VISIR observations

The NACO observations were complemented by mid-infrared images obtained with the VISIR instrument (Lagage et al. 2004). The VISIR observations were performed in July 2009 (MJD 55014.2 and 55015.1, t=877-878d), June and July 2010 (t=1228d and t=1254d) and June 2011 (t=1589d). The atmospheric conditions were stable for all nights. The imager was used in burst mode (DIT ranging from 10 to 25 ms), providing a pixel scale of 0.075" and a field of view of 19.2" x 19.2". The chopping throw was 8 arcsec and the chopping frequency 0.25 Hz. A set of narrow-band filters covering the full N band were used, with integrated total exposure times per filter ranging from 280s to 360s. Several filters were centered on potential spectral features: PAH1, PAH2 and NeII. The exposures taken with the PAH2 (λ=11.25µm, ∆λ=0.59µm) and NeII filters (λ=12.81µm, ∆λ=0.21µm) were immediately followed by exposures taken with a nearby continuum filter called PAH2 2 (λ=11.88µm, ∆λ=0.37µm) and NeII 1 (λ=12.27µm, ∆λ=0.18µm).

The standard chopping/nodding technique was applied to remove the sky contribution. The burst mode provides a large cube of short-exposure frames in which all single chopping and nodding images recorded. The reduction of the VISIR data additionally includes the correction of various defects exhibited by the VISIR detector.

The VISIR burst mode and NACO cube modes are very similar. The principle is to acquire very short exposures (≤50 ms) to keep the complete integration within a fraction of the coherence time (∼300 ms at Paranal in the mid-IR). The burst modes of NACO and VISIR provided large cubes of short exposures that enable the reconstruction of quality-enhanced images using shift-and-add techniques, and selecting the best percentile (lucky imaging) following an algorithm described by Kervella et al. (2009) and Lagadec et al. (2011). The VISIR burst mode images of V1280 Sco were deconvolved using a Richardson-Lucy algorithm. Thirty iterations were sufficient to obtain good quality converged images. The VISIR images are shown in Fig.5 (2009), Fig.6 (2010) and Fig.7 (2011).

Flux calibration was performed using standard aperture photometry methods applied to the science and reference stars. The observations were performed in fairly similar atmospheric conditions and the raw fluxes observed per filter using the same cal-ibrator are similar at better than 5%. Unfortunately, the VISIR PSF calibrator, HD 152934, was chosen for its brightness but was a poorly suited photometric calibrator. HD 152934 is an anonymous S-type AGB star whose spectral type is not well constrained. We used synthetic templates of S-type stars produced by the MARCS codes (Gustafsson et al. 2008) to fit the SED of HD 152934. We then estimated the flux in the VISIR filters using the best synthetic spectrum (T e f f = 3000 K and logg = 0) and calibrated the science object photometrically. This procedure is indirect and leads to a potential calibration bias larger than 10% in some filters for such a complex source.

SINFONI observations

VLT/SINFONI is a near-infrared (1.1 -2.45 µm) integral field spectrograph (IFS) fed by an AO module [START_REF] Eisenhauer | Society of Photo-Optical Instrumentation Engineers (SPIE) Conference Series[END_REF]. Integral field spectroscopy allows one to gather spectra of the sky over a two-dimensional field of view. Final products are data cubes with two dimensions of x, y and a third dimension formed by the wavelengths. During our observations the spectrograph operated in the K and H bands, providing a spectral resolution of 4000 and 3000, respectively. The FOV was cut into 32 slices, and each slice was projected onto 64 detector pixels. The field of view of the data set is 0.8"×0.8" and is composed of spaxels of 12.5×25 mas in the K and H bands. We obtained several VLT/SINFONI integral field spectroscopic data sets for V1280 Sco from May to August 2011. The FOV was oriented such that the major axis of the nebula roughly coincided with the vertical direction. During that period, the central stellar-like source increased considerably in flux, while the bipolar nebula in which this source is embedded had a spatial extent of ≤0.7" which made these observations challenging. The observations were performed in service mode and were repeated several times, but the atmospheric conditions never met the criteria for providing good quality data. We nevertheless worked on the best data of the sample and present the most robust results. The data set of 26 June 2011 exhibited the better signal-to-noise ratio (S/N) although the bipolar nebula was slightly cut in the north. Because of the limited S/N of the observations and the difficult removal of the telluric lines, a differential approach, using a comparison between a given emission line flux and its corresponding adjacent continuum was employed in the reduction and analysis.

Observational results

The dusty nebula

A bipolar nebula with a high aspect ratio is clearly seen in the NACO, SINFONI and VISIR images we obtained, suggesting that the source is seen at high inclination. For our NACO and SINFONI data, the nebula extension was estimated by determining the regions of images with the highest gradient before reaching the noise level (or written differently, by the regions of densest contour levels before reaching the noise level). The estimation was performed on the images with and without PSF subtraction. The extensions measured are reported in Table 2.

The near-IR images were dominated by the central source, which brightened considerably over the three years of observations. The apparent size in the K band was 304 x 191 mas in 2009 (t = 877d). Corrected for the PSF extension, this leads to an angular size of 290±38 x 167±31 mas. After a PSF subtraction, the position angle (PA) of the major axis is as 105 ± 5 • with an axial ratio as ∼ 1.7. However a good estimate of the minor axis extension was difficult to obtain due to the bright central source, and the artifacts remaining after the PSF subtraction.

The NACO observations performed in cube mode in 2010 (t = 1228d) were of excellent quality, and the J, H and K bands can be fully exploited. An elongation was clearly detected in each image at PA=105±3 • , 102±3 • and 107±3 • . For instance, the FWHM of the J band image of V1280 Sco is 61±3mas along the major axis, and 54±3 along the minor axis. The apparent size in the K band was 404 x 260 mas, which, corrected for the FWHM of the PSF, corresponds to 400±30 x 255±24 mas.

The 2011 SINFONI images (t = 1602d) suggest that the source is composed of a bright unresolved source and two apparently well-detached polar caps. There seems to be no indication for extended equatorial material. The extension of the nebula in the direction of the major axis is estimated to be 637±13mas, and the larger extent of the caps perpendicular to the major axis to be 412±13mas (Fig. 9 and Table 2).

The VISIR images also provided robust estimates of the PA at 108.5±2 • in 2009, 109.1±1.6 • in 2010 and 108.9±2.5 • in 2011. The bipolar morphology of our VISIR images was more clearly seen in the most recent images due in part to the expansion of the nebula. Fig. 8 (left) shows a cut of the VISIR images obtained at 8.59 µm in 2009, 2010 and 2011, together with a PSF for comparison. This filter provides the shortest wavelength, hence the best spatial resolution of VISIR and was therefore used for providing the extension estimates (Table 2). The bipolar lobes of V1280 Sco are clearly expanding with time, and in 2011, one could see a double peaked cut generated by the two emission peaks of the bipolar nebula. One-dimensional Gaussian fitting was performed to measure the FWHM of the cuts, and the results are shown in Table 2. After deconvolution, the VISIR images provide a size and an axial ratio equivalent to that seen in the near-IR. The size and appearance of the nebula extension was nearly unchanged from 8 to 13 micron. The deconvolved images confirm that the object is bipolar and expanding. Our most resolved images (from our 2011 dataset) show that the intensities of the two bipolar lobes are different, the southern lobe is brighter. This is probably due to an absorption effect as a consequence of this lobe being closer to us than the northern one. No mid-IR emission is seen between the caps. Fig. 8 shows a radial cut along the poles at 8.59. 8.99, 11.25, and 12.81 µm. The separation between the two lobes does not depend on the wavelength.

The ratio of the intensity between the two peaks increases with wavelength due to absorption and optical depth effects.

Spectroscopy and flux measurements

The V flux has reached a roughly stable plateau at a magnitude of about 10.5 over a long period [START_REF] Naito | The Astronomer's Telegram[END_REF]) and the central source radiates efficiently in the K band. As shown by Lynch et al. (2009), the nova is still powering a strong wind, and extensive nuclear burning is thus thought to have continued.

An unresolved point source dominates the near-IR observations, whereas no point source contribution is observable in the mid-IR for the contemporaneous VISIR measurements. In 2009, the stellar source (i.e. the unresolved flux) accounts for 54±6%, increasing in 2010 to reach 76±6% (NACO data) and in 2011 87±5% (SINFONI) of the total flux. These numbers are potentially biased by the different Strehl ratio reached by the AOs during the observations. These estimates were also possible in the H and J bands for the 2010 NACO observations, with 96±3% and 100±3%, respectively. This increase of the central source contribution indicates a decrease of the circumstellar absorption, related probably to the expansion and dissipation of the nearby dust.

The photometry of the central source and its nearby environment was performed from the NACO, SINFONI and VISIR images and is provided in Table 3. These instruments or the mode used are not necessarily optimized to provide accurate photometry but despite the level of errors such information remains of high interest for these variable sources.

Many lines are detected by SINFONI (t=1609d). Of importance is the simultaneous detection of relatively high-excitation lines (mostly from the Balmer series), and the detection of low ionization species such as NaI and MgI, as already reported by Das et al. (2008) and Sadakane et al. (2010) for this source. The mean FWHM of the Balmer lines is 162±14km • s -1 , and we note that Brγ a broad pedestal (∼700km • s -1 ). Some lines of low excitations are observed, the NaI 2.149 line (FWHM=110±18km•s -1 ), MgI 1.711 (FWHM=144±8km•s -1 ), some are unidentified such at 2.137µm (FWHM=105±8km and some with weak signatures such as MgI 1.576, CI 1.690, as reported by Das et al. (2008). These lines are narrow and almost unresolved given the spectral resolution of these observations (R∼3500 corresponding to a FWHM of 85km • s -1 ).

The presence of the NaI 2.149 line implies that the NaI must be protected from the central source, and is therefore very likely located within the dust. The presence of these low-excitation lines so late (t=1609d) after the outburst highlights how slowly V1280 Sco has evolved, because these lines are not expected at this stage.

For the strongest lines, a SINFONI image was created by integrating the flux within a narrow spectral range, and by subtracting a red-and blue-sided continuum. For the Brγ line, the main source of the emission is observed in the central object, which contributes 98±7% of the total flux. A slightly different behavior may be observed for the He I 2.06 µm (see Fig. 9) line for which the core contributes 93±7%, and for which the lobes seem better contrasted. In the case of V1280 Sco, one may hypothesize that the He I 2.06 µm emission in the caps could arise either from a shock between the fast nova wind and the slowly expanding, dust rich polar caps, or by a radiative interaction between this material and the growing UV flux from the white dwarf.

The PAH 2 filter is designed to detect carbonaceous molecules. This filter is not best suited because the 11.25µm feature in novae is seen at ∼11.4µm (Evans et al. 2005), although most of the flux should be isolated by the filter given the bandwidth (0.59µm). Yet, it is challenging to detect any morphological difference between images in the two filters because of the limited spatial resolution and the weakness of the flux contrast. It is not evident that the regions of PAH emission is distinct from that of the continuum. Spitzer IRS (6 May 2008) and IRTF/Spex (20 May 2009) observations show that dust emission features appeared at 3. 28, 3.4, 8.3, 8.7, and 11.4 microns, the socalled UIRs (unidentified infrared bands). Likely, carbonaceous molecules also formed in situ from condensed dust (Bernstein & Lynch 2009;[START_REF] Evans | [END_REF], 2005, HAC).

The expansion rate

In the near-IR, the datasets are not homogeneous with images taken with NACO in 2009 without cube mode, then in 2010 in cube mode and in 2011 using the SINFONI instrument. We 1 Filter dedicated to continuum measurements close to the PAH2 filter.

did not attempt to derive an expansion rate for the lobes in the K band due to the variances in the observational techniques.

Despite their lower spatial resolution, the images from the VISIR instrument were obtained solely in burst mode. Moreover, in the mid-IR, the central source is too faint to be detected, and the images probe the contribution from the dust alone. The NACO and SINFONI images exhibit a bright stellar contribution that has to be carefully taken into account and removed before any geometrical information can be inferred. The expansion was measured from the raw and deconvolved VISIR images. Fig. 8 shows the cuts of the images along the direction of the major axis and the results are shown in Table 2. Correcting the FWHMs from the FWHM of the PSF at 8.59µm, this leads to an estimate of the expansion rate of 0.36±0.06 (2009), 0.39±0.05 (2010) and 0.40±0.04 (2011) mas per day, from which we infer a global estimate of 0.39±0.03 mas per day. As an independent test, one can compute the separation of the polar caps as measured by SINFONI to be 0.62±0.05"at day 1602, providing an expansion rate very close to the VISIR measurements. This value, measured in the direction of the major axis of the nebula is close to the expansion rate estimate of 0.35±0.03 mas per day from the VLTI in a similar wavelength range. The VLTI baselines used to infer the expansion of the early dusty envelope were in a direction close to the major axis of the nebula (within typically 30 • ), and therefore approximately probed the velocities projected on the sky in that direction. The bias expected from the use of baselines not aligned to the major axis is an underestimate of the expansion rate.

Discussion

The distance and expansion velocities

Using the angular expansions measured from the infrared images, one can try to improve upon the distance estimate for V1280 Sco. The angular expansion inferred from the VLTI observations was 0.35±0.03 mas/d, while the high-quality VISIR images obtained in the study suggest a slightly higher expansion rate in the direction of the major axis of the nebula of 0.39±0.03 mas/d.

Combined with a measure of the ejecta expansion velocity, the angular expansion rate (mas/d) can be used to derive a distance to V1280 Sco, under the hypothesis of a spherical ejection following

D kpc = 1.154 × 10 -3 V e j dθ dt , (1)
where V e j is the velocity of the ejecta in km • s -1 and dθ dt is the rate of expansion in mas per day.

Assuming a velocity V e j for the ejecta of 500±100km.s -1 , [START_REF] Chesneau | Asymmetric Planetary Nebulae 5 Conference[END_REF] estimated the distance to be 1.6±0.4 kpc.

The difficulty of this exercise is to ensure that the Doppler velocities inferred from the emission lines can be linked to the dust expansion measured in the VLTI data or VISIR images. These quantities must be measured at the same wavelengths, if possible centered on an emission line that provides the kinematics. Moreover, the Doppler velocities are translated into ejection velocities assuming a projection factor (the p factor) whose value depends on the kinematics but also on the flux distribution of the source.

The discovery of the strong bipolarity of the source implies a complex velocity field that cannot be directly related to a limited number of velocity measurements. The expansion velocities were measured in the direction of the major axis of the bipolar nebula and we adopted a Doppler velocity of 500km.s -1 , based on several radial velocity measurements from various emission lines. Co-mixing of the dust and gas emitting regions cannot be unequivocally demonstrated, and it is unknown whether the line-forming region coincides with the dust-continuum-forming region. Therefore, it is very difficult to measure the ejection velocity of the dusty nebula.

Hounsell et al. ( 2010) derived a much closer distance estimate, 630±100 pc based on various estimates of intrinsic luminosity of the source. However, despite the quality of their photometry, many parameters are used to infer the distance, some of them raised to high power indices, implying potentially large errors. Hounsell et al. (2010) found Mv = -6.1, yielding a distance of ≤ 1kpc; however, they pointed out that Mv can be equal to -8 based on t 3 estimated as 34 d, where t 3 is the number of days in which a nova wanes by 3 magnitudes after maximum. This value yields a distance of 1.4 kpc, which is within the uncertainty of the first estimate from [START_REF] Chesneau | Asymmetric Planetary Nebulae 5 Conference[END_REF].

If one assumes the distance quoted by Hounsell et al. (2010), the mean dust-forming region velocity is only ∼220km • s -1 . This velocity is the velocity measured in the plane of the sky, in the direction of the major axis. Owing to the high aspect ratio, a high inclination is expected, and this measurement should be considered as close to the upper limit of the velocity field, and one may consider whether such a slowly expanding material may imprint its signature in spectroscopic observations. Sadakane et al. (2010) reported on spectroscopic information of great interest to this problem. They observed absorption in the NaI lines detected between -649km • s -1 to -885km • s -1 with the strongest components at -750 and -800km • s -1 . One may assume that these absorption features provide a good approximation of the dust velocity field because they probe low-excitation cool regions. Das et al. (2008) detected many lines from these low-ionization species (NaI and MgI) at the earliest stages of the event (i.e. at the onset of the dust-formation phase). The impressive narrowness of the features (∼15km • s -1 ) can be interpreted as originating from a cool medium. The measured FWHM can be considered as an upper limit, accounting for the instrumental FWHM of ∼5km • s -1 . Hence, accounting for some turbulence and velocity dispersion of the expanding material in the FWHM broadening, the thermal broadening may originate from material cooler than 1500 K. The Sadakane et al. (2010) lines of OI and SiII. These low velocities were measured around light-curve maximum and increased up to ∼500km • s -1 just before the dust-formation phase. Using the expansion of VISIR of 0.39±0.03 mas/d in the major axis direction and the slowest velocities of the cool material of 350km • s -1 , one still derives a distance estimate close to 1.0kpc. This value is a lower estimate, since the velocities in the direction of the major axis of the bipolar nebula are expected to be higher, while the spectroscopically detected absorption components are seen in the line-of-sight direction. Lacking an accurate estimate of the inclination, we are left with this estimate which is potentially affected by larger errors.

To conclude, despite the significant limitations and errors that may affect the distance estimate of 1.6kpc±0.4 provided by [START_REF] Chesneau | Asymmetric Planetary Nebulae 5 Conference[END_REF], we are reasonably confident that the distance of V1280 Sco is much larger than the distance of 0.6kpc proposed by Hounsell et al. (2010).

Origin of the bipolarity

A growing body of observational evidence clearly suggests that many nova ejectae are highly bipolar in the first days and months after the outburst. The correlation between speed class and ejection velocity, meaning that the faster the expansion speed, the less shaped the remnant (Bode 2002;Downes & Duerbeck 2000), is an argument for supporting the significant effect of the common envelope phase on the shaping. The ejecta from a slow nova would feel the influence of the secondary much longer, leading to a strong common envelope interaction. These effects, involving frictional deposition of energy and angular momentum from the secondary are described by Kato & Hachisu (2011); Porter et al. (1998); Lloyd et al. (1997); Livio et al. (1990). The very slow nova HR Del (Moraes & Diaz 2009;Harman & O'Brien 2003;Solf 1983, t 3 =230d) or V723 Cas (Lyke & Campbell 2009;[START_REF] Evans | Asymmetric Planetary Nebulae 5 Conference Evans[END_REF], t 3 =180d) are striking examples of this phenomenon.

V1280 Sco appears to be at odds with the t 3 / axial ratio relationship shown for instance in Fig. 6 of Bode (2002). The axial ratio of V1280 Sco is high, surely higher than 1.5. Such high axial ratios are exhibited by the slowest novae in the diagram, with t 3 >100d. Hounsell et al. (2010) estimated t 3 = 34 days, yet cautioned that the fidelity of this determination is weak owing to the complexity of the light curve, which is affected by the dust and the secondary maximum. We cannot exclude that t 3 might have been shorter, although given the distance estimate of 1.6kpc that is favored in our study, t 3 = 34 d fits the maximum magnitude-rate of decline relation (MMRD) relatively well (Downes & Duerbeck 2000).

The late SINFONI images suggest that the dust resides only in the polar caps, and that the equatorial regions seem devoid of any dust in contrast to the bipolar helium nova V445 Pup for instance, which exhibits a large and dense dusty waist (Woudt et al. 2009). The VISIR last images confirm this since the two lobes appear to be clearly detached from each other in the deconvolved images without any sign of emission close to the equatorial plane (Fig. 7). [START_REF] Chesneau | Asymmetric Planetary Nebulae 5 Conference[END_REF] reported some evidence for a significant departure from spherical symmetries in the latest VLTI data (t=145d) from a baseline at more than 60 degree from the major axis. A re-inspection of the data shows that contrary to what was suggested by the authors, the measurements are not affected by any instrumental problem. An aspect ratio as high as 1.3 was probably observed implying that the shaping of the bipolar nebula was already well advanced at this time.

Two questions can be related to these observations. How can this polar ejection be the consequence of a common-envelope phase and what was the time scale of the event? With a different time and spatial scale context, the extreme bipolar planetary nebulae Mz3 and M2-9 can provide enlightening comparisons [START_REF] Evans | Asymmetric Planetary Nebulae 5 Conference Evans[END_REF]. Most of the dust in Mz3 and M2-9 was found in the bipolar lobes (Lykou et al. 2011;Chesneau et al. 2007;Smith & Gehrz 2005) and recent observations suggest that the time scale of the formation of these nebulae must have been very short, i.e. a few years at most, in a context of a much slower expected evolution of at least several centuries. Regarding M2-9, it is known that the core harbors a long-period binary (Corradi et al. 2011, P∼90yr). For these events, the small disks at the core of the systems and the dense lobes favor an intrinsically polar ejection. This hypothesis cannot be ruled out with the formation of the caps of V1280 Sco.

Another scenario implies an intrinsically a-spherical ejection that focuses the mass ejection toward the poles, in which a largescale structured magnetic field may be the shaping key agent. The recent outburst of the recurrent nova T Pyx appeared to be highly bipolar (Chesneau et al. 2011). T Pyx shows a very stable photometric wave that may betray the existence of a magnetic field (Patterson et al. 1998). The presence of a strong magnetic field of about a few MG might have had a non-negligible effect on the eruption, which is also highly suspected for V1500 Cyg stars [START_REF] Oksanen | [END_REF].

Conclusion

We monitored the evolution of the circumstellar material around the classical nova V1280 Sco using high angular resolution infrared observations with the VLT. We resolved it for the first time with a single telescope with the NACO/VLT instrument in the near-infrared and VISIR/VLT in the mid-infrared in 2009, two years after the outburst.

These observations revealed a bipolar-shaped nebula around the nova, with dust present mostly in the lobes of the nebula. The high aspect ratio of the nebula suggests that it is seen at high inclination.

More similar VISIR observations were taken in 2010 and 2011, together with IFU observations with SINFONI/VLT in 2011. This allowed us to observe the expansion of the dusty nebula around V1280 Sco with a rate of 0.39±0,03 milliarcsec per day along the major axis of the nebula. Assuming that the dust we observed with these infrared observations expands at the same speed as the low-excitation ejecta detected via spectroscopy, this suggests that the distance to V1280 Sco is at least 1kpc.

Finally, these observations enabled us to study the temporal evolution of a dusty bipolar source. Further yearly monitoring of V1280 Sco will be easier because the nebula is expanding, and will provide an excellent test-case for the study of the envelope dust and gas content.

  on 2007 February 04.86 (JD = 2 454 136.85), about 12 days before reaching its maximum in visual Send offprint requests to: O. Chesneau ⋆ Based on observations made with ESO telescopes at the Silla Paranal Observatory under programme IDs 283.D-5020, 385.D-0076, 087.D-0074

Fig. 1 .

 1 Fig. 1. Visual light curve of V1280 Sco from AFOEV and ASAS-3 data with the dates of the VLTI and VLT observations.

Fig. 2 .

 2 Fig. 2. Left: Normalized 2009 K band NACO image of V1280 Sco in logarithmic scale. Right: Same for the calibrator.

Fig. 3 .

 3 Fig. 3. Upper left: Normalized 2010 J band NACO image of V1280 Sco in logarithmic scale. Upper right: 2010 J band NACO image of the calibrator. Middle: Same in the H band. Bottom: Same in the K band. A significant brightening of the central source (unresolved) is observed compared to 2009 (Fig.2).

Fig. 4 .

 4 Fig. 4. 2010 NACO K band image after a PSF subtraction. The color table is inverted.

Fig. 6 .

 6 Fig. 6. Same for the VISIR observations performed in 2010 (t = 1254d)

Fig. 7 .Fig. 8 .

 78 Fig. 7. Same for the VISIR observations performed in 2011 (t = 1589d)

  observations and the 2009 NACO/VISIR images were obtained almost at the same epoch in mid 2009. The slower absorbing material in the line of sight was moving in 2009 at a velocity of about 650km • s -1 . Naito et al. (2012) reported on much lower velocities, 350±160km • s -1 , measured from blue-shifted absorption

Fig. 9 .

 9 Fig. 9. Left: Continuum flux from a narrow spectral channel (2.7nm) close to the Brγ line. Middle: same from a channel centered on the Brγ line emission from which the nearby continuum shown on the left was removed. Right: same from a channel centered on the HeI2.05 line emission isolated from a nearby continuum. The Brγ line forming region is centered on the outbursting source, whereas some intrinsic HeI2.05 emission seems to originate from the lobes.

Table 1 .

 1 NACO/VLT, VISIR/VLT and SINFONI/VLT observing logs.

	Date	day 1	SCI/CAL	Inst.	Filters
	2009.07.02	877	V1280 Sco	NACO	J, H, K
	2009.07.02	877	PSF 1 2	NACO	J, H, K
	2009.07.03	878	V1280 Sco	VISIR 4	N band filters 5
	2009.07.03	878	PSF 2 3	VISIR 4	N band filters 5
	2010.06.17	1228 V1280 Sco	NACO 4	J, H, K
	2010.06.17	1228	PSF 1 3	NACO 4	J, H, K
	2010.07.28	1254 V1280 Sco	VISIR 4	N band filters 5
	2010.07.28	1254	PSF 2 3	VISIR 4	N band filters 5
	2011.05.21	1566 V1280 Sco SINFONI	H, K
	2011.06.13	1589 V1280 Sco	VISIR 4	N band filters 5
	2011.06.13	1589	PSF 2 3	VISIR 4	N band filters 5
	2011.06.26	1602 V1280 Sco SINFONI	K
	2011.06.27	1603 V1280 Sco SINFONI	K
	2011.07.03	1609 V1280 Sco SINFONI	H, K
	2011.07.03	1609 V1280 Sco SINFONI	H, K
	2011.08.26	1663 V1280 Sco SINFONI	H, K
	2011.08.28	1664 V1280 Sco SINFONI	H, K
	1 Day after outburst
	2 GSC 07364-01316, J=8.29±0.03, H=7.68±0.07, 7.48±0.02
	3 HD152934, M5V, V=11, K=2.46, IRAS f12= 8.19 Jy
	4 Burst mode
	5 PAH1, λ=8.59µm, ∆λ=0.42µm; ArIII, λ=8.99µm, ∆λ=0.14µm;
	PAH2, λ=11.25µm, ∆λ=0.59µm; PAH2 2, λ=11.88µm,
	∆λ=0.37µm; NeII, λ=12.81µm, ∆λ=0.21µm; NeII 1, λ=12.27µm,
	∆λ=0.18µm

Table 2 .

 2 Extensions of the bipolar nebulae.

	Filter	Major axis 1 Minor axis 1	PSF FWHM
		2009
	K	0.29±0.04" 0.17±0.03" 0.092±0.005"
	PAH2 0.31±0.04"	-	0.23±0.03"
		2010
	K	0.40±0.03" 0.26±0.02" 0.057±0.003"
	PAH2 0.49±0.05" 0.33±0.05"	0.23±0.03"
		2011
	K	0.62±0.05" 0.39±0.01"	0.13±0.02"
	PAH2 0.64±0.06" 0.37±0.05"	0.23±0.03"

1 Corrected for the FWHM of the PSF

  •s -1 )

	V 1280 Sco Deconvolved
	V 1280 Sco
	PSF
	8.59 microns	8.99 microns	11.25 microns	12.81 microns
	V 1280 Sco Deconvolved
	V 1280 Sco
	PSF
	8.59 microns	8.99 microns	11.25 microns	12.81 microns

Fig. 5. Burst-mode VISIR observations performed in 2009 (t=878d) with four different filters (see description in Table

1

). Up: Deconvolved images of V1280 Sco. Middle: Mean images generated from the best short exposures of V1280 Sco. Bottom: Mean images of the PSF with the same treatment.

Table 3 .

 3 Photometry of the total source.

	Filter	λ 0	t = 878d	t = 1254d t = 1589
		[micron]	[Jy]	[Jy]	[Jy]
	J	1.05	-	0.38±0.07	-
	H	1.65	-	0.44±0.04	-
	K	2.1	0.67±0.04 0.44±0.02	-
	PAH 1	8.59	47±6	34 ± 4	28± 4
	ArIII	8.99	43±5	32 ± 4	26 ± 3
	PAH 2	11.25	33±3	25±3	20±3
	PAH 2 2 1	11.88	31±4	23±3	19±3
	NeII	12.81	26±3	20±3	16±2

Acknowledgements. We thank Lowell Tacconi-Garman, Mario van den Ancker and Elena Valenti, among others, for their invaluable assistance with the preparation of these observations. Part of this work was carried out within the framework of the European Associated Laboratory "Astrophysics Poland-France". We thank Pierre Cruzalèbes for his advice on the MARCS synthetic models.