Marion Robin

Abdul Raouf Issa

Cristiana C Santos

Francesco Napoletano

Céline Petitgas

Gilles Chatelain

Mathilde Ruby

Ludivine Walter

Serge Birman

Pedro M Domingos

Brian R Calvi

Bertrand Mollereau
email: bertrand.mollereau@ens-lyon.fr

Drosophila p53 integrates the antagonism between autophagy and apoptosis in response to stress

Keywords: Apoptosis, caspase, Drosophila melanogaster, macroautophagy, neurodegenerative disease, oxidative stress, p53, Parkinson disease, photoreceptor

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Introduction

Since its discovery more than 30 years ago, TP53/TRP53/ p53 has been recognized to be a major tumor suppressor protein that functions principally through effects on apoptosis, cell cycle arrest, and DNA repair [START_REF] Vousden | p53 in health and disease[END_REF][START_REF] Brady | p53 at a glance[END_REF]. Although noncanonical functions of TP53/TRP53/p53 in cell metabolism, autophagy, necrosis, and proliferation have recently been studied in detail, their roles in tumorigenesis have yet to be determined [START_REF] Napoletano | p53-dependent programmed necrosis controls germ cell homeostasis during spermatogenesis[END_REF][START_REF] Hager | Understanding the non-canonical pathways involved in p53-mediated tumor suppression[END_REF][START_REF] Mollereau | The p53 control of apoptosis and proliferation: lessons from Drosophila[END_REF].

Autophagy is an intracellular multistep process responsible for the degradation and recycling of cytoplasmic contents by lysosomal proteases [START_REF] He | Regulation mechanisms and signaling pathways of autophagy[END_REF]. The first step in autophagy is formation of a double-membrane structure that engulfs cytoplasmic components, such as protein aggregates and damaged organelles, and matures to form a vesicle known as an autophagosome. In turn, the autophagosome fuses with a lysosome to form an autolysosome. This process culminates in degradation of the vesicle contents, a phenomenon known as autophagic flux. For proper functioning, autophagy requires both vesicle formation and autophagic flux [START_REF] Klionsky | Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)[END_REF]. Autophagy is a prosurvival mechanism required for the removal of damaged organelles or misfolded proteins and the impairment of autophagic flux is commonly observed in neurodegenerative diseases [START_REF] Hetz | Disturbance of endoplasmic reticulum proteostasis in neurodegenerative diseases[END_REF].

One of the key unanswered questions in TP53/TRP53/p53 biology concerns the mechanisms by which TP53/TRP53/p53 induces different responses following exposure to stress. The answer may lie in the multiple transcriptional targets and cofactors differentially activated by TP53/TRP53/p53, depending on the cellular and environmental context. It has been suggested that TP53/TRP53/p53 target genes induce reactive oxygen species (ROS) detoxification and DNA repair in lowstress conditions and induce senescence and apoptosis in high-stress conditions [START_REF] Vousden | p53 in health and disease[END_REF]. The study of TP53/TRP53/p53 isoforms provides some interesting clues on how TP53/TRP53/ p53 achieves specific cellular responses [START_REF] Marcel | Biological functions of p53 isoforms through evolution: lessons from animal and cellular models[END_REF]. In humans, the TP53/p53 protein is expressed as 12 isoforms that are generated by 2 internal promoters and alternative spicing and codon initiation sites [START_REF] Khoury | The Isoforms of the p53 Protein[END_REF][START_REF] Bourdon | p53 Family isoforms[END_REF]. In Drosophila, genome annotations have predicted the existence of 3 Drosophila p53 protein isoforms (FlyBase FBgn0039044) (Figure S1A) [START_REF] Marygold | Using FlyBase, a Database of Drosophila Genes and Genomes[END_REF]. The beststudied isoform is ∆Np53 (p53A), which has a 110-amino acid deletion in its transactivation domain and is structurally analogous to the N-terminally truncated human TP53/p53 isoforms [START_REF] Ollmann | Drosophila p53 is a structural and functional homolog of the tumor suppressor p53[END_REF][START_REF] Jin | Identification and characterization of a p53 homologue in Drosophila melanogaster[END_REF][START_REF] Brodsky | Drosophila p53 binds a damage response element at the reaper locus[END_REF]. The p53 (p53B) isoform refers to the fulllength p53 protein that has an intact transactivation domain and is structurally analogous to the human full-length TP53/ p53 [START_REF] Marcel | Biological functions of p53 isoforms through evolution: lessons from animal and cellular models[END_REF][START_REF] Bourdon | p53 isoforms can regulate p53 transcriptional activity[END_REF][START_REF] Dichtel-Danjoy | Drosophila p53 isoforms differentially regulate apoptosis and apoptosis-induced proliferation[END_REF]. The third, most recent discovered isoform, p53E, is a 334-amino acid protein with a unique 10-amino acid transactivation domain which presumably acts as a dominant negative [START_REF] Zhang | The function of Drosophila p53 isoforms in apoptosis[END_REF].

An alternative possibility to explain TP53/TRP53/p53 pleiotropic functions is that TP53/TRP53/p53 may induce multiple pathways simultaneously. For example, the TP53/ TRP53/p53 target DRAM is involved in the regulation of both autophagy and apoptosis [START_REF] Crighton | DRAM links autophagy to p53 and programmed cell death[END_REF][START_REF] Crighton | DRAM, a p53-induced modulator of autophagy, is critical for apoptosis[END_REF]. However, the molecular mechanisms enabling a single TP53/Trp53/p53 gene to regulate 2 apparently incompatible processesautophagy (prosurvival) and apoptosis (prodeath) -in the same cellular context remain unclear. Presumably, there must be antagonism between the pathways to ensure that TP53/TRP53/p53 does not induce conflicting signals, but instead favors the most response appropriate for the particular cell type and stress intensity. Simple organisms, such as Drosophila, have proved useful for studying the primordial functions of TP53/ TRP53/p53, such as induction of apoptosis in response to irradiation [START_REF] Ollmann | Drosophila p53 is a structural and functional homolog of the tumor suppressor p53[END_REF][START_REF] Jin | Identification and characterization of a p53 homologue in Drosophila melanogaster[END_REF][START_REF] Brodsky | Drosophila p53 binds a damage response element at the reaper locus[END_REF][START_REF] Mollereau | Cell death: what can we learn from flies? Editorial for the special review issue on Drosophila apoptosis[END_REF], and for examining a wide range of less-studied functions, including differentiation, growth, neuroprotection, proliferation, and meiotic recombination [START_REF] Mollereau | The p53 control of apoptosis and proliferation: lessons from Drosophila[END_REF][START_REF] Dichtel-Danjoy | Drosophila p53 isoforms differentially regulate apoptosis and apoptosis-induced proliferation[END_REF][START_REF] Fan | Dual roles of Drosophila p53 in cell death and cell differentiation[END_REF][START_REF] Mesquita | A dp53-dependent mechanism involved in coordinating tissue growth in Drosophila[END_REF][START_REF] Lu | Meiotic recombination provokes functional activation of the p53 regulatory network[END_REF][START_REF] De La Cova | Supercompetitor status of drosophila myc cells requires p53 as a fitness sensor to reprogram metabolism and promote viability[END_REF][START_REF] Nakamura | Mitochondrial defects trigger proliferation of neighbouring cells via a senescence-associated secretory phenotype in Drosophila[END_REF][START_REF] Merlo | p53 prevents neurodegeneration by regulating synaptic genes[END_REF].

In this study, we investigated the relationship between p53regulated autophagy and apoptosis in Drosophila. We found that p53 protected the organism from ROS. Interestingly, Drosophila lacking p53 displayed impaired autophagic flux, higher caspase activation and mortality levels upon paraquat treatment. We also found that autophagy and apoptosis were differentially regulated by the p53 isoforms. Whereas the p53 (p53B) isoform induced protective autophagy in adult neurons, the ∆Np53 (p53A) isoform inhibited autophagy by activating the caspases Dronc, Drice, and Dcp-1. These findings uncover a novel layer of p53-mediated regulation of life and death via differential activation of autophagy and apoptosis.

Results

Redundant roles of p53 isoforms in the resistance to ROS

To investigate the importance of p53 in the stress response at the whole-organism level, we first exposed wild-type (WT, w 1118 ) and p53 null (p53 5A-1-4 , null mutant for all the isoforms) flies to ROS by feeding them with 20 mM paraquat (PQ)-containing medium. PQ is a free radicalinducing agent that has been widely used to induce ROS in cells and organisms [START_REF] Arking | Elevated paraquat resistance can be used as a bioassay for longevity in a genetically based long-lived strain of Drosophila[END_REF][START_REF] Wang | JNK signaling confers tolerance to oxidative stress and extends lifespan in Drosophila[END_REF][START_REF] Girardot | Genome wide analysis of common and specific stress responses in adult drosophila melanogaster[END_REF]. We found that p53 null flies were more sensitive than control flies to PQ-induced ROS, with the median time to 50% lethality being reached at ~20 h compared with ~48 to 72 h (Figure 1(a)). Furthermore, p53 null flies rescued by a genomic construct that encompasses the p53 locus (p53Bac, p53 5A1-4 ) showed a PQ sensitivity similar to that of WT flies (Figure 1(a)), which confirms that PQ-sensitivity maps to the p53 locus. To study the physiological function of the ∆Np53 (p53A) and p53 (p53B) isoforms under conditions of oxidative stress, we first examined the levels of ∆Np53 (p53A) and p53 (p53B) transcripts in animals exposed to PQ (Figure S1B). We observed that while ∆Np53 (p53A) transcripts were unchanged by PQ, p53 (p53B) transcripts were increased 3-fold, suggesting that p53 (p53B) may play an important role in the PQ response. Next, we examined transcript levels in 2 characterized p53 isoform mutants [START_REF] Zhang | The function of Drosophila p53 isoforms in apoptosis[END_REF]. Ch-p53B STOP; p53 5A-1-4 (here named Ch-p53B STOP) which is mutant for p53 (p53B) but encodes the ∆Np53 (p53A) isoform and GFP-p53A STOP; p53 5A-1-4 (here named GFP-p53A STOP) which is mutant for ∆Np53 (p53A) but encodes p53 (p53B) isoform. The p53E isoform lacks most of the transactivation domain, having a dominant negative effect; therefore, we excluded it from our study. As predicted, Ch-p53B STOP flies do not express p53 (p53B) but had normal levels of ∆Np53 (p53A) transcripts (Figure S1C), which is consistent with previous data [START_REF] Zhang | The function of Drosophila p53 isoforms in apoptosis[END_REF]. To our surprise, the GFP-p53A STOP mutant lacked both p53 (p53B) and ∆Np53 (p53A) transcripts, which is not observed in the earlier study for technical reasons [START_REF] Zhang | The function of Drosophila p53 isoforms in apoptosis[END_REF]. We thus excluded the GFP-p53A STOP mutant from further analyses and focused on the p53 (p53B) mutant (Ch-p53B STOP) in which ∆Np53 (p53A) is functional. We compared PQ toxicity in Ch-p53B STOP, p53 null , and WT flies and observed that while p53 null mutant showed an increase sensitivity to PQ there was no difference in the survival of Ch-p53B STOP and WT flies (Figure S1D). To further assess the role of ∆Np53 (p53A) in the resistance of flies to PQ treatment, we used CRISPR/Cas9 method to generate ∆Np53 (p53A) mutant flies, named the p53 A2.3 mutant line, carrying a small deletion removing the coding and splice donor of the ∆Np53 (p53A) unique exon, which impairs the ∆Np53 (p53A) isoform (Figure S2). We thus compared PQ toxicity in p53 A2.3 mutant, p53 null , and WT flies and observed no difference in the survival of p53 A2. 3 and WT flies (Figure S2C). Together, these results show that the inactivation of both ∆Np53 (p53A) and p53 (p53B) isoforms in p53 null mutant flies, but not the individual loss of ∆Np53 (p53A) (p53 A2.3 ) or p53 (p53B) (Ch-p53B STOP, p53 -/-), confers an increased sensitivity to PQ treatment. This indicates that ∆Np53 (p53A) and p53 (p53B) isoforms play redundant functions in the resistance to PQ.

p53 null mutant flies have a defective autophagic response in response to paraquat

The sensitivity of p53 null flies to PQ is similar to that of autophagy-defective mutant flies, which are extremely sensitive to ROS (Figure 1(b)) [START_REF] Juhasz | Atg7-dependent autophagy promotes neuronal health, stress tolerance, and longevity but is dispensable for metamorphosis in Drosophila[END_REF][START_REF] Simonsen | Promoting basal levels of autophagy in the nervous system enhances longevity and oxidant resistance in adult Drosophila[END_REF]. Together with the fact that TP53/TRP53/p53 is a known regulator of autophagy [START_REF] Crighton | DRAM, a p53-induced modulator of autophagy, is critical for apoptosis[END_REF][START_REF] Crighton | DRAM links autophagy to p53 and programmed cell death[END_REF], it raises the possibility that autophagy may be defective in p53 null flies. To investigate this, we examined the expression of ref(2)P/SQSTM1/p62, a multifunctional scaffold protein that is degraded during autophagy and accumulates in the cytoplasm of autophagy-defective atg8a mutants [START_REF] Nezis | Ref(2)P, the Drosophila melanogaster homologue of mammalian p62, is required for the formation of protein aggregates in adult brain[END_REF]. We found that ref(2)P levels were higher in p53 null flies than control flies exposed to PQ (Figure 1(c) and S3A). ref(2)P accumulation was also observed in atg8a -/-flies, as previously described [START_REF] Nezis | Ref(2)P, the Drosophila melanogaster homologue of mammalian p62, is required for the formation of protein aggregates in adult brain[END_REF]. The accumulation of ref(2)P could be due a reduced activation of autophagy in flies lacking p53. However, we did not observe a p53-dependent induction of the Atg8a-II:Atg8a-I ratio in WT flies after PQ treatment. In contrast, the Atg8a-II:Atg8a-I ratio was lower in p53 null compared to control flies exposed to PQ, indicating that p53 was not activating autophagy but rather that basal autophagy levels were reduced in the p53 mutant (Figure 1(d)). Thus, p53 null flies exhibit an increased sensitivity to ROS, which is associated with a reduced basal autophagy and an increased ref(2)P levels, suggesting an impairment in the autophagic flux.

Next, we asked whether the increased sensitivity to PQ is associated with increased caspase activation in p53 null flies. Since one of the primary targets of PQ toxicity is the nervous system [START_REF] Cassar | A dopamine receptor contributes to paraquat-induced neurotoxicity in Drosophila[END_REF], we investigated whether loss of p53 resulted in caspase activation in whole flies or fly brains after exposure to PQ. Western blot analysis and immunostaining using specific antibodies [START_REF] Dichtel-Danjoy | Drosophila p53 isoforms differentially regulate apoptosis and apoptosis-induced proliferation[END_REF][START_REF] Querenet | Spen is required for pigment cell survival during pupal development in Drosophila[END_REF] revealed a strong increase in the cleaved form of Dcp-1 and immunoreactivity detected by an anti-cleaved human CASP3 antibody in p53 null and atg8a -/-mutant flies following exposure to PQ in 4-day-old flies (Figure 2(a-h) and S3B). The anti-cleaved human CASP3 antibody, which recognizes the cleaved effector caspases, Drice and Dcp-1 reflects Dronc activity [START_REF] Fan | The cleaved-Caspase-3 antibody is a marker of Caspase-9-like DRONC activity in Drosophila[END_REF]. This increase was evident by western blotting of whole flies (Figure 2(a,b)), fly heads (Figure S3B) and by immunostaining of adult brains (Figure 2(c-h)). In the brain, cells positive for immunoreactivity detected with an anticleaved, human CASP3 antibody, could be detected in the whole brain cortex, particularly in the dorsolateral and dorsomedial protocerebrum, after PQ treatment (Figure 2(c-h)). Although caspase activation was not detected in WT whole flies by western blotting, it could be observed in dissected brains by immunostaining (Figures 2(f,h)). Collectively, these results suggest that the elevated sensitivity of p53 null mutant flies to oxidative stress injury leads to an increase in apoptosis. The observation that p53 null flies display defective autophagic flux and increased apoptosis following exposure to PQ prompted us to study the regulation of autophagy and apoptosis by p53 at the cell and tissue level. We used Drosophila photoreceptor cell (PR) neurons as a model to overexpress ∆Np53 (p53A) and p53 (p53B) in a WT background [START_REF] Mollereau | Photoreceptor differentiation in Drosophila: from immature neurons to functional photoreceptors[END_REF]. Because the isoforms differentially regulate caspase activation, this system allowed us to directly examine the influence of p53 isoforms on caspase activation and the autophagic response in adult PRs (Figure S4) [START_REF] Mendes | ER stress protects from retinal degeneration[END_REF]. When the isoforms were ectopically expressed in adult PRs, we found that ∆Np53 (p53A), similar to the apoptosis protein Rpr (Reaper), induced robust activation of Dcp-1 and apoptosis, whereas p53 (p53B) had no detectable induction of Dcp-1 compared to control (Figure S4A to D). In agreement with the lack of activated Dcp-1 induced by p53 (p53B), the expression of the caspase inhibitor p35 only suppressed Dcp-1 staining and PR degeneration induced by Rpr and ∆Np53 (p53A) but not PR degeneration induced by p53 (p53B). (Figure S4E-I). These results indicate that ∆Np53 (p53A) induces PR apoptosis while p53 (p53B) promotes caspaseindependent PR degeneration.

To assess whether the expression of p53 (p53B) and ∆Np53 (p53A) induces autophagy, we visually monitored the formation of autophagy vesicles in the PRs using a GFP-tagged form of Atg8a/LC3 (GFP-Atg8a) as a reporter. Overexpression of either p53 (p53B) or ∆Np53 (p53A) in PRs increased the number of GFP-Atg8a dots compared with cells overexpressing a control protein, indicating a greater abundance of autophagy vesicles in the cytoplasm (Figure S5A to C and S5G). The induction of autophagy by Drosophila p53 isoforms was also confirmed by the presence of autophagic vacuoles visualized by transmission electron microscopy (TEM) in PR expressing p53 (p53B) or ∆Np53 (p53A) (Figure S5H and S5I).

The increase in autophagic vesicles could result from activation of autophagy, leading to an increase in autophagosome formation, and/or from inhibition of autophagic flux, leading to an accumulation of unprocessed vesicles, as observed in flies overexpressing the polyglutamine protein, Gug/Atro (termed Gug/Atro75QN subsequently in the text) [START_REF] Nisoli | Neurodegeneration by polyglutamine Atrophin is not rescued by induction of autophagy[END_REF]. To distinguish between these 2 possibilities, we assessed autophagic flux by expressing GFP-ref( 2)P and mCherry-GFP-Atg8a reporters in PRs (Figures 3 and4). GFP-ref(2)P offers an advantage over immunostaining of endogenous ref(2)P because its accumulation is independent of ref(2)P promoter regulation by ROS [START_REF] Jain | p62/Sequestosome-1, Autophagy-related Gene 8, and Autophagy in Drosophila Are Regulated by Nuclear Factor Erythroid 2-related Factor 2 (NRF2), Independent of Transcription Factor TFEB[END_REF] and it therefore unambiguously reflects autophagy impairment. As expected, PRs overexpressing Gug/Atro75QN accumulated more GFP-ref(2)P than did control PRs, indicating that autophagic flux was blocked (Figure 3(a,d,g)). We observed a clear difference in autophagic flux between cells overexpressing p53 (p53B) and ∆Np53 (p53A). GFP-ref(2)P levels were unchanged by p53 (p53B) overexpression compared to control (Figure 3(a,c,g)), indicating normal autophagic flux, whereas overexpression of ∆Np53 (p53A) led to an accumulation of GFP-ref(2)P similar to what was observed in PRs overexpressing Gug/Atro75QN (Figure 3 (a,b,d,g)). The same effects of p53 (p53B) and ∆Np53 (p53A) overexpression were observed when autophagic flux was measured using the mCherry-GFP-Atg8a reporter (Figure 4). In PRs expressing this reporter, autophagosomes appear yellow since they emit both GFP and mCherry fluorescence, while autolysosomes emit only mCherry fluorescence due to quenching of eGFP in the acidic environment [START_REF] Klionsky | Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)[END_REF][START_REF] Nagy | How and why to study autophagy in Drosophila: it's more than just a garbage chute[END_REF][START_REF] Nezis | Autophagy as a trigger for cell death: autophagic degradation of inhibitor of apoptosis dBruce controls DNA fragmentation during late oogenesis in Drosophila[END_REF]. We observed more mCherry than GFP fluorescence in PRs expressing p53 (p53B), indicating the presence of autolysosomes and an intact autophagic flux (Figure 4(a,c)). In contrast, mCherry was more colocalized with GFP in PR cells overexpressing ∆Np53 (p53A) compared to p53 (p53B) (Figure 4(a-c)). The perdurance of both GFP and Cherry staining in autophagic vacuoles, reflects a relative paucity of autolysosomes and an inhibition of autophagic flux by ∆Np53 (p53A), perhaps due to a failure of autophagosomes and lysosomes to fuse or a lack of degradative enzymes in autolysosomes (Figures 4(b,c,g). Taken together, these results demonstrate that ∆Np53 (p53A), but not p53 (p53B), inhibits the autophagic flux in PRs.

We hypothesized that the differential effects of ∆Np53 (p53A) and p53 (p53B) on autophagic flux may be due to differences in caspase activation by the 2 isoforms (Figure S4C and S4D). To test this hypothesis, we compared levels of GFP-Atg8a, GFP-ref( 2)P, and mCherry-GFP-Atg8a in cells concomitantly expressing either p53 (p53B) or ∆Np53 (p53A) and the effector caspase inhibitor p35. We found that concomitant expression of ∆Np53 (p53A) and p35 resulted in lower levels of GFP-Atg8a (Figure S5A, S5B, S5E and S5G) and of GFP-ref (2)P (Figure 3(a,b,e,g)) compared to PRs overexpressing ∆Np53 (p53A) alone. This indicates that the inhibition of caspases by p35 restores the defective autophagic flux in PR expressing ∆Np53 (p53A). Similarly, concomitant expression of ∆Np53 (p53A) and p35 reduced the levels of GFP and the colocalization of GFP and Cherry, compared to ∆Np53 (p53A)-overexpressing PRs carrying mCherry-GFP-Atg8a (Figure 4(a,b,d,e,g)). In contrast, p35 had no effect on GFP-Atg8a, GFP-ref( 2)P and mCherry-GFP-Atg8a levels when coexpressed with p53 (p53B) (Figures S5F, S5G, 3F, 3G, 4F and4G). This was expected because p53 (p53B) expression does not induce caspase activation (Figure S4D). Expression of p35 alone induced a reduction of basal GFP-Atg8a and GFP-ref(2)P levels in control PRs suggesting that low level of caspase activation, which is not detected with the antibody against cleaved Dcp-1, inhibits autophagic flux in wild type retina (Figure S5J staining in PRs expressing ∆Np53 (p53A) (Figure S6C and S6D) and decreased GFP-ref( 2)P to the level seen in control PRs (Figure 5(h,j,p)). RNAi-mediated drice knockdown also led to a decrease in GFP-ref(2)P levels in PRs expressing ∆Np53 (p53A) (Figure 5(h,k,p)). As a control, knockdown of dcp-1 or drice had no significant effects on GFP-ref(2)P levels in PRs expressing p53 (p53B) or in control PRs (Figure 5(m,n,o,p) and S6E). Thus, activation of Dronc, Dcp-1, and Drice is required for ∆Np53 (p53A) to inhibit processing of autophagy vesicles.

Induction of autophagy protects PRs from p53 (p53B)-induced death

We showed that the overexpression of p53 (p53B) does not induce caspase activation but instead induces progressive caspase-independent PR death and concomitant activation of functional autophagy. By contrast, ∆Np53 (p53A) overexpression induces caspase activation, subsequent inhibition of autophagic flux and PR death. We therefore investigated the role of p53 (p53B)-dependent autophagy in PR survival. We overexpressed p53 (p53B) or ∆Np53 (p53A) in retinas carrying atg1 mutant clones by using the Tomato-GFP-FLP-FRT method, and evaluated PR survival in young flies at a time before extensive cell death has occurred [START_REF] Gambis | Two-color in vivo imaging of photoreceptor apoptosis and development in Drosophila[END_REF][START_REF] Dourlen | Drosophila fatty acid transport protein regulates rhodopsin-1 metabolism and is required for photoreceptor neuron survival[END_REF]. Expression of ∆Np53 (p53A) resulted in similar levels of cell death in WT (labeled with tomato and GFP) and atg1 mutant (labeled GFP and the absence of tomato) PRs (Figure 6(c,e)). This finding was expected because autophagy is impaired by expression of ∆Np53 (p53A) in WT retinas (Figures 3 and4). By contrast, the rate of p53 (p53B)-induced PR death was higher in atg1 mutant cells compared to the surrounding area of WT PRs (Figure 6(d,e)). Overall, these results indicate that p53 (p53B) induces functional autophagy that protects PRs from p53 (p53B)-induced neurodegeneration.

Discussion

To gain a better understanding on how p53 can orchestrate cell survival and cell death through various and antagonistic pathways, we have studied the consequences of p53 neuronal gain of function and of paraquat-induced oxidative stress in presence or absence of p53. Neuronal expressions of ∆Np53 (p53A) or p53 (p53B), 2 protein isoforms of Drosophila p53, are both efficient inducers of autophagy but each isoform induces a differential regulation of autophagy (Figure 7): p53 (p53B) induces a functional autophagy, while ∆Np53 (p53A) induces a defective autophagic flux. Indeed, the net contribution of ∆Np53 (p53A) on autophagy is reduced compared to that of p53 (p53B) by the concomitant activation of caspases, which inhibits the autophagic flux. These results also indicate that there must be antagonism between apoptosis and autophagy to ensure that p53 does not induce conflicting signals, and instead promotes the appropriate response for the particular cell type and stress intensity. For example, we, and others, have shown that autophagy protects against neuronal death by inhibiting apoptosis [START_REF] Ravikumar | Rapamycin pre-treatment protects against apoptosis[END_REF][START_REF] Yang | Neuronal apoptosis and autophagy cross talk in aging PS/APP mice, a model of Alzheimer's disease[END_REF][START_REF] Fouillet | ER stress inhibits neuronal death by promoting autophagy[END_REF]. Here, we observed the reverse effect, with caspase activation leading to the inhibition of autophagic flux. Specifically, we showed that the initiator caspase Dronc and the effector caspases Drice and Dcp-1 are required to inhibit the autophagic flux. Thus, the mutual antagonism between apoptosis and autophagy ensures that one of these responses is preferentially induced depending on the cellular context. Two studies have shown that Dcp-1 regulates autophagy in Drosophila [START_REF] Nandi | Acinus integrates AKT1 and subapoptotic caspase activities to regulate basal autophagy[END_REF][START_REF] Devorkin | The Drosophila effector caspase Dcp-1 regulates mitochondrial dynamics and autophagic flux via SesB[END_REF]. In the first study, Kramer et al. show that Dcp-1 inhibits quality control autophagy in developing PRs [START_REF] Nandi | Acinus integrates AKT1 and subapoptotic caspase activities to regulate basal autophagy[END_REF]. In contrast to our finding that Dcp-1 inhibits autophagy by blocking autophagic flux, they show that Dcp-1 inhibits the induction of autophagy in the WT retina by cleaving Acn (Acinus). We thus favor the hypothesis that Dcp-1 has different and currently unidentified targets for regulating autophagic flux in differentiated PRs. In the second study, the Gorski group examines starvationinduced degeneration of the mid-stage egg chamber; in this case, Dcp-1 is required for cell death and autophagy [START_REF] Devorkin | The Drosophila effector caspase Dcp-1 regulates mitochondrial dynamics and autophagic flux via SesB[END_REF][START_REF] Hou | Effector caspase Dcp-1 and IAP protein Bruce regulate starvation-induced autophagy during Drosophila melanogaster oogenesis[END_REF]. Also in contrast to our finding that Dcp-1 inhibits autophagic flux, they show that Dcp-1 increases autophagic flux by interacting in a proteolysis-independent manner with mitochondrial adenine nucleotide translocase stress-sensitive B (SesB). Collectively, these findings and our own show that Dcp-1 can regulate the induction of autophagy and autophagic flux in multiple ways depending on the cell type and developmental stage. Thus, the outcomecell survival or deathdepends not only on the differential expression of p53 isoforms but also on their ability to activate 2 mutually antagonistic responses, autophagy or apoptosis, that are independently responsive to the prevailing conditions.

The differential regulation of apoptosis by TRP73/p73 isoforms has also been studied in mouse neuronal culture submitted to neurotoxic expression of Aβ protein [START_REF] Benosman | Complex regulation of p73 isoforms after alteration of amyloid precursor polypeptide (APP) function and DNA damage in neurons[END_REF]. In this model of neurodegeneration, TRP73β promotes while ∆NTRP73 inhibits apoptosis, which is opposite to our observation that ∆Np53 promoted apoptosis while p53-induced caspase-independent cell death in flies. Furthermore, the differential expression of human TP53 isoforms ∆133TP53 and TP53β in astrocytes is important to confer neuroprotection in cellular models of Alzheimer disease and amyotrophic lateral sclerosis [START_REF] Turnquist | p53 isoforms regulate astrocyte-mediated neuroprotection and neurodegeneration[END_REF]. Thus, further work is needed to define the This study reveals the importance of ∆Np53 (p53A) and p53 (p53B) in response to OS. It also shows that both ∆Np53 (p53A) and p53 (p53B) confer redundant resistance to OS induced by PQ. This indicates that physiological levels of both p53 isoforms are required for the organismal survival. A physiological role of both ∆Np53 (p53A) and p53 (p53B) isoforms in the resistance to OS is novel and contrasts with the fact that most of p53 functions are attributed to the ∆Np53 (p53A) isoform in Drosophila [START_REF] Mollereau | The p53 control of apoptosis and proliferation: lessons from Drosophila[END_REF]. Indeed, ∆Np53 (p53A) is the main isoform expressed in developing and normal fly tissues and is responsible for the induction of apoptosis in response to irradiation (FlyBase FBgn0039044 and [START_REF] Zhang | The function of Drosophila p53 isoforms in apoptosis[END_REF]). In contrast, p53 (p53B) expression is mainly restricted to reproductive organs where it is required for programmed necrosis during spermatogenesis [START_REF] Napoletano | p53-dependent programmed necrosis controls germ cell homeostasis during spermatogenesis[END_REF]. Thus, depending on tissue type and stress, a single or both isoforms are required to induce an appropriate p53 biological response. Our results also suggest that p53 is required for protection against OS by maintaining functional levels of basal autophagy in Drosophila. Indeed, p53 null mutant flies exhibit a decreased resistance to PQ that is associated with an impaired autophagic flux. The defective autophagy could be a direct consequence of reduced autophagy activation in flies lacking p53. However, we did not observe a p53-dependent activation of autophagy after PQ treatment. In contrast, the basal autophagy was reduced in p53 null mutant flies treated with PQ, indicating that p53 is not activating autophagy but rather that basal autophagy is impaired in the absence of p53. Thus, the defective autophagy is not due to a decrease of autophagy induction but could reflect a caspase-dependent inhibition of autophagic flux in p53 null mutant exposed to PQ. To reconcile that p53 is protective in flies treated with PQ with the fact that overexpression of ∆Np53 (p53A) or p53 (p53B) induces cell death when overexpressed in PRs, we propose that p53 responses and the outcome survival or death depends on p53 levels of expression (Figure 7). In PQtreated flies, p53 is induced at physiological level, and is required for cell and organismal survival possibly by promoting an anti-oxidant response as previously proposed [START_REF] Sablina | The antioxidant function of the p53 tumor suppressor[END_REF][START_REF] Nam | p53 promotes cellular survival in a context-dependent manner by directly inducing the expression of haeme-oxygenase-1[END_REF]. In contrast, overexpression of ∆Np53 (p53A) or p53 (p53B), under the strong ninaE/Rh1 driver, activates autophagy and caspase-dependent or caspase-independent cell death, respectively, ultimately leading to cell demise. These results reveal distinct p53 responses, with the outcomesurvival or deathdepending not only on the intensity and duration of stress as previously proposed [START_REF] Oren | Decision making by p53: life, death and cancer[END_REF][START_REF] Horn | Coping with stress: multiple ways to activate p53[END_REF] but also on the levels of each p53 isoform. In conclusion, our data bring novel insights to the model on the flexibility of the pleiotropic p53 gene in inducing a single context-appropriate pathway to protect the organism [START_REF] Mollereau | The p53 control of apoptosis and proliferation: lessons from Drosophila[END_REF].

Materials and methods

Fly strains and genetic manipulations

Flies were reared at 25°C in a 12 h/12 h light-dark cycle. The following lines were used: w 1118 (WT), p53 5A1-4 null allele [START_REF] Rong | Targeted mutagenesis by homologous recombination in D. melanogaster[END_REF] (Bloomington Stock center, BL6815), atg8a KG07569 null allele (Bloomington stock center, BL14639 [START_REF] Scott | Direct induction of autophagy by atg1 inhibits cell growth and induces apoptotic cell death[END_REF]), and dronc I29 (gift of Dr. A. Bergman). In overexpression experiments, ninaE/rh1-gal4 line (gift of J. Treisman [START_REF] Mollereau | A green fluorescent protein enhancer trap screen in Drosophila photoreceptor cells[END_REF]) or the da-gal4 line (Bloomington stock center) were crossed with UAS-GFPref( 2)P (gift of T. Neufeld [START_REF] Chang | An Atg1/Atg13 complex with multiple roles in TOR-mediated autophagy regulation[END_REF]), UAS-GFP-LC3 (UAS-GFP-Atg8a) gift of H. Stenmark (Department of Molecular Cell Biology, Institute for Cancer Research, Oslo, Norway) [START_REF] Rusten | Programmed autophagy in the Drosophila fat body is induced by ecdysone through regulation of the PI3K pathway[END_REF]), UAS-mCherry-GFP-Atg8a (gift of I. Nezis (School of Life Sciences, University of Warwick, Coventry, United Kingdom) [START_REF] Nezis | Autophagy as a trigger for cell death: autophagic degradation of inhibitor of apoptosis dBruce controls DNA fragmentation during late oogenesis in Drosophila[END_REF]), UAS-mCD8-RFP, UAS-LacZ (Bloomington Stock center), UAS-p53, UAS-∆Np53 [START_REF] Dichtel-Danjoy | Drosophila p53 isoforms differentially regulate apoptosis and apoptosis-induced proliferation[END_REF], UAS-p35 (Bloomington stock center), UAS-luciferase IR (Bloomington stock center), UAS-dcp-1 IR (VDRC34328), UAS-drice IR (VDRC28064), UAS-p53 IR, (VDRC10692), or UAS-p53 IR (VDRC45138). The ey-flp, ninaE/rh1-GAL4;UAS-GFP;FRT80 UAS-tdTomato line was described previously [START_REF] Gambis | Two-color in vivo imaging of photoreceptor apoptosis and development in Drosophila[END_REF][START_REF] Dourlen | Drosophila fatty acid transport protein regulates rhodopsin-1 metabolism and is required for photoreceptor neuron survival[END_REF] and was crossed with the ninaE/rh1-gal4 line to generate clones with atg1Δ3D FRT80 stocks (gift from T. Neufeld (Department of Genetics, University of Minnesota, Minneapolis, USA) [START_REF] Scott | Role and regulation of starvation-induced autophagy in the Drosophila fat body[END_REF]). Cherry-p53B STOP; p53 5A-1-4 (FlyBase FBal0318401) (here named Ch-p53B STOP) and GFP-p53A STOP; p53 5A-1-4 (FlyBase FBal0318403) (here named GFP-p53A STOP) were previously reported [START_REF] Zhang | The function of Drosophila p53 isoforms in apoptosis[END_REF].

Transformation with p53BAC and generations of ∆Np53 (p53A) alleles by CRISPR-Cas9 p53BAC flies carrying a wild-type p53 genomic BAC clone of 21-kilobase in the P[acman] system [START_REF] Venken | acman] BAC libraries for transgenesis studies in Drosophila melanogaster[END_REF] were injected to generate transgenic lines using ΦC31 integrase-mediated transgenesis (Best Gene, Inc. Chino Hills, CA, USA). Vector DNA was injected in embryos carrying attP docking sites on the second chromosome (strain 9736 at 53B2).

The ∆Np53 (p53A) isoform-specific alleles were made using CRISPR-Cas9 methods as described [START_REF] Gratz | Genome engineering of Drosophila with the CRISPR RNA-guided Cas9 nuclease[END_REF]. The p53 A2.3 allele is a 23 bp deletion that spans the first ∆Np53 (p53A) transcript exon/intron junction (coordinates 23053346--23053368 in the D. melanogaster genome version 6), with a 7 base-pair insertion of TACTGCT. This allele deletes part of the unique ∆Np53 (p53A) coding sequence and the splice donor site. The p53 A39.4 allele is a 29 base pair deletion (coordinates 23053338-23053366), which also spans the first ∆Np53 (p53A) exon/intron junction, deleting part of the first unique exon that encodes ∆Np53 (p53A) and the RNA splice donor site.

Chemical treatments

Paraquat (Sigma, 36541) was freshly added to phosphatebuffered saline (PBS Dubelccos; Invitrogen Life Technologies, 14200067) containing 0.8% low melting agarose (Sigma, A9414) and 10% sucrose (Sigma, S0389) shortly before the experiment. Twenty flies (3 d of age) were fed with 20 mM paraquat (Sigma, 36541)-containing medium for 5-7 d (survival experiments) or 21 h (protein or RNA extractions and brain dissections). Unless otherwise indicated, flies were kept at 25°C throughout the experiment.

Live fluorescence imaging of PRs CO 2 -anesthetized flies were placed in a 35-mm cell culture dish half-filled with 1% agarose, covered with water at 4°C, and observed using a confocal microscope (LSM700; Carl Zeiss Microscopy GmbH, Göttingen, Germany) as described previously [START_REF] Gambis | Two-color in vivo imaging of photoreceptor apoptosis and development in Drosophila[END_REF][START_REF] Dourlen | The Tomato/GFP-FLP/FRT method for live imaging of mosaic adult drosophila photoreceptor cells[END_REF].

Retina dissection and immunostaining

Retinas of 3-day-old flies were dissected following the standard procedure described in [START_REF] Domingos | Regulation of R7 and R8 differentiation by the spalt genes[END_REF]. Briefly, heads were kept in a drop of PBS to avoid drying. One eye was then cut off using a scalpel and the cuticle around the eye removed. Finally, the brain parts attached to the retina were carefully removed using forceps. Samples were conserved in 4% paraformaldehyde (PFA)-PBS on ice until all the retinas had been dissected, and they were then fixed for 20 min at room temperature (RT). After fixing, the samples were washed 3 times in 0.3% Triton X-100 (Sigma, T8787) in PBS (0.3% PBS-T) and incubated with polyclonal rabbit anti-GFP (Invitrogen, A6455; 1:200) or monoclonal rabbit anti-cleaved Dcp-1 (Cell Signaling Technology, 9578S; 1:300) in 0.1% Triton -100, 4% normal goat serum (Sigma, G9023), PBS for 16 h at 4°C. The samples were washed 3 times in washing solution and incubated with anti-rabbit Alexa Fluor 488 (Invitrogen, A21206: 1:500) or anti-rabbit Alexa Fluor 546 (Invitrogen, A10040: 1:500) secondary antibody, as appropriate. Samples were also incubated with phalloidin-rhodamine (Sigma, 77,418: 1:400) or phalloidin-633 (Sigma, 68,825: 1:400) to better visualize the PR cells. Finally, samples were washed 3 times and embedded in Vectashield medium containing DAPI (Vectashield, AbCys, H1200), mounted, and stored at 4°C until visualization. For the analysis of the mCherry-GFP-Atg 8a signal, samples were washed in 0.1% PBS-T after fixation and immediately mounted for visualization.

Brain dissection and immunostaining

Brains of paraquat (PQ)-or control-treated flies were dissected at RT in Ringer solution (Sigma, 96724) and fixed for 2 h on ice in 4% PFA in PBS. After 3 15-min washes in 0.5% PBS-T, tissues were blocked for 2 h in 2% bovine serum albumin fraction V (Sigma, A2153) in 0.5% PBS-T. Brains were incubated overnight at 4°C in blocking solution containing rabbit anti-cleaved human CASP3/caspase 3 (Asp175) mAb (1:100; Cell Signaling Technology, 9661). After 3 15-min washes in 0.5% PBS-T, brains were incubated for 2 h at RT with Alexa Fluor 488 antirabbit (1:1000; Invitrogen, A21206). Tissues were washed 3 times in 0.5% PBS-T for 15 min and then mounted in ProLong Gold Antifade Mountant (Life Technologies, P36930).

Transmission electron microscopy

Dissected Drosophila eyes were fixed in 0.1 M cacodylate buffer, 2.5% glutaraldehyde, and 2 mM CaCl 2 for 16 h at 4°C. After rinsing with 0.1 M cacodylate buffer at RT, the eyes were incubated with 1% OsO 4 in 0.1 M cacodylate buffer for 2 h at RT. Tissues were then progressively dehydrated in acetone at RT and mounted in 100% epoxy resin (Epon 812; Electronic Microscopy Sciences, 14120) in silicone-embedding molds. After resin polymerization for 48 h at 60°C, samples were sliced into 60-nm sections, which were stained with lead citrate and examined with a Philips CM120 (Thermo Fischer Scientific, Eindhoven, The Netherlands) transmission electron microscope (TEM) operating at 80 kV.

Image processing

Images were acquired at the imaging facility (PLATIM, UMS3444) at the UMR8249 imaging facility and analyzed with ImageJ (National Institutes of Health) or with Fiji software. For retina immunostaining, images were acquired on acousto-optical beam splitter confocal laser-scanning microscopes (SP5; Leica Microsystems, Wetzlar, Germany) with an HCX Plan Apochromat 63 × 1.4-0.6 oil (numerical aperture: 1.4) objective using the acquisition software LAS AF (Leica). For whole-mount brain staining, images were acquired on an A1R confocal microscope (Nikon Instrument, Tokyo, Japan) with a 40× objective using NIS-Elements Advanced Research software (Nikon). For live imaging of PRs, images were acquired on a confocal laser-scanning microscope (LSM700; Carl Zeiss Microscopy GmbH, Göttingen, Germany) with an immersion 40 × 0.75 water (numerical aperture: 0.75) objective using the acquisition software Zen (Zeiss).

Protein extracts and western blotting analysis

Proteins were extracted from 3 or 4 whole animals after treatment (4-day-old flies) using a standard protocol. Briefly, anesthetized or frozen flies were squashed in extraction buffer (1% NP40; abcam ab142227), 20 mM Tris HCl, pH 7.5, 100 mM NaCl, 2 mM DTT, and protease inhibitor cocktail (Roche, 11836153001), incubated on ice for 30 min, and centrifuged for 30 min at 12,000 g. An aliquot (1 μl) of the supernatant was then tested in a Bradford protein assay reaction (Bio-Rad, 500-0201). The samples were boiled, and 50 μg of protein per lane were resolved by SDS-PAGE using 15% gels for the analysis of endogenous Atg8 and cleaved Dcp-1 and 4-20% gels (Bio-Rad, 456-1093) for detection of ref( 2)P, endogenous Atg8, and cleaved Dcp-1 simultaneously. After electrophoresis, proteins were transferred to PVDF (for endogenous Atg8) or nitrocellulose membranes, blocked for 1 h in TBS (50 mM Tris-HCL pH 7, 0.15 M NaCl in ddH 2 O) containing 0.1% Tween 20 (Sigma, P7949) (0.1% TBS-T) and 5% milk and then incubated at 4°C for 16 h in 0.1% TBS-Tween, 1% milk containing rabbit anti-cleaved Dcp-1 (1:1000; Cell Signaling Technology, 9578S), rabbit anti-cleaved human CASP3 (Asp175) (1:1000; Cell Signaling Technology, 9661), mouse anti-TUBA/α-tubulin (1:1000; Sigma, T6199), anti-Atg8 (1:5,000; gift of G. Juhasz (Department of Anatomy, University of Budapest, Hungary) [START_REF] Nagy | Atg17/FIP200 localizes to perilysosomal Ref(2)P aggregates and promotes autophagy by activation of Atg1 in Drosophila[END_REF], or rabbit anti-ref(2)P (1:500; gift of S. Gaumer (Laboratoire de Génétique et de Biologie Cellulaire, Université de Saint-Quentin-en-Yvelines, Montigny le Bretonneux, France) [START_REF] Nezis | Ref(2)P, the Drosophila melanogaster homologue of mammalian p62, is required for the formation of protein aggregates in adult brain[END_REF]. Membranes were washed 3 times in 0.1% TBS-T for 10 min and incubated at RT for 2 h in 0.1% TBS-T/1% milk containing anti-rabbit HRP (1:10,000; GE Healthcare, NA9340) or anti-mouse HRP (1:10,000; GE Healthcare, NA9310) secondary antibody, as appropriate. After 3 washes, proteins were revealed using ECL prime (GE Healthcare, RPN2232) according to the kit protocol. Western blot analysis of the p53 CRISPR alleles was performed with antihuman TP/p53 (Santa Cruz Biotechnology, C11) on protein extracts from adult flies 4 h after irradiation with 4000 rads of gamma rays as described previously [START_REF] Zhang | The function of Drosophila p53 isoforms in apoptosis[END_REF][START_REF] Zhang | Low levels of p53 protein and chromatin silencing of p53 target genes repress apoptosis in drosophila endocycling cells[END_REF].

RT-PCR

Expression of p53 (p53B) and ∆Np53 (p53A) were monitored by RT-PCR. RNA was extracted from adult flies using TRIzol reagent (Invitrogen, 15596026) and cDNA was produced using the SuperScript III First Strand Synthesis System (Invitrogen, 18080044) with random hexamers.

PCR was performed using the following primers: p53 (p53B) forward: GGACACAAATCGCAACTGCT, ∆Np53 (p53A) forward: CACAGCCAATGTCGTGGCAC, and a common reverse primer for p53 (p53B) and ∆Np53 (p53A): GGCCATGGGTTCCGTGGTCA. Amplicons of ∆Np53 (p53A) (129 base pairs [bp]) and p53 (p53B) (519 bp) were visualized after 30 and 35 cycles, respectively, with a melting temperature of 60°C. Samples were resolved on a 2% agarose gel. RpL32/rp49 was used as an internal control for RNA extraction and RT-PCR efficiency and was amplified using forward: ATCGTGAAGAAGCGCACCAAG and reverse: ACCAGGAACTTCTTGAATCCG primers. Amplicons of RpL32/rp49 (203 bp) were visualized after 25 cycles with a melting temperature of 60°C.

Image and statistical analyses

All microscopy and western blot images were analyzed using ImageJ software, with the exception of the western blot shown in Figure S1C, which was analyzed using Fiji. Quantification of GFP-ref( 2)P and GFP-Atg8a was performed using the Find Maxima function of ImageJ. Western blots were quantified densitometrically.

Statistical analyses were performed using Prism software (GraphPad). In all figures, bar graphs represent the mean ± SEM. For survival experiments, the results were analyzed using two-way ANOVA with the Tukey multiple comparison post hoc test. Quantification of western blots and caspase staining in the brain was analyzed using one-way ANOVA and the Bonferroni post hoc test. For all other experiments, the results were analyzed using the Student t test.

List of abbreviations

Figure 1 .

 1 Figure 1. p53-and autophagy-mutant flies show increased sensitivity to paraquat. (a and b) Survival curves of wild-type and mutant Drosophila fed with normal or 20 mM paraquat (PQ)-containing media (A, n = 4 and B, n = 5). 1) Control, w1118 flies, 2) p53 null (p53 5A1-4 mutants), 3) p53BAC, p53 null flies carrying a wild-type p53 genomic rescue BAC clone on the second chromosome and 4) atg8 -/-, (atg8 KG07569 mutants). (c) Densitometric quantification of endogenous ref(2)P levels in whole flies fed with normal or PQ-containing media. Data are the mean ± SEM of 5 independent blots and are presented as the ratio of ref(2)P:tubulin in experimental animals relative to the ratio in control non-treated flies. *P < 0.05, **P < 0.01 by one-way ANOVA followed by the Bonferroni post hoc test. (d) Quantification of Atg8-II:Atg8-I from western blots of control and p53 null flies treated or non-treated with PQ. Data are the mean ± SEM of n = 3 blots and are presented as expression levels relative to that in non-treated control flies.

Figure 2 .

 2 Figure 2. p53-and autophagy-defective flies show increased caspase activation after paraquat treatment. (a and b) Representative western blot and densitometric quantification of endogenous cleaved Dcp-1 in whole flies. Data are the mean ± SEM of 3 independent blots and are expressed as the ratio of cleaved Dcp-1:tubulin. ***P < 0.001 by one-way ANOVA followed by the Bonferroni post hoc test. (c) Dorsomedial protocerebrum area of the fly brain selected for quantification of immunoreactivity to an anti-cleaved human CASP3 antibody (yellow box). (d-g) Representative images of the above-mentioned cleaved human CASP3 immunostaining in the dorsomedial protocerebrum of untreated control (d) and p53 -/-(e) flies or PQ-treated control (f) and p53 -/-(g) flies. Scale bars: 25 µm. White arrows show cells positively stained for cleaved human CASP3. (h) Quantification of staining with immunoreactivity against cleaved human CASP3 in the dorsomedial protocerebrum from untreated (n = 8) and PQ-treated (n = 6) control flies, or untreated (n = 5) and PQ-treated (n = 5) p53 -/-flies. Data are the mean ± SEM of the number of dots of immunoreactivity against cleaved human Caspase 3 counted in the dorsomedial protocerebrum. **P < 0.01, ***P < 0.001 by one-way ANOVA followed by the Bonferroni post hoc test.

  and S5K). Collectively, our results indicate that ∆Np53 (p53A)-dependent activation of caspases inhibits autophagic flux in adult Drosophila PRs. Dronc, Dcp-1, and Drice are required for inhibition of autophagic flux by ∆Np53 (p53A) Having shown that caspase activation induced by ∆Np53 (p53A) impairs autophagic flux, we next asked whether the

Figure 3 .

 3 Figure 3. Differential ref(2)P processing in photoreceptors expressing p53 (p53B) or ∆Np53 (p53B). (a-f) Representative fluorescence microscopy images of the retinas of adult flies expressing autophagic flux reporter GFP-ref(2)P in photoreceptors (PRs). The nuclei were stained with DAPI (blue). Flies also overexpressed mCD8-RFP (control protein), ∆Np53 (p53A), p53 (p53B), gug/atro75QN, ∆Np53 (p53A) and p35, or p53 (p53B) and p35 in PRs under the control of ninaE/rh1-GAL4. Actin was visualized by staining with phalloidin (red). Scale bars: 20 µm. (g) Quantification of GFP-ref(2)P dots per retina in the strains represented in (a-f) using the Find Maxima function of ImageJ software. Data are the mean ± SEM of n = 8 to 15 retinas. *P < 0.05, **P < 0.01 by the Student t test.

Figure 4 .

 4 Figure 4. Differential processing of mCherry-GFP-Atg8 in p53 (p53B)-and ∆Np53 (p53A)-expressing flies. (a-f). Representative fluorescence microscopy images of the retina of adult flies overexpressing the autophagy reporter mCherry-GFP-Atg8 plus LacZ (control) ± p35, ∆Np53 (p53A) ± p35, and p53 (p53B) ± p35 driven by ninaE/ rh1-GAL4. mCherry in red and GFP is green. Staining from n = 2 to 10 retinas. Arrowheads show high levels of GFP-positive dots. Scale bars: 10 μm. (g) Quantification of colocalization of cherry and GFP staining by the Pearson coefficient from A to F, n = 15-26 retinas *P < 0.05, **P < 0.01 by one way Anova followed by the Tukey multiple comparison test.

Figure 5 .

 5 Figure 5. Inhibition of Dronc, Drice, or Dcp-1 restores ref(2)P processing in photoreceptors expressing ∆Np53 (p53A). (a-f) Representative fluorescence microscopy images of the retinas of adult flies expressing the autophagic flux reporter GFP-ref(2)P in photoreceptors (PRs). Flies also overexpressed mCD8-RFP (control protein), ∆Np53 (p53A), p53 (p53B), gug/atro75QN, ∆Np53 (p53A) and dronc I29 , or p53 (p53B) and dronc I29 in PRs under the control of ninaE/rh1-GAL4. Dronc I29 flies carry one dronc I29 mutant allele. Actin and cell nuclei were visualized by staining with phalloidin (red) and DAPI (blue), respectively. Scale bars: 20 µm. (g) Quantification of GFP-ref(2)P dots per retina area in the strains represented in (a-f). Data are the mean ± SEM of n = 8 to 17 retinas. *P < 00.5 by the Student t test. (h-o) Representative images of the retina of adult flies expressing the autophagic flux reporter GFP-ref(2)P in PRs. Flies also overexpressed mCD8-RFP (control), ∆Np53 (p53A), ∆Np53 (p53A) with dcp-1RNAi, ∆Np53 (p53A) with drice-1RNAi, gug/atro75QN, p53 (p53B), p53 (p53B) with dcp-1RNAi, or p53 (p53B) with drice-1RNAi under the control of ninaE/rh1-GAL4. Actin and cell nuclei were visualized by staining with phalloidin (red) or DAPI (blue), respectively. Scale bars: 20 µm. (p) Quantification of GFP-ref(2)P dots per retina area in the strains represented in (h-o). Data are the mean ± SEM of n = 8 to 15 retinas. *P < 0.05 by the Student t test.

Figure 7 .

 7 Figure 7. Life and death: levels of p53 isoforms matter. (Left) When p53 isoforms are overexpressed in photoreceptor neurons at high levels, p53 (p53B) induces autophagy and caspase-independent cell death, while ∆Np53 (p53A) induces caspase activation and inhibits autophagic flux. (Right) Physiological levels of p53 isoforms confer redundant resistance to reactive oxygen species (ROS) and a functional autophagic flux. Pink schematic labeled photoreceptors on the left represents one ommatidium containing 8 photoreceptor neurons and accessory cells.

Figure 6 .

 6 Figure 6. atg1 mutant photoreceptors show increased sensitivity to p53 (p53B)-induced, but not ∆Np53 (p53A)-induced death. Ommatidia from flies overexpressing LacZ (control), ∆Np53 (p53A), or p53 (p53B) in PRs under the control of ninaE/rh1-GAL4. (a) Representative mosaic ommatidia with wild-type (ey-flp, ninaE/rh1-GAL4; UAS-GFP; FRT80 atg1Δ3D/FRT80 ninaE/Rh1-tomato) and atg1 -/-mutant (ey-flp, ninaE/rh1-GAL4;UAS-GFP; FRT80 atg1Δ3D/FRT80 atg1Δ3D) PRs, as generated for (b-d). Numbers 1 to 6 indicate the outer PRs (R1-6). Wild-type PRs carry 2 copies of Tomato in addition to GFP and thus appear yellow Mutant clones, in green, were generated by mitotic recombination and thus carry GFP but not the Tomato reporter. (b-d) Representative retinas showing atg1∆3D mutant clone size). Scale bars: 20 µm. (e) Quantification of PR loss in wild-type and mutant clones from n = 12 to 14 retinas. Data are shown as global PR loss per fly strain. ***P < 0.001 by the Student t test.

Acknowledgments

We thank Dali Ma for for generating unpublished material related to this work, Michael Dixon for technical assistance, Daan Van Den Brink and Charlotte Scholtes for art work. We also thank A. Bergmann, J. Treisman, T. Neufeld, H. Stenmark, H.D. Ryoo, I. Nezis; the Bloomington, DGRC Kyoto, and VDRC stock centers; and DSHB for fly stocks and reagents. We thank the ARTHRO-TOOL fly, PLATIM (UMS3444 Biosciences Lyon) and the Centre Technologique des Microstructures CTμ (Lyon 1) microscopy facilities. The authors declare no conflicts of interest.

Disclosure statement

No potential conflict of interest was reported by the authors.

Funding

This work was supported by ANR Ire1-PD (13-ISV4-0003-01) and ARC foundation, la Ligue contre le Cancer, Rhône, France 705 Parkinson, FINOVI (2017-01 26-A011) grants to BM, a Fondation de France (2012-00034435) grant to SB and BM, a Labex MemoLife (ANR-10-LABX-54 MEMO LIFE) grant to SB, and an NIH 1R01GM113107 to B.R.C. PMD was supported by FCT grants LISBOA-01-0145-FEDER-007660, FCT-ANR/NEU-NMC/0006/2013, 710 PTDC/NEU-NMC/2459/2014, and IF/ 00697/2014.


Olivier Krebs

Emile Benjamin

Aristide Lemaître

Magnetic anisotropy of singly Mn-doped InAs/GaAs quantum dots

Keywords: number͑s͒: 72.25.Fe, 71.35.Pq, 72.25.Rb, 78.67.Hc

We report on the microphotoluminescence spectroscopy of InAs/GaAs quantum dots ͑QDs͒ doped by a single Mn atom in a magnetic field either longitudinal or perpendicular to the optical axis. In both cases the spectral features of positive trion ͑X + ͒ are found to split into strongly circularly polarized components, an effect very surprising in a perpendicular magnetic field. The field-induced splitting is ascribed to the transverse Zeeman splitting of the neutral acceptor complex A 0 issued by the Mn impurity, whereas the circular optical selection rules result from the p-d exchange which acts as a very strong longitudinal magnetic field inhibiting the spin mixing by the transverse field of the QD heavy-hole ground state. A theoretical model of the spin interactions which includes ͑i͒ the local strain anisotropy experienced by the acceptor level and ͑ii͒ the anisotropic exchange due to the out-of-center Mn position provides a very good agreement with our observations.

Doping a semiconductor quantum dot ͑QD͒ with a single Mn atom brings up remarkable spin-related properties due to the sp-d exchange interactions between the confined carriers ͑electron and hole͒ and the magnetic impurity. In the last few years Mn-doped CdTe QD's have been extensively studied by microphotoluminescence ͑-PL͒ spectroscopy in an external magnetic field. [1][2][3][4] Most of the observations were very well interpreted by assuming a 5/2 spin for the Mn ion acting on the carriers confined in a quantum dot through Heisenberg Hamiltonians. Yet, the strong vertical confinement of QD's along their growth axis, as well as their in-plane biaxial strain were shown to modify significantly the spectral features because of the resulting heavy-hole nature of the valence-band ground state.

The quite specific signature of InAs/GaAs quantum dots doped with a single Mn atom has been recently uncovered in -PL spectroscopy. 5 In this system, the Mn impurity acts as an effective J = 1 spin with a noticeable fine-structure splitting in zero magnetic field. This results from the neutral acceptor ͑A 0 ͒ complex formed by Mn in a III-V matrix, namely, a negatively charged center A -and a bound hole h 1 . [6][7][8][9] The J = 1 spin corresponds to the ground state of the 3d 5 Mn spin S =5/ 2 and the bound hole total angular momentum J h 1 =3/ 2 which interact via the antiferromagnetic p-d exchange. Its zero-field splitting results from some local anisotropy of the potential experienced by the bound hole. 5,10,11 Within this interpretation, the anisotropy of the A 0 complex does not affect the optical selection rules of the QD interband transitions which still involve a conduction electron ͑e͒ and a valence-band hole ͑h 2 ͒ essentially of heavyhole character both with S-like orbital. This was shown in Ref. 5 where a longitudinal magnetic field split all the optical transitions into their circularly polarized ͑ Ϯ ͒ components. In this paper, we show that the optical selection rules in a transverse magnetic field are in contrast deeply affected by the anisotropy of the A 0 effective spin, besides in a rather nonintuitive way. Indeed, in a magnetic field perpendicular to the optical axis, the optical transitions which are expected to be linearly polarized as usually encountered in undoped InAs QDs, [12][13][14] exhibit for Mn-doped InAs QDs a strong circular polarization ͑ + or -͒. We show that this effect results from the A 0 spin anisotropy which enables to split by Zeeman effect the ferromagnetic ͑FM͒ and antiferromagnetic ͑AFM͒ configurations of the h 2 -A 0 complex, while the heavy-hole h 2 keeps a well-defined pseudospin

⇑͑J h 2 ,z =+3/ 2͒ or ⇓͑J h 2 ,z =-3/ 2͒.
We studied a sample grown by molecular-beam epitaxy on a semi-insulating GaAs ͓001͔ substrate which consists of a single layer of InAs/GaAs QD's randomly doped by a single Mn atom ͑see Ref. 5 for details͒. We estimate that ϳ0.1-1% of the quantum dots are effectively doped by a single Mn atom. The -PL spectroscopy of individual Mndoped InAs QDs was carried out with a split-coil magnetooptic cryostat. A 2 mm focal-length aspheric lens ͑NA 0.5͒ actuated by piezo motors was used to focus the He-Ne excitation laser and to collect the PL from the sample. This compact microscope, which integrates both the sample and the optical lens, can be rotated about the vertical axis of the cryostat in order to change the magnetic field direction with respect to the optical axis from parallel ͑Faraday configura-tion͒ to perpendicular ͑Voigt configuration͒. Relying on in situ sample imaging we could therefore study the same quantum dot in both configurations. All measurements presented here were performed at low temperature ͑T =2 K͒. The collected PL was dispersed by a 0.6 m focal-length double spectrometer and detected by a nitrogen-cooled CCD array camera.

Figures 1 and2 report on the optical spectroscopy of a charged exciton X + in the same Mn-doped InAs QD ͑QD1͒ measured, respectively, in Faraday and Voigt configuration. Let us first comment on the results shown in Fig. 1. As evidenced in Ref. 5, the Mn-doped InAs QD is identified by its spectral features in zero magnetic field shown in Fig. 1͑b͒. It consists of two doublets separated by the exchange energy ⌬ between the FM and AFM configurations of h 2 -A 0 , plus a weaker line denoted O which corresponds to the transition involving the A 0 state J z = 0. Another specific feature is the equal splitting ␦ of both FM and AFM doublets which is ascribed to the fine structure of A 0 in its anisotropic environment.

When the magnetic field is applied parallel to the optical axis z the quantum levels involved in these optical transitions are split due to the Zeeman effect ͓see Fig. 1͑c͔͒, which gives rise to a very distinctive contour plot of the PL intensity as shown in Fig. 1͑d͒. This "magneto-PL" image is composed of a series of spectra measured in both + and -polarizations, while the magnetic field was changed step by step with an increment of 50 mT from 0 to 5 T. The PL intensity is plotted against a linear color scale, and interpolation was used for a better graphical rendering. We observe a characteristic crosslike pattern, quite similar to the one reported in Ref. 5 for a negatively charged exciton ͑X -͒, with yet a no-ticeable difference. Here the cross shows up in negative fields ͑i.e., in -polarization͒ while it was previously found in positive fields. This kind of mirror symmetry is similar to that observed in Refs. 2 and 5 between an exciton and a biexciton. Here, it corresponds to the symmetry between a positive trion ͑X + ͒ and a negative trion ͑X -͒. Indeed, both involve the same spin configurations either in the final or initial state of the optical recombination with yet orthogonal selection rules due to the Pauli principle. ͑For example, for

X + ͉⇑⇓↑͘→ - ͉ ⇑ ͘ while for X -͉↑↓⇑͘→ + ͉ ↑ ͘.͒ Note that
in this sample most of the quantum dots ͑with or without Mn dop-ing͒ were found as positively charged by an excess hole, after measuring the sign of the Overhauser shift ͑namely, the sign of nuclear polarization͒ generated under quasiresonant circularly polarized excitation. 15,16 The A 0 exchange anisotropy originates from the position of the Mn atom out of the QD center. One can distinguish two main contributions: ͑i͒ the anisotropy of the local potential experienced by the h 1 bound hole which lifts the degeneracy of the J = 1 state of A 0 ͑Refs. 5, 7, and 9͒ and ͑ii͒ the anisotropic part of the exchange coupling between the h 2 hole and the out-of-center Mn spin. 17 The first contribution is responsible for the coupling of the ͉J z = Ϯ 1͘ A 0 states which gives rise to their anticrossing ␦ when they are brought into coincidence. This occurs in zero magnetic field for the X + -A 0 ϵ e-A 0 levels because the effective exchange with X + ͑namely, with a single e spin͒ is essentially zero here, and at B z Ϸ Ϯ 2 T for the h 2 -A 0 levels ͓see Fig. 1͑c͔͒. More generally, this anisotropy is also responsible for the forbidden transitions corresponding to ͉⌬J z ͉ = 2 which form the cross pattern and which are partially permitted because J z is not a ͑Color online͒ QD1 ͑a͒ schematics of an InAs QD doped by a single Mn atom forming a neutral acceptor A 0 . ͑b͒ PL spectrum of a charged exciton X + in such a QD at zero magnetic field. ͑c͒ Diagram of energy levels involved in the X + -A 0 → h 2 -A 0 transition. Solid ͑dashed͒ lines correspond to the levels with ↓͑ ↑ ͒e spin and ⇓͑ ⇑ ͒h 2 pseudospin connected via a + ͑ -͒ photon. ͑d͒ Contour plot of QD1 PL around E 0 = 1.355 eV as a function of longitudinal magnetic field measured in -and + circular polarizations ͑left͒ and theoretical simulation ͑right͒. The diamagnetic shift is subtracted so that the linear dependence on B z due to the Zeeman effect produces straight lines. σ- good quantum number. The second contribution is responsible for the weaker lines in Fig. 1͑d͒ which gives rise to the anticrossings denoted ⌬ a . As discussed below, they result from a mixing of the ͉⇑ , Ϯ 1͘ and ͉⇓ , Ϯ 1͘ h 2 -A 0 states which makes visible the normally forbidden transitions ͉↑ , Ϯ 1͘ → ͉⇓ , Ϯ 1͘. Note however that all the lines keep a very strong circular polarization, because the e-A 0 initial states still have pure electron spin ↑ or ↓.

z σ+ σ- x (a) y z'' InAs QD QD QD QD D D D D Q QD Q QD QD QD QD D y y y y QD QD D QD Q Q QD D QD QD D D D QD D QD D QD D y y y QD A 0 =h 1 +Mn B z z' q f q f a a s s -1 1 0 E-E 0 (meV) Magnetic Field B z (T) 0 4 2 1 3 5 4 2 1 3 5 0 4 2 1 3 5 -4 -2 -1 -3 -5 Magnetic Field B z (T) Experience Model (d) σ + σ + σ- FM AFM a a a a Bz σ + |∞,1Ú |AE,1Ú |∞,ø1Ú |AE,ø1Ú |fi,0Ú | ‡,0Ú |AE,0Ú |∞,0Ú |fi,1Ú | ‡,1Ú |fi,ø1Ú | ‡,ø1Ú δ δ AFM O FM a a X + ∞ fi ‡ h 2 ‡ A 0 A 0 h 2 -A 0 X + -A 0
Magnetic Field B x (T) σ + σ- (a) B x <0 B x >0 0 4 2 6 -4 -2 -6 Magnetic Field B x (T) σ + σ+ -1 1 0 E-E 0 (meV) Model (c) E-E 0 (meV)
In Voigt configuration, the A 0 exchange anisotropy gives rise to a very surprising signature. When the transverse magnetic field is increased each line originating from the zerofield doublets splits into four lines with a strong + or - circular polarization as reported in Fig. 2͑b͒. Such a polarization is quite unexpected because, due to the mixing of the spin states for both the electron and hole by the field, the optical transitions of trions should become linearly polarized as observed in nonmagnetic QDs. [12][13][14]18 Note the timereversal symmetry is yet well respected here since changing the sign of the applied magnetic field reverses the circular polarization from Ϯ to ϯ ͓see Fig. 2͑a͔͒. This clearly implies that the PL spectra depend on the magnetic field direction in the xy plane because changing the sign of B x can be achieved by a rotation about z, which points out the role of A 0 in-plane anisotropy.

We have modeled these magneto-PL images by considering the spin Hamiltonians of the four involved particles ͑Mn, h 1 , e, and h 2 ͒ assumed to occupy the ground state of their respective confinement potential. e and h 2 have essentially the S-like character of the respective conduction and valence QD ground state, while h 1 is assumed to be strongly localized onto the Mn site which lies itself at a certain distance from the QD center ͓see Fig. 1͑a͔͒. The theoretical indiscernibility between both overlapping holes is phenomenologically treated by introducing an exchange Hamiltonian between their spins ͑see below͒, which amounts to treat this issue in the Heitler-London scheme. [START_REF] Fazekas | Lecture Notes on Electron Correlation and Magnetism[END_REF] This approach enables us to restrict ourselves to the spin degree of freedom of the four involved particles ͑S Mn =5/ 2, J h 1 =3/ 2, S e =1/ 2, and J h 2 =3/ 2͒, with interactions described using only their respective spin operators. The single-particle Hamiltonians read

H ˆMn = g Mn B S ˆMn • B, H ˆh1 = g h 1 B J ˆh1 • B + H ˆs, H ˆh2 = g h B J ˆh2 • B + H ˆHL , H ˆe = g e B S ˆe • B, ͑1͒
where B is the Bohr magneton and g ␣ denotes the Landé factor of the particle ␣ taken to the first order as a scalar for Mn, h 1 and h 2 , while for e a longitudinal ͑g e,z ͒ and transverse ͑g e,Ќ ͒ factor will be used. In this formalism, the well-known anisotropy of the Landé factor for the hole states will naturally result from their splitting into heavy-and light-hole states. The potential anisotropy H ˆs experienced by h 1 can be described via an effective strain tensor with three main axes that, in general, differ from the crystallographic axes. 20 In that aim, we introduced as the dominant term a compressive strain ⑀ ʈ along the zЈ direction defined by two angles s and s ͓see Fig. 1͑a͔͒ plus an in-plane shear strain ⑀ Ќ with xЈ and yЈ main axes defined by an angle s in the plane perpendicular to zЈ. H ˆs reads then

H ˆs = - ⑀ ʈ 3 ͫ J ˆh1 ,z Ј 2 - 1 2 ͑J ˆh1 ,x Ј 2 + J ˆh1 ,y Ј 2 ͒ ͬ + ⑀ Ќ 2 ͑J ˆh1 ,x Ј 2 -J ˆh1 ,y Ј 2 ͒,

͑2͒

where the J ˆh1 ,␣ Ј 2 's deduce from the J ˆh1 ,␣ 2 operators by the three successive rotations transforming z into zЈ, namely, via the passage matrix e -i s J ˆh1 ,z e -i s J ˆh1 ,y e -i s J ˆh1 ,z . For the h 2 hole confined in the QD, we used a similar description, with ⌬ HL ϳ 30 meV the splitting between the heavy-hole and light-hole states due to the stronger confinement along z. The corresponding term reads

H ˆHL = - ⌬ HL 3 ͫ J ˆh2 ,z 2 - 1 2 ͑J ˆh2 ,x 2 + J ˆh2 ,y 2 ͒ ͬ . ͑3͒
We assumed that the exchange interactions between the different spins take the form of Heisenberg Hamiltonians, 8,11 with besides a specific anisotropic part H a for the p-d exchange between the out-of-center Mn and h 2 . 17 Since the e-A 0 exchange turns out to be negligible in our experimental observations, which could be due to weak overlap with the A 0 impurity, we only consider here the exchange interactions involving the Mn, h 1 and h 2 spins. The corresponding exchange Hamiltonian reads

H ˆX = Mn-h 1 S ˆMn • J ˆh1 + Mn-h 2 S ˆMn • J ˆh2 + h 1 -h 2 J ˆh1 • J ˆh2 + H ˆa,

͑4͒

where the two-spin exchange energies ␣-␣ Ј are considered as fitting parameters since they depend on the actual overlap between the particles. Yet, we expect the Mn-hole exchange interaction to be antiferromagnetic as usually reported in literature. The anisotropic part H ˆa in Eq. ͑4͒ has been derived in Ref. 17 in the case of a spherical quantum dot. To the first order, it depends linearly on a parameter which depends itself on the Mn position and vanishes when it lies at the QD center. Although our InAs quantum dots are lens-shaped with no well-defined center, we assumed here the same expression for H ˆa as follows:

H ˆa = Mn-h 2 ͑J ˆh2 ,z Љ 2 S ˆMn • J ˆh2 + S ˆMn • J ˆh2 J ˆh2 ,z Љ 2 ͒, ͑5͒
where the angular momentum operator J ˆh2 ,z Љ along the direction zЉ ͓defined by two angles a and a , see Fig. 1͑a͔͒ refers to the position of the Mn atom with respect to some effective QD center, e.g., the maximum of the h 2 S-like envelope function.

To calculate the theoretical PL emission spectra, the Hamiltonians of the initial and final states in X + transition are first diagonalized. In practice we restricted ourselves to the first A 0 levels J =1 ͓as represented in Fig. 1͑c͔͒ and J =2, where J ˆ= S ˆMn + J ˆh1 . The PL intensity emitted in the transition from ͉i͘ to ͉f͘ is calculated by taking into account both the oscillator strength ϰ͉͗f͉P ˆ+͉i͉͘ 2 ͑where P ˆ+ is the dipolar operator for a + polarization͒ and the population ii of the initial state ͉i͘. As He-Ne excitation produces nonpolarized carriers and electron spin relaxation may be considered negligible in QDs during the radiative lifetime of X + ͑Ϸ1 ns͒, the ii 's are determined only by the populations in the different states of A 0 . Assuming that under weak optical excitation density, thermalization occurs mostly in the final state ͑FS͒ of the system ͑here the h 2 -A 0 complex͒ to a certain equilibrium temperature T FS , we deduced the ii 's from the partial trace of the FS density matrix

FS = exp͑-H ˆFS / k B T FS ͒ / Tr͓exp͑-H ˆFS / k B T FS ͔͒,
where k B is the Boltzmann factor. Eventually, to compare with the experiments we applied to the calculated transitions a Lorentzian broadening of FWHM= 25 eV.

The above model enabled us to reproduce quite well both magneto-PL images performed in Faraday and Voigt configurations by using the same set of parameters ͑see Table I͒. The electron and hole Landé factors were determined in order to reproduce the linear slopes due to the Zeeman effect. We found very similar values for the three quantum dots investigated here. Remarkably, thanks to the strong exchange field experienced by h 2 , the transverse electron Landé factor g e,Ќ could be determined unambiguously from the common splittings ͑ϳ125 eV at 6 T͒ of the transitions measured in circular polarization, see Fig. 2. The parameters describing the local anisotropy ͓Eq. ͑2͔͒, the exchange strength ͓Eq. ͑4͔͒, and the A 0 position anisotropy ͓Eq. ͑5͔͒ were manually adjusted to reproduce the most remarkable features of the experimental images: e.g., the zero-field anticrossing ␦, the FM-AFM exchange ⌬, the anticrossing ⌬ a or the + / -Zeeman splitting in Voigt configuration. In contrast to our previous assumption in Ref. 5 and to the geometrical effects discussed in the case of II-VI QDs, 4 we did not include any heavy-hole light-hole mixing due to QD in-plane asymmetry to reproduce the anticrossing ⌬ a . Actually, the latter can be fully ascribed to the A 0 position anisotropy, since ⌬ a ϰ Mn-h 2 sin 2 a . The heavy-light hole splitting ⌬ HL was assumed to amount to a few 10 meV, so that the h 2 hole has a dominant heavy-hole character in agreement with the PL polarization in Fig. 1. [START_REF]This parameter cannot be determined with precision because it has basically no[END_REF] Besides, the strong circular polarization still measured in Voigt ͓up to 90%, see Fig. 2͑b͔͒ indicates that the h 2 hole spin states ͉ ⇑ ͘ and ͉ ⇓ ͘ are essentially not mixed by the magnetic field, in contrast to undoped InAs QD's where an effective transverse g-factor g h Ќ ϳ 0.3 is found. 13,[START_REF] Bayer | [END_REF] We ascribe this effect to the strong FM-AFM exchange ⌬ which inhibits this coupling for magnetic fields as long as ͉B x ͉ Ͻ⌬/ ͑g h Ќ B ͒ϳ50 T. We still have to explain the splitting between the + and -components, namely, between the ͉⇑ , Ϯ 1͘ and ͉⇓ , Ϯ 1͘ states. This effect which is well reproduced by our model ͓see Fig. 2͑c͔͒ results from the local anisotropy of A 0 which yields a finite in-plane spin projection of the ͉Ϯ1͘ states. The latter experience therefore a Zeeman effect in a transverse magnetic field dragging along the almost pure hole spin states ⇑ and ⇓. The resulting splitting ⌬ ⇑,⇓ of the FM and AFM h 2 -A 0 levels calculated within second-order perturbation theory reads 23

⌬ ⇑,⇓ = 7g Mn -3g h 1 5 cos s sin 2 s ͑⑀ Ќ + ⑀ ʈ ͒ 6 h 1 -h 2 -14 Mn-h 2 B B x . ͑6͒
As logically expected, the splitting ⌬ ⇑,⇓ vanishes for a magnetic field perpendicular to zЈ ͑ s = Ϯ / 2͒ or for s =0 or / 2 when cylindrical symmetry is restored. Experimentally, we indeed observed a large variety of magneto-PL images in Voigt configuration when measuring different Mndoped InAs QDs. QD2 shown in Fig. 3 is very similar to QD1 in Faraday configuration but exhibits a very distinctive behavior in Voigt. The Voigt magneto-PL image is almost symmetrical between + and -polarizations, which means that individual lines are now weakly circularly polarized. Such a situation is expected when the main strain axis zЈ is almost orthogonal to the field direction. We could indeed reproduce QD2 magneto-PL images by taking s = -93°, whereas other fitting parameters were found close to those of QD1. Note, however, that the anisotropic exchange parameter is almost twice that for QD1. This was found necessary to reproduce a third zero-field doublet ͓denoted OЈ in Fig. 3͑a͔͒ with the same ␦ splitting as the FM and AFM doublets. This doublet corresponds to the transition from the two e-A 0 lower states ͓split by ␦ in zero field, see Fig. 1͑c͔͒ to the J z =0 h 2 -A 0 level. This normally forbidden transition becomes permitted here thanks to the strong anisotropic exchange ϰ.

Remarkably, the relative position of these three doublets reflects thus directly the level fan chart of h 2 -A 0 in zero field. Figure 4 illustrates another interesting case ͑QD3͒, where the strain anisotropy experienced by h 1 is no longer dominated by a strong uniaxial strain ⑀ ʈ along zЈ. As shown in Fig. 4͑a͒, the zero-field spectrum of QD3 strongly deviates from our usual observation of Mn-doped QDs. Up to nine lines are visible, but no doublet structures can be clearly perceived. By performing the autocorrelation of these normalized peaks, we found that these lines perfectly reflect all 3 ϫ 3 possible transitions from e-A 0 to h 2 -A 0 as indicated by i᭤ j labels ͑i , j =1,2,3͒ in the figure. This is evidenced by the horizontal bars in Fig. 4͑a͒ which show that all two-level splittings ͑in the initial or final shells͒ appear three times in the measured spectrum. The images in Voigt and Faraday showing both many anticrossings and nonlinear field dependencies confirm that these lines originate from the same Mndoped QD, even though the crosslike pattern usually observed is now hardly perceptible. Quite remarkably, our model enables us to reproduce still fairly well the experimental magneto-PL images. We essentially had to reduce the strength of ⑀ ʈ ͑see Table I͒ while keeping the other parameters close to those of QD1. This indeed leads to eigenstates with A 0 angular momentum very different in the initial ͑e-A 0 ͒ and final ͑h 2 -A 0 ͒ shells, so that all transitions become partially allowed. In Voigt configuration, we still observe, such as for QD1, a marked field-induced splitting due to the transverse Zeeman effect of the A 0 complex. However, the circular polarization of the transitions is now much weaker ͑Ͻ ϳ 30%͒ than for QD1. This is due to the mixing of the h 2 spin states by the anisotropic exchange H ˆa, which is appreciably enhanced with respect to QD1 because of the smaller strain-induced splittings of A 0 levels. Our experimental observations and numerical simulations indicate that so far we have likely studied Mn-doped InAs QDs with strongly uncentered Mn atom. This sounds compatible with the fundamental issue raised in Ref. 24 where a singlet configuration in a common S-like orbital is predicted for the h 1 -h 2 ground state when the Mn atom lies exactly at the QD center. Our model describes instead two holes occupying distinct orbitals at different positions a finite overlap between them giving rise to the AFM coupling as for electrons in the H 2 molecule. [START_REF] Fazekas | Lecture Notes on Electron Correlation and Magnetism[END_REF] In conclusion, the -PL investigation in both a longitudinal and transverse magnetic field of individual singly Mndoped InAs QDs reveals remarkable insights into the spin interactions between carriers and a Mn impurity in a III-V matrix. The explicit anisotropic part to the p-d exchange 17 due to the Mn position with respect to the QD center explains better than the QD geometrical anisotropy 4 certain forbidden transitions and anticrossings observed in Faraday configuration. More spectacular is still the conservation of circularly polarized selection rules along with Zeeman splitting in a transverse magnetic field. This nonintuitive result is remarkably well interpreted by considering pure heavy-hole states in the quantum dot and local potential anisotropy ex- (a)

E-E 0 (meV) perienced by the acceptor level bound to the Mn impurity. The latter plays a crucial role to explain the dependence on the in-plane ͑azimuthal͒ magnetic field angle, as well as the effective optical selection rules. Our results which validate the picture of a Mn impurity keeping a tightly bound hole in spite of the QD strain and composition, 24 opens the way toward resonant experiments similar to those recently achieved in semiconductor QD molecule 25 in order to optically prepare and read out a single spin.

-1 2 0 1 -2 σ - σ + B x = 8T E-E 0 (meV) -1 2 0 1 -2 AFM FM O δ δ O' X + E-E 0 (
We acknowledge fruitful discussions with A. K. Bhattachargee and S. Chutia. This work was partly supported by the Région Ile-de-France and ANR contracts BOITQUANT and MOMES.

  FIG.1. ͑Color online͒ QD1 ͑a͒ schematics of an InAs QD doped by a single Mn atom forming a neutral acceptor A 0 . ͑b͒ PL spectrum of a charged exciton X + in such a QD at zero magnetic field. ͑c͒ Diagram of energy levels involved in the X + -A 0 → h 2 -A 0 transition. Solid ͑dashed͒ lines correspond to the levels with ↓͑ ↑ ͒e spin and ⇓͑ ⇑ ͒h 2 pseudospin connected via a + ͑ -͒ photon. ͑d͒ Contour plot of QD1 PL around E 0 = 1.355 eV as a function of longitudinal magnetic field measured in -and + circular polarizations ͑left͒ and theoretical simulation ͑right͒. The diamagnetic shift is subtracted so that the linear dependence on B z due to the Zeeman effect produces straight lines.

FIG. 2 .

 2 FIG.2. ͑Color online͒ QD1 ͑a͒ PL Contour plot of the same X + of Fig.1as a function of transverse magnetic field B x positive ͑right͒ or negative ͑left͒. In both cases, the PL has been measured in -and + circular polarizations as indicated. The diamagnetic shift ͑half of that in Faraday configuration͒ has been subtracted. ͑b͒ PL spectra at B x = -5 T in both + and -polarizations. ͑c͒ Theoretical simulation of the PL contour plot ͑see text͒ with the same parameters as in Fig.1.

2 FIG. 3 .

 23 FIG. 3. ͑Color online͒ QD2. ͑a͒ PL spectra of an X + trion at B = 0 T and B x = 8 T centered at E 0 = 1.355 eV. ͑b͒ PL Contour plot as a function of transverse ͑left͒ and longitudinal ͑right͒ magnetic fields in -and + circular polarizations as indicated. The diamagnetic shift is subtracted. Additional spectral features likely due to different charge states of QD2 are also visible ͑c͒ Theoretical simulation of the PL contour plot.

FIG. 4 .

 4 FIG. 4. ͑Color online͒ QD3 ͑a͒ PL spectra of an X + trion at B = 0 T and B x = 8 T centered at E 0 = 1.3525 eV. Inset at zero field shows the 3 ϫ 3 level fan chart responsible for the observed line splittings. ͑b͒ PL Contour plot as a function of transverse ͑left͒ and longitudinal ͑right͒ magnetic fields in -and + circular polarizations as indicated. The diamagnetic shift is subtracted. ͑c͒ Theoretical simulation of the PL contour plot.

TABLE I .

 I Parameters used for theoretical simulations. Strains ͑⑀ ʈ , ⑀ Ќ ͒ and exchange energies ͑ Mn-h 1 , Mn-h 2 , h 1 -h 2 ͒ are given in meV.

		g Mn	g h 1	g h 2	g e,z	g e,Ќ	T FS ͑K͒
		2	0.8	0.85	-0.6	-0.35	15
		⑀ ʈ	⑀ Ќ	s ͑deg͒ s ͑deg͒ s ͑deg͒
	QD1	6.95	2.27	36°-120°49°Q
	D2	6.75	2.55	35°-93°55°Q
	D3	3.6	2	50°55°40°
		Mn-h 1	Mn-h 2	h 1 -h 2		a ͑deg͒ a ͑deg͒
	QD1	4.2	0.6	1.2	-0.1	81°-120°Q
	D2	4.5	0.63	1.35	-0.19	85°-123°Q
	D3	5	0.85	1.8	-0.12	102°55°K