Emilie Aponte

Marie Lafitte

Audrey Sirvent

Valerie Simon

Maud Barbery

Elise Fourgous

Mariano Maffei

Florence Armand

Romain Hamelin

Julie Pannequin

Valérie Simon

Philippe Fort

Miquel Pons
email: mpons@ub.edu

Serge Roche
email: serge.roche@crbm.cnrs.fr

Regulation of Src tumor activity by its N-terminal intrinsically disordered region

Keywords: cell signaling, intrinsically disordered region, protein kinase, cell transformation

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INTRODUCTION

Src, originally identified as an oncogene, is a membrane-anchored tyrosine kinase, which mediates signaling induced by a wide range of cell surface receptors, leading to cell growth and adhesion [START_REF] Yeatman | A renaissance for SRC[END_REF]. Src deregulation is associated with cancer development, although the underlying mechanisms are not fully understood [START_REF] Summy | Src family kinases in tumor progression and metastasis[END_REF][START_REF] Sirvent | Src Family Tyrosine Kinases in Intestinal Homeostasis, Regeneration and Tumorigenesis[END_REF]. Src shares with the other Src Family Kinases (SFKs) a common modular structure formed by the membrane-anchoring SH4 region followed by an intrinsically disordered region (IDR) named unique domain (UD), and the SH3, SH2, and kinase domains [START_REF] Summy | Src family kinases in tumor progression and metastasis[END_REF]. Our knowledge of Src regulation relies on crystallographic data that revealed SH2 and SH3-dependent intramolecular interactions that control Src catalytic activity [START_REF] Boggon | Structure and regulation of Src family kinases[END_REF]. However, the functions of the SH4-UD module have often been disregarded because of X-ray invisibility. UD is the mostly divergent part of SFK proteins, which supported the idea of a unique function among SFKs [START_REF] Arbesú | Intramolecular Fuzzy Interactions Involving Intrinsically Disordered Domains[END_REF][START_REF] Amata | Phosphorylation of unique domains of Src family kinases[END_REF]. However, early studies reported that the whole UD deletion does not affect Src oncogenic activity [START_REF] Cross | A short sequence in the p60src N terminus is required for p60src myristylation and membrane association and for cell transformation[END_REF], which suggests that this region may not play an important role in Src signaling. While considerable insight into Src regulation has been provided since the discovery of the Src oncogene, the functional role of its unstructured region remains unclear.

IDRs are highly prevalent in proteins regulating essential cell processes, such as transcription or signaling that are implicated in human diseases [START_REF] Wright | Intrinsically Disordered Proteins in Cellular Signaling and Regulation[END_REF]. The integration of multiple weak interactions is crucial for the increasingly recognized role of IDRs in the formation of membrane-less organelles, through liquid-liquid phase separation [START_REF] Wright | Intrinsically Disordered Proteins in Cellular Signaling and Regulation[END_REF]. Multiple, rapidly exchanging weak contacts are also at the basis of the formation of the so called "fuzzy complexes" by IDRs, in which the IDR remains disordered, but the complex is stabilized by multiple transient contacts [START_REF] Arbesú | Intramolecular Fuzzy Interactions Involving Intrinsically Disordered Domains[END_REF][START_REF] Fuxreiter | Fuzziness: linking regulation to protein dynamics[END_REF]. Intramolecular fuzzy complexes involve a similar fuzzy interaction between an IDR and a globular domain in the same protein to form a relatively compact structure [START_REF] Arbesú | Intramolecular Fuzzy Interactions Involving Intrinsically Disordered Domains[END_REF][START_REF] Fuxreiter | Fuzziness: linking regulation to protein dynamics[END_REF]. Intramolecular fuzzy complexes may regulate the communication between the disordered and globular regions of a signaling protein and sense the environment (e.g. membrane lipids) that will influence the activity of folded domains, such as kinase activity or the binding capacity of SH2 and SH3 domains [START_REF] Arbesú | Intramolecular Fuzzy Interactions Involving Intrinsically Disordered Domains[END_REF][START_REF] Fuxreiter | Fuzziness: linking regulation to protein dynamics[END_REF]. Thus, IDRs provide a unique mechanism of protein regulation by the local environment. In line with this, recent molecular studies uncovered key features of such a new Src-UD regulatory mechanism [START_REF] Pérez | Lipid binding by the Unique and SH3 domains of c-Src suggests a new regulatory mechanism[END_REF][START_REF] Maffei | The SH3 Domain Acts as a Scaffold for the N-Terminal Intrinsically Disordered Regions of c-Src[END_REF][START_REF] Spassov | A Dimerization Function in the Intrinsically Disordered N-Terminal Region of Src[END_REF][START_REF] Roux | A Myristoyl-Binding Site in the SH3 Domain Modulates c-Src Membrane Anchoring[END_REF]. Specifically, Src-UD forms an intramolecular fuzzy complex, where its conformational freedom is restricted by multiple contacts with the globular SH3 domain [START_REF] Maffei | The SH3 Domain Acts as a Scaffold for the N-Terminal Intrinsically Disordered Regions of c-Src[END_REF]. Although their primary sequences are divergent, UD fuzzy complexes are present in other SFKs, suggesting that this IDR defines a central regulatory mechanism in SFKs [START_REF] Arbesú | The Unique Domain Forms a Fuzzy Intramolecular Complex in Src Family Kinases[END_REF][START_REF] Teixeira | The Two Isoforms of Lyn Display Different Intramolecular Fuzzy Complexes with the SH3 Domain[END_REF]. Our molecular studies on Src identified a conserved region that contributes to the interaction of the UD with the RT and nSrc loops of the SH3 domain [START_REF] Pérez | Lipid binding by the Unique and SH3 domains of c-Src suggests a new regulatory mechanism[END_REF][START_REF] Maffei | The SH3 Domain Acts as a Scaffold for the N-Terminal Intrinsically Disordered Regions of c-Src[END_REF]. This region displays affinity with phospholipids and was named Unique Lipid Binding Region (ULBR) [START_REF] Pérez | Lipid binding by the Unique and SH3 domains of c-Src suggests a new regulatory mechanism[END_REF]. Further in vitro studies showed that ULBR participates in the interaction between the N-terminal myristoyl group and the SH3 domain and contributes to the modulation of membrane anchoring [START_REF] Roux | A Myristoyl-Binding Site in the SH3 Domain Modulates c-Src Membrane Anchoring[END_REF], suggesting that it could also be involved in substrate selection and signaling. Here we start addressing the oncogenic relevance of the Src UD by focusing on ULBR.

RESULTS

Evolutionary conservation of ULBR in SFK-UD

Although Src-UD is thought to be evolutionary conserved, we noticed a strong sequence divergence between mammalian and invertebrates SFKs. To clarify this point, we readdressed SFK sequence conservation by comparing the selective constraints exerted along SFK sequences. We computed ω, the ratio of non-synonymous (dN) to silent (dS) mutations from a multiple alignment of Src, Yes and Fyn coding sequences of seven primate species, covering 74 million years of evolution. In such framework, neutral selection is associated with an ω-value close to 1, while values <1 or >1 indicate purifying and diversifying selection, respectively. As shown in Figure 1a, all domains produced ω-values <1. However, UD ω-values indicated variable selection levels depending on SFK members. Notably, the heaviest selection was exerted on Fyn UD (ω = 0.0001), while the selection exerted on Src UD was moderate (ω = 0.126) and even lighter for Yes (ω = 0.363). This indicates that UD sequences are not under neutral selection and supports the notion that these sequences play essential roles in SFKs functions. Consistent with this idea, a comparative sequence analysis of SFK-UD IDR sequences from 10 vertebrate species revealed that residues 64-Phe-Gly-Gly-66 (human Src numbering) are highly conserved across species and also across the three functional related Src, Fyn and Yes. They are part of the conserved Src ULBR (residues 60-67) highlighting an important function for this small region (Fig 1b).

ULBR inactivation affects Src oncogenic activity

We next explored the functional role of Src-ULBR on cell transformation. For this, ULBR was inactivated by replacement of residues 63-65 (Leu-Phe-Gly) by three alanines (named Src3A), known to affect Src-UD molecular properties (i.e. binding to phospholipids and SH3, Src membrane anchoring) [START_REF] Pérez | Lipid binding by the Unique and SH3 domains of c-Src suggests a new regulatory mechanism[END_REF][START_REF] Maffei | The SH3 Domain Acts as a Scaffold for the N-Terminal Intrinsically Disordered Regions of c-Src[END_REF][START_REF] Roux | A Myristoyl-Binding Site in the SH3 Domain Modulates c-Src Membrane Anchoring[END_REF]. Since these ULBR properties can be regulated by phosphorylation of surrounding Ser69 and Ser75 [START_REF] Pérez | Lipid binding by the Unique and SH3 domains of c-Src suggests a new regulatory mechanism[END_REF][START_REF] Amata | Multi-phosphorylation of the Intrinsically Disordered Unique Domain of c-Src Studied by In-Cell and Real-Time NMR Spectroscopy[END_REF] to an autoregulatory mechanism mediated by the substrate and E3 ligase Cbl [START_REF] Yokouchi | Srccatalyzed phosphorylation of c-Cbl leads to the interdependent ubiquitination of both proteins[END_REF]. Despite this and unlike wild-type Src, oncogenic SrcY530F expression induced anchorage-independent growth as measured by the number of colonies in soft agar. Interestingly, ULBR inactive mutations strongly reduced this transforming effect, suggesting that an intact ULBR is required for oncogenic Src activity (Fig 2c). The invasive properties of NIH3T3 cells were also reduced, as assayed in Boyden chambers coated with matrigel (Fig 2d). However, other transformation related properties induced by SrcY530F, such as dissolution of F-actin bundles causing actin cytoskeletal rearrangement (Fig S1c) [START_REF] Manes | Slap negatively regulates Src mitogenic function but does not revert Src-induced cell morphology changes[END_REF], were retained in ULBR mutants. This indicates that ULBR selectively regulates some of Src transforming activities.

We next addressed the importance of ULBR in Src transforming activity in human cancer. In spite of the fact that SRC somatic mutations are rarely detected in human malignancies, aberrant Src activity, resulting from pathological deregulation, is a bad prognosis maker in epithelial tumors and has important roles during tumor development/progression [START_REF] Summy | Src family kinases in tumor progression and metastasis[END_REF][START_REF] Sirvent | Src Family Tyrosine Kinases in Intestinal Homeostasis, Regeneration and Tumorigenesis[END_REF]. In colon cancer cells, regulation of Src signaling is highly perturbed due to defects in the regulation of its catalytic activity due to CSK inactivation [START_REF] Sirvent | Src family tyrosine kinases-driven colon cancer cell invasion is induced by Csk membrane delocalization[END_REF], which mediates Src-Tyr530 phosphorylation [START_REF] Boggon | Structure and regulation of Src family kinases[END_REF]. Defects in the regulation of Src substrates degradation, due to inactivation of the inhibitory signaling protein SLAP in these tumor cells also participates in Src oncogenic signaling [START_REF] Naudin | SLAP displays tumour suppressor functions in colorectal cancer via destabilization of the SRC substrate EPHA2[END_REF].

As a result, ectopic expression of wild type Src in SW620 colon cancer cells, which originate from a lymph node metastasis and express low levels of endogenous Src, strongly increases their growth and invasive and abilities [START_REF] Leroy | Quantitative phosphoproteomics reveals a cluster of tyrosine kinases that mediates SRC invasive activity in advanced colon carcinoma cells[END_REF][START_REF] Sirvent | Analysis of SRC oncogenic signaling in colorectal cancer by stable isotope labeling with heavy amino acids in mouse xenografts[END_REF]

ULBR inactivation affects Src oncogenic signaling

We next investigated the mechanisms involved in Src-ULBR function. Oncogenic SrcY530F induced a large increase of protein tyrosine phosphorylation in NIH3T3 cells, which was substantially reduced upon ULBR inactivation (Fig S1a). ULBR mutants also showed a reduction in SFKs activity, as measured by the level of their conserved tyrosine phosphorylation localized in the activation loop (i.e. pTyr419 in Src) (Fig S1a ), with respect to the one observed in SrcY530F. This suggests that, in the context of oncogenic SrcY530F in NIH3T3 cells, ULBR regulates both substrate phosphorylation and SFK activity. Similar results were observed in HEK293T cells, in which transiently expressed Src resulted in high level of Src activity due to high ectopic kinase expression and low endogenous CSK levels [START_REF] Lecointre | Dimerization of the Pragmin Pseudo-Kinase Regulates Protein Tyrosine Phosphorylation[END_REF]. In this context, Src We next searched for the molecular mechanisms implicated in ULBR-dependent Src substrates phosphorylation. The Src-UD has been suggested to participate in protein dimerization, enabling kinase activation [START_REF] Spassov | A Dimerization Function in the Intrinsically Disordered N-Terminal Region of Src[END_REF][START_REF] Roux | Kinetics characterization of c-Src binding to lipid membranes: Switching from labile to persistent binding[END_REF]. We tested the possible ULBR contribution to this molecular process. For this, Src constructs tagged with a hemagglutinin (HA) or a FLAG sequence at the C terminus were co-transfected in HEK293T cells, and Src self-association was assessed by coimmunoprecipitation. Src dimerization was not detected, unless when using stringent lysis conditions (ie RIPA buffer) [START_REF] Sirvent | Src family tyrosine kinases-driven colon cancer cell invasion is induced by Csk membrane delocalization[END_REF][START_REF] Veracini | Two distinct pools of Src family tyrosine kinases regulate PDGF-induced DNA synthesis and actin dorsal ruffles[END_REF]. This suggests that dimerization may occur in cholesterolenriched membrane domains, consistent with lipid binding-induced dimerization/oligomerization observed in SH4 myristoylated Src derivatives [START_REF] Roux | Kinetics characterization of c-Src binding to lipid membranes: Switching from labile to persistent binding[END_REF][START_REF] Roux | Single molecule fluorescence reveals dimerization of myristoylated Src N-terminal region on supported lipid bilayers[END_REF]. Using these conditions, we did not detect any effect of ULBR inactivation on Src self-association, indicating that this conserved region may not be involved in kinase dimerization (Fig 4c). We next evaluated the role of ULBR on Src subcellular localization. For this, Src constructs (wildtype or ULBR mutants) were generated with either a GFP or mCherry tag at the C terminus together with a spacer (GluX3) for molecular constraint limitation between GFP (or mCherry)

and Src. Wild-type Src fused to mCherry was co-expressed with Src-ULBR mutants fused to GFP in HEK293T cells and their co-localization was analyzed by direct fluorescent microscopy. We found an almost strict co-localization between wild-type Src and ULBR-Src mutants, i.e. at perinuclear membranes, endocytic vesicles and membrane cell periphery, suggesting that ULBR inactivation had no effect on Src sub-cellular localization (Fig 4d). Since the cellular Src kinase activity was affected by ULBR inactivation, we analyzed the impact of ULBR inactivation on Src kinase activity in vitro using enolase, or EPHA2 as substrates [START_REF] Leroy | Quantitative phosphoproteomics reveals a cluster of tyrosine kinases that mediates SRC invasive activity in advanced colon carcinoma cells[END_REF].

No difference between purified Src-GFP and Src3A

Phospho-proteomic analyses of ULBR-Src signaling in tumor cells

We next characterized ULBR-dependent Src phospho-signaling by proteomic methods. SW620 cancer cells were retrovirally transduced with mock (control), wild-type Src and Src3A constructs. A global tyrosine phospho-proteomic analysis was first performed by phosphotyrosine peptide immune-purification from trypsin-digested cell lysates followed by label-free mass spectrometry-based quantification [START_REF] Jeitany | Inhibition of DDR1-BCR signalling by nilotinib as a new therapeutic strategy for metastatic colorectal cancer[END_REF]. From this analysis, we detected 279 phosphopeptides in control cells with a log2 fold change (FC) ≥1 upon Src (or Src3A) expression (table S1). These Src substrates were essentially composed of signaling proteins, regulators of cell adhesion, trafficking, mRNA maturation, protein synthesis and cell metabolism (table S1), consistent with previous studies [START_REF] Leroy | Quantitative phosphoproteomics reveals a cluster of tyrosine kinases that mediates SRC invasive activity in advanced colon carcinoma cells[END_REF][START_REF] Sirvent | Analysis of SRC oncogenic signaling in colorectal cancer by stable isotope labeling with heavy amino acids in mouse xenografts[END_REF]. We next examined how these peptides distribute into wild type Src and Src3A expressing samples, using a log2 FC ≥ 2 threshold. Src-and Src3Ainduced protein tyrosine phosphorylation showed very similar profiles (Fig 5a experiments centered in this region did not reveal any clear oncogenic activity [START_REF] Cross | A short sequence in the p60src N terminus is required for p60src myristylation and membrane association and for cell transformation[END_REF], possibly because Src-IDR may contain opposite regulatory sequences that would not be detected with this approach. Consistent with this idea, our mutagenesis analysis guided from our NMR data led to specifically inactivate the ULBR, a small region conserved in Src-UD, which revealed its essential role in Src tumor activity. This sequence may however not regulate all Src transforming functions, as suggested from Src-induced morphological changes in mouse fibroblasts. Actually, Src-ULBR may act as a fine-tuning mechanism, which may be exacerbated upon Src overactivation to promote cancer development. This fine-tuning mechanism is supported by previous works on the Src capacity to induce Xenopus oocytes maturation [START_REF] Pérez | Lipid binding by the Unique and SH3 domains of c-Src suggests a new regulatory mechanism[END_REF], Src regulation of retinal ganglion cell survival or postmitotic neuron function by Ser75 phosphorylation [START_REF] Kashiwagi | A Ser75-to-Asp phospho-mimicking mutation in Src accelerates ageing-related loss of retinal ganglion cells in mice[END_REF][START_REF] Kato | Nonphosphorylatable Src Ser75 Mutation Increases Ethanol Preference and Consumption in Mice[END_REF] and is consistent with a recent Src optogenetic study [START_REF] Kerjouan | Control of SRC molecular dynamics encodes distinct cytoskeletal responses by specifying signaling pathway usage[END_REF].

Our study also brings valuable molecular insight into the mechanism of Src regulation by its unstructured region. Previous studies reported that Lck-IDR can mediate protein interaction by adopting an organized structure [START_REF] Kim | A zinc clasp structure tethers Lck to T cell coreceptors CD4 and CD8[END_REF]. Whether IDR of other SFKs display similar molecular property is not known, although Src-UD was involved in protein interactions [START_REF] Yu | NMDA channel regulation by channelassociated protein tyrosine kinase Src[END_REF].

IDRs adopt multiple conformations that are sensitive to the environment and, through multiple weak interactions in a fuzzy complex, may direct the activity of folded domains of signaling proteins towards different pathways [START_REF] Wright | Intrinsically Disordered Proteins in Cellular Signaling and Regulation[END_REF]. Our results support such a Src-IDR regulatory mechanism at the plasma membrane since ULBR regulates Src membrane anchoring and phosphorylation of essential membrane localized substrates of tumor signaling, notably RTKs and SFKs. An unsuspected finding from this work is the Src capacity to activate additional SFKs, such as Fyn, to promote oncogenic signaling. This result suggests the existence of a SFK network involved in cancer development and uncovers a novel layer of Src signaling complexity, which deserves further investigation.

By focusing on ULBR, this work started addressing the biological role of Src-IDR but existing molecular studies uncovered additional regions involved in the fuzzy complex made by SFKs-IDR, which would also contribute to Src regulation. Unravelling the biological role of these regions may bring a more complete view on the complexity of Src regulation by its IDR. Finally, Src has been identified as an attractive target in oncology but Src inhibitors developed for the clinic gave disappointing results in colon cancer, probably because of high toxicity and inefficient Src signaling inhibition (3). Interestingly, structural analyses of noncatalytic domains of TKs have revealed unique modes of kinase regulation [START_REF] Nagar | Structural basis for the autoinhibition of c-Abl tyrosine kinase[END_REF], which resulted 13 in the development of allosteric inhibitors with improved anti-tumor activities, as reported for asciminib in chronic myeloid leukemia [START_REF] Eide | Combining the Allosteric Inhibitor Asciminib with Ponatinib Suppresses Emergence of and Restores Efficacy against Highly Resistant BCR-ABL1 Mutants[END_REF]. We thus propose that targeting the IDR fuzzy complex with small molecules would circumvent some of these issues and therefore may define an attractive strategy to block Src tumor activity in human cancer.

MATERIAL AND METHODS

Antibodies: anti-p42/44 MAPKs, anti-p42/44 MAPKs pT202/Y204, anti-p38 MAPK, anti-p38 MAPK pT180/Y182, anti-AKT, anti-AKT pS473, anti-EPHA2 pY594, anti-Src pY418, anti-Stat3 pY705, anti-Cyclin D3 (Cell Signaling Technology), anti-EPHA2, anti-Stat3, anti-Myc (Santa Cruz Biotechnology), anti-Src specific (2.17 Phylogenetic analyses: Nucleic and protein SFK sequences were retrieved from NCBI annotated nr database (http://www.ncbi.nlm.nih.gov). Accessions are listed Table S1. Protein sequences were aligned using MAAFT v7.450 [START_REF] Katoh | MAFFT multiple sequence alignment software version 7: improvements in performance and usability[END_REF]. Nucleic sequence alignments were based on protein alignments. Phylogenetic trees were estimated by PhyML (40), using the General Time Reversible (GTR) model with invariant and gamma decorations. Nonsynonymous versus synonymous substitution ratios (ω= dN/dS) were calculated using PAML 4.4 [START_REF] Yang | PAML 4: phylogenetic analysis by maximum likelihood[END_REF]. We compared the ''one-ratio'' model (a single ω ratio for the entire tree) and the ''two-ratio'' model (distinct ω values for each of the Src, Fyn and Yes branches) by using the likelihood ratio test.

Plasmids: pMX-pS-CESAR retroviral vector expressing human Src was described in [START_REF] Naudin | SLAP displays tumour suppressor functions in colorectal cancer via destabilization of the SRC substrate EPHA2[END_REF].

pMX-Src L63A/F54A/G65A (Src 3A) was described in [START_REF] Maffei | The SH3 Domain Acts as a Scaffold for the N-Terminal Intrinsically Disordered Regions of c-Src[END_REF]. The other plasmids were obtained Biochemistry. Immunoprecipitation and immunoblotting were performed as described in [START_REF] Naudin | SLAP displays tumour suppressor functions in colorectal cancer via destabilization of the SRC substrate EPHA2[END_REF].

Kinase assays were performed as described in [START_REF] Naudin | SLAP displays tumour suppressor functions in colorectal cancer via destabilization of the SRC substrate EPHA2[END_REF] RNA extraction and RT-quantitative PCR. mRNA was extracted from cell lines and tissue samples using the RNeasy plus mini kit (Qiagen) according to the manufacturer's instructions.

RNA (1µg) was reverse transcribed with the SuperScript VILO cDNA Synthesis Kit (Invitrogen). Quantitative PCR (qPCR) was performed with the SyBR Green Master Mix in a LightCycler 480 (Roche). Expression levels were normalized with the Tubulin human housekeeping gene. Primers used for qPCR: Tubulin, Forward-5'CCGGACAGTGTGGCAA CCAGATCGG3', Reverse-5'TGGCCAAAAGGACCTGAGCG AACGG3'; Fyn, Forward-5'TGACCTCCATCCCCAACTA3', Reverse-5'TTCCCACCAATCTCCTTCC3'; EPHA2, Forward-5'GGGACCTGATGCAGAACATC3', Reverse-5'AGTTGGTGCGGAGCCAGT3'.

Cell imaging: HEK293T cells plated on glass coverslips coated with fibronectin were transfected with Src-GFP and Src-mCherry constructs for 24 hours and subcellular Src distribution was analyzed after cell fixation (4% paraformaldehyde) by direct fluorescence using confocal microscopy. Src-transformed NIH3T3 cells were plated on glass coverslip coated with fibronectin for 24 hours and actin was visualized with Texas red-conjugated phalloidin (1:200 dilution) after cell fixation (4% paraformaldehyde) and permeabilization (0.05% TRITON for 10 min at room temperature).

Soft agar colony formation and cell invasion assay. Colonies formation: 1 000 cells per well were seeded in 12-well plates in 1ml DMEM containing 10% FCS and 0.33% agar on a layer of 1ml of the same medium containing 0.7% agar. After 18-21 days, colonies with > 50 cells were scored as positive. Cell invasion assay was performed as described in (30) using Fluoroblok invasion chambers (BD Bioscience) in the presence of 100 µl of 1-1.2 mg/ml Matrigel (BD Bioscience).

Phospho-proteomic analyses. Quantitative phosphoproteomics was performed as in [START_REF] Jeitany | Inhibition of DDR1-BCR signalling by nilotinib as a new therapeutic strategy for metastatic colorectal cancer[END_REF].

Briefly, SW620 cells were lysed in urea buffer (8M urea in 200mM ammonium bicarbonate pH 7.5). Phosphopeptides were purified after tryptic digestion of 20 mg (for cells) or of 35 mg (for mouse tumors) total proteins using the PTMScan ® Phospho-Tyrosine Rabbit mAb (P-Tyr-1000) Kit (Cell Signaling Technology), according to manufacturer's protocol. An additional enrichment step using the IMAC-Select Affinity Gel (Sigma Aldrich) was performed to increase the phosphopeptide enrichment. Purified phosphopeptides were resuspended in 10% formic acid and two technical replicates for each sample were analyzed. Phospho-kinase arrays:

proteome profiler human phospho-kinase array and human phospho-RTK array kits including 49 RTKs were purchased from R&D Systems. Indicated SW620 cells were lysed, and 300 μg of protein lysates were subjected to western blotting according to the manufacturer's protocol.

Signals on membranes were quantified using the Amersham Imager 600 (GE Healthcare) from 2 independent biological replicates.

In vivo experiments and Immunohistochemistry (IHC).

In vivo experiments were performed in compliance with the French guidelines for experimental animal studies (Direction des services vétérinaires, ministère de l'agriculture, agreement B 34-172-27). 2x10 6 SW620 cells (or derivatives) were subcutaneously injected in the flank of 5-week-old female athymic nude mice (Envigo). Tumor volumes were measured as the indicated intervals using calipers. After 24 days, tumors were excised, weighed and cryopreserved or processed for subsequent immunohistochemistry analysis as described in [START_REF] Sirvent | Analysis of SRC oncogenic signaling in colorectal cancer by stable isotope labeling with heavy amino acids in mouse xenografts[END_REF].

Statistical analysis. All analyses were performed using GraphPad Prism. Data are presented as the mean ± SEM. When distribution was normal (assessed with the Shapiro Wilk test), the twotailed t test was used for between-group comparisons. In the other cases, the Mann-Whitney

  , we also inactivated ULBR by replacement of Ser69 or Ser75 by the phospho-mimicking glutamic acid (ie SrcS69E and SrcS75E) (Fig 2a). These ULBR mutations were incorporated in the oncogenic SrcY530F mutant, in which the pTyr530-SH2 interaction is destabilized, thereby inducing an active and open Src conformation (4). Transforming activity was then assessed upon retroviral transduction in immortalized mouse embryonic fibroblasts NIH3T3 (Fig 2b & c). SrcY530F protein levels were reduced compared to regulated Src (Fig 2b), which was previously attributed

  (Fig 2e-g). Expression of ULBR mutated Src produced similar results as in fibroblasts, i.e. a strong diminution of both anchorageindependent cell growth and cell invasion (Fig 2f&g). Importantly, subcutaneous injection of these tumor cells in nude mice produced similar results. In this experimental in vivo cancer model, Src expression enhanced tumor development by 8-fold as compared to control cells, while this effect was reduced by 60% upon Src3A mutant expression (Fig 3a &b). Immunohistochemical analysis of tumor sections showed a significant reduction in colon cancer cell proliferation in Src3A samples (Fig 3c). In contrast to wild-type Src, Src3A significantly increased tumor cells apoptosis (Fig 3d). Src also induced a substantial increase in tumor angiogenesis, important for tumor progression. While the overall length of tumor vessels was not significantly modified by ULBR inactivation, we noted a significant diminution of nascent tumor vessels in Src3A expressing tumors, suggesting an implication of this region during tumor angiogenesis (Fig 3e).

  induced a large increase in protein tyrosine phosphorylation, which was dependent upon a functional ULBR (Fig4a). Src3A also showed a 20% reduction in SFK activity as compared to wild-type Src (Fig4a). We next searched for a similar mechanism operating in human cancer.Consistent with a robust transforming activity, retroviral transduction of wild-type Src in SW620 cells increased protein tyrosine phosphorylation. As in HEK293T cells, 3A ULBR mutation caused a reduction in substrate phosphorylation but a reduction in active Src level was not evident (Fig4b). The regulatory role of ULBR on Src signaling was next confirmed on MAPK activity, an important downstream effector of Src transforming activity in epithelial cells[START_REF] Sirvent | Oncogenic signaling by tyrosine kinases of the SRC family in advanced colorectal cancer[END_REF]. ULBR mutations reduced Src-induced p42/44 MAPKs activation both in HEK293T cells and SW620 tumor cells (Fig4a & b). A similar effect of ULBR inactivation was observed on oncogenic Src signaling in NIH3T3 cells. Src has been shown to induce fibroblasts cell transformation by a p38 MAPK and Stat3-dependent signaling mechanism[START_REF] Turkson | Requirement for Ras/Rac1-mediated p38 and c-Jun N-terminal kinase signaling in Stat3 transcriptional activity induced by the Src oncoprotein[END_REF][START_REF] Bowman | Stat3mediated Myc expression is required for Src transformation and PDGF-induced mitogenesis[END_REF].Accordingly, SrcY530F transforming activity was accompanied by an increase in pTyr507-Stat3 and p38 signaling, which was reduced upon ULBR inactivation (FigS1b).

EPHA2

  and Fyn as important mediators of Src-ULBR signaling in tumor cellsFinally, we aimed at validating these results functionally. ULBR inactivation reduced Srcinduced p42/44 MAPK and Akt signaling leading to SW620 cell invasion (Fig 2fand 4b) and we interrogate the role of two ULBR-dependent Src substrates on this signaling response, i.e. EPHA2 and Fyn. EPHA2 is aberrantly stabilized in colon cancer cells by a Src-dependent mechanism implicating inactivation of the SLAP-UBE4A ubiquitination complex[START_REF] Naudin | SLAP displays tumour suppressor functions in colorectal cancer via destabilization of the SRC substrate EPHA2[END_REF]. Moreover, EPHA2 phosphorylation on Tyr594 by Src amplifies Akt signaling, leading to colon cancer cell invasion (20). Consistent with our proteomic analyses, Src-ULBR regulated EPHA2-Tyr594 phosphorylation (Fig S3a). At the functional level, EPHA2 silencing inhibited Src signaling leading to Akt and p42/44 MAPK activation and cell invasion (Fig 5d, f and S4); however, it had no clear effect on Src3A signaling responses. EPHA2 is thus an important mediator of ULBR-Src signaling in SW620 cells. The fact that Src induces activation of other SFKs, e.g. Fyn, in colon cancer cells raises the idea that Src interacts with other SFKs to induce oncogenic signaling. Consistently, Src expression increased Fyn protein levels (Fig 5e), likely via a posttranscriptional mechanism because it had no impact on FYN transcript level (Fig S4b). Functionally, Fyn depletion specifically inhibited Src-induced p42/44 MAPK activation and cell invasion (Fig 5f and S4). However, no such inhibitory effect was observed on Src3A signaling placing Fyn as an additional effector of Src-ULBR signaling. The interaction between Src and Fyn signaling was next confirmed by showing a potentiating effect of Fyn on Srcinduced p42/44 MAPK activation in HEK293T cells (Fig S4d). However, this signaling response was reduced upon Src-ULBR mutation, supporting further a role for Src-UD in Src-Fyn signaling (Fig S4d). Overall, our results point to an important role of Src-ULBR in the regulation of membrane substrates phosphorylation, essential for Src tumor signaling. DISCUSSION Most eukaryotic proteins have intrinsically disordered regions (IDRs) that challenge the classical structure-function paradigm. Here, we uncover an important role of this molecular property made by Src-UD in cancer development. The biological roles of IDRs contained in SFK have remained unclear, despite strong insight into Src regulation. Previous mutagenesis

  ) antibody was a generous gift from Dr S Parsons (University of Virginia, VA, USA), anti-pTyr clone pY1000 Sepharose bead conjugated (PTM Scan, Cell Signaling Technology); anti-FLAG (Sigma Aldrich), and anti-GFP (Chromotek), anti-tubulin (gift from N. Morin, CRBM, Montpellier, France); anti-pTyr 4G10 (gift from P. Mangeat, CRBM, Montpellier, France); anti-cst1 (that recognizes Src, Fyn and Yes) was described in[START_REF] Roche | Requirement for Src family protein tyrosine kinases in G2 for fibroblast cell division[END_REF]. Anti-rabbit IgG-HRP and anti-mouse IgG-HRP (GE Healthcare). Anti-Mib1h (Dako)(KI67), anti-active-caspase3 (AP175; cell signaling), anti-CD31 (Ab28364, Abcam).

  by PCR using the QuickChange Site-Directed Mutagenesis Kit (Stratagene) using specific oligonucleotides as follows: Src S69E, Forward-5'CGGAGGCTTCAACGCCTCG GACACCGT3', Reverse-5'ACGGTGTCCGAGGCGTTGAAGCCTCCG3'; Src S75E, Forward-5'GACACCGTCACCGCCCCGCAGAGGG3', Reverse-5'CCCTCTGCGGGGCG GTGACGGTGTC3'; Src Y530F (Src YF), Forward-5'CGGGCTGGAACTGGGGCTCGGTG G3', Reverse-5'CCACCGAGCCCCAGTTCCAGCCCG3'. Each of the Y530F counterparts Src L63A/F54A/G65A/Y530F (SrcYF 3A), Src S69E/Y530F (SrcYF S69E); Src S75E/Y530F (SrcYF S75E) were obtained by adding the Y530F mutation using the following oligonucleotides Forward-5' TCTCGAGCTCAAGCTTAGTACCCTTCACCATGGGTAGC AACAA3', Reverse-5' GGCGACCGGTGGATCCGAGCCGGAGCCGAGGTTCTCCCCGG GCTGGTA3'. Src-GFP and Src-mCherry constructs were obtained by insertion of Src sequence (or Src mutants) in pEGFP-N1 and pmCherry N1 respectively including a GluX3 spacer. Src-Flag and Src-Myc constructs were obtained by inserting Src and Src3A sequences in pcDNA3 vectors. pSG5 Fyn construct was described in[START_REF] Manes | Slap negatively regulates Src mitogenic function but does not revert Src-induced cell morphology changes[END_REF].Cell cultures, retroviral infections and transfections. Cell lines (NIH3T3, HEK293T and SW620 cells) (ATCC, Rockville, MD) were cultured, transfected and infected as described in[START_REF] Naudin | SLAP displays tumour suppressor functions in colorectal cancer via destabilization of the SRC substrate EPHA2[END_REF]. Stable cell lines were obtained by fluorescence-activated cell sorting. For siRNA transfection, 2.10 5 cells were seeded in 6-well plates and transfected with 20 nmol of siRNA and 9 µl of Lipofectamine RNAi Max according to the manufacturer's protocol (ThermoFisher Scientific). A scramble siRNA (siMock) 5'TTCTCCGAACGTGTCACGTTT3' was used as a negative control (Eurofins). The following siRNAs were used for functional assays: siRNA

  using 200ng of purified EPHA2 recombinant protein (OriGen Technologies) or indicated concentration of purified Enolase (Sigma Aldrich), in the absence or presence of about 50ng of purified Src-GFP (or Src3A-GFP as indicated) in the presence of 0.1mM ATP Lithium Salt (Roche Diagnostics) in kinase buffer (20mM Hepes pH6.5, 10mM MnCl2, 1mM DTT) for indicated time at 30°C. Src-GFP purification was performed by anti-GFP immunoprecipitation from HEK293T cells transfected with Src-GFP (or Src3A-GFP) construct.

Figure 1 .

 1 FIGURES LEGENDS Figure 1. Evolutionary conservation of the UD in SFKs. a: Analysis of dN/dS ratios (ω) of the SH4, UD, SH3, SH2 and kinase domains in primate SFKs. Nucleic sequences coding the different domains were aligned based on translation and processed by codeml to estimated ωvalues. The phylogenetic tree on the left was generated by PhyML analysis of a multiple alignment of full-length SFK sequences. b: Multiple alignment of UD sequences of primate SFKs. Sequences were aligned with MAFFT. The most conserved residues are indicated above the alignment. The strictly conserved FGG of the ULBR is framed. Numbering corresponds to human c-Src sequence. H sap: Homo sapiens; P tro: Pan troglodytes; G gor: Gorilla gorilla; P abe: Pongo abelii; M mul: Macaca mulatta; C jac: Callithrix jacchus; O gar: Otolemur garnettii; G gal: Gallus gallus; X tro: Xenopus tropicalis; D rer: Danio rerio.

Figure 2 .

 2 Figure 2. ULBR inactivation affects Src oncogenic activity. a: Strategy of ULBR inactivation. b: the level of Src expression in NIH3T3 cells transduced with indicated Src constructs. c, d: anchorage independent cell growth (c) and invasion (d) of indicated Srctransformed NIH3T3 cells. e: the level of Src expression and activity in SW620 colon cancer cells transduced with indicated Src constructs. f, g: anchorage independent growth (f) and invasion (g) of SW620 cancer cells expressing indicated Src constructs. The histograms show the percentage of colonies in soft agar normalized to the maximal condition set a 100% (colonies) and the percentage of migrating cells in the matrigel matrix normalized to control condition set at 100% (cell invasion). Is shown the mean ± SEM; n>3; ns: p>0.05; *p<0.05; **p<0.01; p<0.001; Student's t test.

Figure 3 .

 3 Figure 3. ULBR inactive mutations affects Src tumor activity in nude mice. a, b: timecourse of tumor development in nude mice subcutaneously inoculated with SW620 tumor cells

Figure 4 .

 4 Figure 4. ULBR inactive mutation inhibits MAPK signaling without affecting Src localization and kinase activity. a, b: ULBR regulates Src-induced protein tyrosine phosphorylation and of p42/44 MAPK activation. Immunoblots of whole-cell lysates showing cellular protein tyrosine phosphorylation and of p42/44 MAPK activity in SW620 cells (a) and HEK293T cells (b) transduced with indicated Src constructs. c: Src dimerization is not affected by ULBR inactivation. HEK293T cells were transfected with the indicated constructs. Src-Myc proteins were immunoprecipitated (IP) from cell lysates and immunoblotted with the indicated antibodies. Immunoblots of whole-cell lysates were also performed as indicated. d: Representative confocal image of direct fluorescence of HEK293T cells co-expressing Src-mCherry and indicated Src-GFP ULBR mutants. The overlay is also shown. e: In vitro kinase assay of purified Src-GFP and Src3A-GFP that were expressed in HEK293T cells using indicated substrate. The level of immunoprecipitated Src-GFP proteins and tyrosine phosphorylation of indicated substrate is shown.

Figure 5 :

 5 Figure 5: phospho-proteomic analysis of Src-ULBR signaling in SW620 cancer cells. a: A label-free quantitative phospho-proteomic analysis centered on tyrosine phosphorylation. A Veen diagram where quantified phospho-peptides were sorted as differentially phosphorylated

  Figure 1

  -GFP kinase activity was detected in respect to enolase substrate concentration or kinase duration. (Fig 4e & S2). Similarly, no effect of ULBR inactivation was observed with EPHA2, suggesting that the lack of effect of ULBR mutation in vitro is not substrate specific. Altogether, these data indicate that the observed in vivo effects with ULBR mutants depend on Src membrane anchoring but not on cell compartmentalization or lipid-induced kinase dimerization.

  phosphorylation of RTKs (>2-fold increase for 13/49 RTKs), including adhesive receptors DDR2 and EPHs, growth factors receptors EGFR, MET, FGFR3 and AXL an RYK, a co-receptor of the Wnt/beta-catenin signaling. Interestingly, Src-ULBR had a

	modulatory role on Src-induced RTKs phosphorylation, i.e. a positive effect on EGFR, MET,
	FGFR3, RYK and EPHA2 phosphorylation and an inhibitory effect on EPHA5, EPHB2, B4
	and B6 phosphorylation (Fig 5b, S3a & b). We also complemented this analysis by probing a
	phospho-kinase antibody array (Fig 5c), which revealed that Src expression activates Stats (2,
	5 and 6) and SFKs (Fyn, Yes, Lck, Lyn and Hck) signaling proteins. Interestingly, while Src-

and table

S1

), indicating that ULBR has no major impact on Src substrate phosphorylation and/or this region may regulate subtle phosphorylation changes that could not be detected from the label free MS analysis We next profiled ULBR signaling by probing a phospho-receptor tyrosine kinase (RTK) antibody array (Fig

5b & S3

). This biochemical survey revealed that Src has a large impact on tyrosine ULBR was dispensable for most of the probed phospho-signaling activities, it was required for Src-induced Fyn activation (i.e. pTyr420-Fyn level) and activation of the downstream effectors p42/44 MAPKs (i.e. pThr202/pTyr204 level) in these cancer cells (Fig

5c & S3b

). We thus concluded that ULBR modulates tyrosine phosphorylation of specific membrane-localized substrates, e.g. RTKs and SFKs.

  *p<0.01; p<0.001; Student's t test.

	H sap
	P tro
	G gor
	P abe
	M mul
	C jac
	O gar
	G gal
	X tro
	D rer
	H sap
	P tro
	G gor
	P abe
	M mul
	C jac
	O gar
	G gal
	X tro
	D rer
	H sap
	P tro
	G gor
	P abe
	M mul
	C jac
	O gar
	G gal
	X tro
	D rer

ACKNOWLEDGEMENTS

We thank the Montpellier RIO Imaging and RHEM platforms for imaging and immunohistochemistry analyses, and the Proteomic Core Platform of EPFL for proteomic analyses. This work was supported by fundacion Marato TV3, La Ligue Nationale Contre le Cancer (LNCC), Montpellier SIRIC Grant «INCa-DGOS-Inserm 6045», CNRS, and the University of Montpellier. RHEM facility supported by SIRIC Montpellier Cancer Grant INCa_Inserm_DGOS_12553, the european regional development foundation and the occitanian region (FEDER-FSE 2014-2020 Languedoc Roussillon), IBiSA and Ligue contre le cancer for processing our animal tissues and histology technics. ML is supported by the Fondation pour la Recherche Médicale (FRM) and the Fondation de France. SR is an INSERM investigator.

AUTHORS CONTRIBUTION

All authors contributed extensively to the work presented in this paper. Experimental analysis and Data acquisition: EA, ML, MB, VS, BR, EF, FA, RH, JP and AS. Bioinformatics analysis: PF, MA and RH. Writing of the paper: SR and MP. Project supervision: SR.

CONFLICT OF INTEREST

The authors declare no conflict of interest.


Sophie Colson
email: colson.sophie@tohoku.ac.jp

Manuel Kuhni

Akihiro Hayakawa

Hideaki Kobayashi

Cédric Galizzi

Dany Escudié

Stabilization mechanisms of an ammonia/methane non-premixed jet flame up to liftoff

Keywords: non-premixed jet flame, stabilization, ammonia/methane, flame-burner thermal interaction, liftoff process

Ammonia is a promising alternative fuel for CO 2 emission mitigation. The use of ammonia blends allows for more flexibility compared to pure ammonia fuel and is often considered for immediate CO 2 emission reduction in existing facilities running on natural gas.

However, fundamental studies on these fuel mixtures remain scarce. This study thus focuses on ammonia/methane blend fuels. The effect of ammonia on methane jet flame stabilization is investigated using a non-premixed jet flame configuration to observe the flame stabilization mechanisms, the flame-burner interactions and how ammonia addition affects the attached flame stabilization up to liftoff. The flame tip position was observed using CH* chemiluminescence. Heat transfer to the burner was monitored by temperature measurement at

Le F Fuel Lewis number

Le eff

Effective Lewis number as defined in [START_REF] Park | Effects of Lewis number and preferential diffusion on flame characteristics in 80%H2/20%CO syngas counterflow diffusion flames diluted with He and Ar[END_REF] for diffusion flames

Z st

Stochiometric mixing fraction

MR

Momentum Ratio

Re J Jet Reynolds number

Re lip

Local Reynolds number in the wake of the burner lip

Introduction

Ammonia has attracted recent attention for its use as a carbon-free energy carrier [START_REF] Iea | The Future of Hydrogen[END_REF][START_REF] Kobayashi | Science and technology of ammonia combustion[END_REF]. Indeed, ammonia presents interesting features that make it both cost-effective and fastly and broadly deployable for the energy industry [START_REF] Valera-Medina | Ammonia for power[END_REF][START_REF] Vezina | Key Life Cycle Assessment Numbers for NH3, Green and Brown Energy[END_REF]. Though its production is for now mainly carbon-based, green-ammonia production is possible using water and nitrogen, employing water electrolysis and Haber-Bosh process as a way to store energy from renewable resources. This green ammonia can be later used as a fuel in various combustion situations ranging from SI engines to gas turbine applications [START_REF] Mounaïm-Rousselle | Ammonia as Fuel for Low-Carbon Spark-Ignition Engines of Tomorrow's Passenger Cars[END_REF][START_REF] Kurata | Development of a wide range-operable, rich-lean low-NOx combustor for NH3 fuel gas-turbine power generation[END_REF][START_REF] Lesmana | NH3 as a transport fuel in internal combustion engines: A technical review[END_REF]. However, some major challenges remain including the NOx emission and flame stabilization issues. Regarding the stabilization issue, the use of fuel blends can be considered, such as ammonia/hydrogen mixtures or ammonia/hydrocarbons mixtures, with a greater reactivity than the pure ammonia, therefore easing stabilization. Among those blends, ammonia/hydrocarbons mixing are essential for short-term CO 2 emission mitigation strategy through the addition of ammonia in existing facilities currently running on hydrocarbons. The combustion properties of those blends had thus recently attracted attention [START_REF] Okafor | Experimental and numerical study of the laminar burning velocity of CH4-NH3-air premixed flames[END_REF][START_REF] Ichikawa | Burning velocity and flame structure of CH 4 /NH 3 /air turbulent premixed flames at high pressure[END_REF][START_REF] Han | Experimental and kinetic modeling study of laminar burning velocities of NH3/air, NH3/H2/air, NH3/CO/air and NH3/CH4/air premixed flames[END_REF][START_REF] Colson | Experimental and Numerical Study of NH3 /CH4 Counterflow Premixed and Non-premixed Flames for Various NH 3 Mixing Ratios[END_REF]. However, studies on those blend fuel flame stabilization mechanisms remain scarce.

Among them, Hayakawa et al. [START_REF] Hayakawa | Experimental investigation of stabilization and emission characteristics of ammonia/air premixed flames in a swirl combustor[END_REF] studied the flame stabilization domain in a swirl burner for premixed ammonia flame. A more fundamental approach was taken by Rohdes et al. [START_REF] Rohde | The stabilization of turbulent ammonia-air flames on flameholders[END_REF] observing a premixed ammonia turbulent flame stabilized on a flame holder, doped with O 2 .

This work focuses on the stabilization mechanisms and the flame-burner interactions of an ammonia/methane flame using a non-premixed jet flame configuration in air coflow. A local approach is considered in the present work, focusing on the stabilization mechanisms up to liftoff, and completing the global approach and the characterization of the jet stabilization domain performed in a previous study [START_REF] Colson | Study of the Combined Effect of Ammonia Addition and Air Coflow Velocity on a Non-premixed Methane Jet Flame Stabilization[END_REF].

The non-premixed jet flame configuration has been used in many fundamental studies on flame stabilization as reviewed by Lyons or Lawn [START_REF] Lyons | Toward an understanding of the stabilization mechanisms of lifted turbulent jet flames: Experiments[END_REF][START_REF] Lawn | Lifted flames on fuel jets in co-flowing air[END_REF]. This academic configuration is relatively simple and allows to look at the detail of the mechanisms of flame stabilization, from a global approach [START_REF] Takahashi | Lifting criteria of jet diffusion flames[END_REF][START_REF] Takahashi | Lifting mechanism of free jet diffusion flames[END_REF] to a more local approach close to flame tip [START_REF] Takahashi | Near-field turbulent structures and the local extinction of jet diffusion flames[END_REF][START_REF] Takahashi | Attachment mechanisms of diffusion flames[END_REF][START_REF] Lamige | On preheating and dilution effects in non-premixed jet flame stabilization[END_REF][START_REF] Otakeyama | Stability of CH4-N2/air jet diffusion flame for various burner rim thicknesses[END_REF][START_REF] Marin | Experimental study of the lifting characteristics of the leading-edge of an attached non-premixed jet-flame: Air-side or fuel-side dilution[END_REF]. In the present configuration, and as developed in our previous work [START_REF] Colson | Study of the Combined Effect of Ammonia Addition and Air Coflow Velocity on a Non-premixed Methane Jet Flame Stabilization[END_REF] we describe the evolution of the stabilization domain of a methane flame with ammonia addition. In the following, we will refer to "liftoff" as the transition between attached and lifted flames, "blowout" as the transition from lifted flames to extinction, "re-attachment" as the transition from lifted flames to attached flames and finally "blowoff" as direct extinction from attached flames. In our previous work [START_REF] Colson | Study of the Combined Effect of Ammonia Addition and Air Coflow Velocity on a Non-premixed Methane Jet Flame Stabilization[END_REF], ammonia addition led to a particularly large decrease of the liftoff limit, with direct blowoff observed close to 50% of ammonia in terms of mole fraction in the fuel mixture.

Chemical effects are expected to be dominant, as observed from the particularly large decrease in the laminar burning velocity when ammonia is added to the fuel mixture. Nonetheless, the predominance of the chemical aspects on flame stabilization may depend on the ammonia content in the mixture, relatively to other parameters such as thermal or diffusive ones. The study of global parameters alone could not explain satisfactorily the large decrease observed, and a more local and dynamic approach, focusing on the flame tip (leading edge), is thus considered in the present work to observe the evolution with ammonia addition of the aerothermo-chemical coupling at the burner lip up to liftoff.

The objective of the present work is to clarify how flame stabilization is impacted by the addition of ammonia in the fuel mixture by looking at the evolution of the flame local stabilization dynamics and its interaction with the burner when adding ammonia, completing our previous global approach [START_REF] Colson | Study of the Combined Effect of Ammonia Addition and Air Coflow Velocity on a Non-premixed Methane Jet Flame Stabilization[END_REF].

Experimental setup

2.1.Setup Overview

The flame stabilization was studied using a non-premixed jet flame configuration as represented in Fig. 1. The setup is constituted of a pipe for fuel injection of inner diameter, D i , 6 mm, and lip thickness, e, of 2 mm. Pipe length, L, is 950 mm, ensuring a ratio L/D i > 150. This pipe exits in a closed squared chamber of 250 x 250 mm 2 . The oxidizer, air in this study, is going through perforated plates and a convergent to insure a well-relaminarized profile at the burner outlet.

Velocity profiles at the burner outlet were observed using laser doppler anemometry measurements in previous work [START_REF] Lamige | On preheating and dilution effects in non-premixed jet flame stabilization[END_REF] and the mean jet velocity, U J , obtained from the mass flow rate was shown to be in satisfactory agreement with those measurements (within 6 % error). In the present work the coflow velocity, U co , was set to 0.2 ms -1 , corresponding to the largest stabilization domain up to blowoff in the range of U co = 0 -0.3 ms -1 accessible [START_REF] Colson | Study of the Combined Effect of Ammonia Addition and Air Coflow Velocity on a Non-premixed Methane Jet Flame Stabilization[END_REF].

Methane and ammonia are mixed before injection in the setup. The ratio of ammonia to methane in the fuel mixture is defined in Eq. ( 1) using the ammonia mixing ratio, E, as in previous work [START_REF] Okafor | Experimental and numerical study of the laminar burning velocity of CH4-NH3-air premixed flames[END_REF][START_REF] Ichikawa | Burning velocity and flame structure of CH 4 /NH 3 /air turbulent premixed flames at high pressure[END_REF],

𝐸    ( 1 )
where X i is the mole fraction of fuel i in the fuel mixture and LHV i is the lower heating value of i, with LHV CH4 = 802.3 kJmol -1 and LHV NH3 = 316.8 kJmol -1 .

The flow rate of each gas is controlled and monitored by thermal mass flow controllers (SLA5850 series, Brooks Instrument Co., Hatfield, PA, USA) with a precision of 0.7 % of the mass flow rate, more 0.2 % of the full range, leading by propagation to a maximal uncertainty of 2 % on the velocities and 3 % on the ammonia mixing ratio, E. In this study, the dynamics of the attached flame up to liftoff was observed for E varying between 0 and 0.3, near the direct blowoff case as developed in previous work [START_REF] Colson | Study of the Combined Effect of Ammonia Addition and Air Coflow Velocity on a Non-premixed Methane Jet Flame Stabilization[END_REF]. The range of jet velocity, U J , explored thus cover values from 0.05 ms -1 to 16 ms -1 .

Observations at the burner lip.

To analyze the local dynamics of the attached flame and how the aero-thermo-chemical balance is broken at liftoff, several local measurements were performed at the burner lip. The attached flame position was observed using CH* chemiluminescence imaging. CH* is known to be a good marker of the largest heat release rate region within the flame front and has been used in several works on methane/hydrocarbons flame [START_REF] Child | Spectrophotometric studies of laminar flames-I the decay of radical radiation[END_REF]. Besides, from 1D simulations on The CH* chemiluminescence was collected using an intensified camera (Teledyne Princeton Instruments PI-MAX 2) with an exposure time of 7 ms and a gain of 120. Series of 300 images were collected for each condition.

When introducing ammonia in the flame, the CH* chemiluminescence intensity is expected to decrease due to the smaller amount of CH* in the reaction region. Thus, the intensity was normalized by its maximum value on each image before post-processing. The 2D image of the front was retrieved from the 3D CH* chemiluminescence using Abel deconvolution [START_REF] Giassi | Analysis of CH* concentration and flame heat release rate in laminar coflow diffusion flames under microgravity and normal gravity[END_REF].

Similar filtering was applied to all images. Detection of the flame attachment position was then performed by detection of a threshold of 45 % for the attachment height, H a . Then at height H a , the attachment radius, R a , was detected with a threshold of and 40 %. These threshold values were selected to ensure proper detection of the attachment position for all the ammonia mixing ratio, E, and jet velocity, U J , conditions investigated, including the cases of lower signal to noise ratio due to lower CH* emissions. An example of a raw image and a post-processed image is In addition to the attached flame position dynamics, the thermal interactions with the burner were monitored by the mean of two K-thermocouples (Chromel -Alumel, 75 μm diameter in a sleeve of 0.5 mm) inside the burner lip as represented in Fig. 3. Those thermocouples enable to monitor the temperature at the lip surface, T lip , and the one 6 mm under the lip surface, T 6mm , and the calculation of the conductive heat flux in the burner, Φ. Assuming unidirectional conduction, this heat flux, Φ, is obtained using the following Eq. ( 2)

represented in Fig 2.
𝛷 𝜆▪ ( 2
)
Where λ is the conductivity of the burner lip (Al 2 O 3 ) at (T lip + T 6mm )/2 and Δz = 6 mm is the distance between the hot junctions of the two thermocouples. Finally, a temperature mapping of the flame was performed using an R-type thermocouple (Pt-Pt/Rh13%, 50 μm diameter). This temperature mapping was performed in the direct vicinity of the burner lip at a height, z, between 2 mm and 8 mm above the burner lip, and a radius, r, between 0 and 15 mm. This mapping was used to observe the flame temperature variations and temperature distribution near the burner and how they varied with ammonia addition and jet

Results and discussion

Flame local stabilization dynamics

Local study of the flame stabilization in the present configuration was done in previous work [START_REF] Lamige | Burner lip temperature and stabilization of a non-premixed jet flame[END_REF] in the case of a methane flame with a focus on thermal aspects, with preheating of the oxidizer, from low jet velocity in the laminar regime and up to liftoff. This previous work describes the evolution of the flame tip position, burner temperature and heat flux up to liftoff, and defined 3 main regimes of stabilization as briefly recalled in the following and summarized in Fig. 4:

 Regime I: the flame moves toward the oxidizer and downward as the jet velocity is increased up to an extremum position (R E , H E ). This first regime corresponds to the gradual expansion of the jet as U J is increased up to a momentum ratio, MR, in the range 3-7, corresponding to the extremum position.

 Regime II: From the extremum position (R E , H E ), if U J is further increased, the flame will move back toward the fuel jet, as an increased jet velocity drags air in the wake of the burner lip, changing the local fuel and oxidizer concentration, and moving the stoichiometric line Z = Z st closer to the jet centreline. In this regime, the temperature at the burner lip keeps decreasing due to enhanced heat transfer.

 Regime III: The last regime observed before liftoff corresponds to laminar to turbulent transition in the pipe, Re J = U J • D i / ν, with ν the kinematic viscosity of the fuel mixture.

From that transition point (R L-T , H L-T ), the flame tip keeps moving toward the centreline as more air is dragged in the wake of the burner lip, but flame attachment height slightly decreases. The temperature at the burner lip further drops as convection is emphasized.

Figure 4. Sketch representing the attached flame stabilization regimes as introduced by Lamige et al. [START_REF] Lamige | Burner lip temperature and stabilization of a non-premixed jet flame[END_REF].

(R E , H E ) (R L-T , H L-T )
Burner axis

Regime I Regime II Regime III

Fuel Air

Burner lip

The regime I can be further subdivided into two regimes Ia and Ib, corresponding to a change in the thermal interaction with the burner lip. In region Ia, the flow is assumed to be purely viscous in the wake of the burner lip and heat transfer dominated by conduction. As the jet velocity, U J , is increased, heat transfer by convection becomes more important, corresponding to regime Ib.

This change is observable in the evolution of the temperature at the burner lip, T lip , and the conductive heat flux in the burner lip, Φ [START_REF] Lamige | Burner lip temperature and stabilization of a non-premixed jet flame[END_REF]. When U J is increased, T lip increases and reaches a maximum before decreasing. This maximum corresponds to the transition from Ia to Ib. The conductive heat flux Φ in the burner calculated from the thermocouples measurements confirms this analysis with an increase in Φ in regime Ia, stabilizing in region Ib before decreasing in regime II, for which air recirculation appears, and further decreasing when entering regime III, as the jet becomes turbulent. A local Reynolds number, Re lip = U J • e / ν lip , was thus introduced, characterizing the transition Ia to Ib, with U J , the jet velocity, e, the burner lip thickness and ν lip the local viscosity calculated for a stoichiometric mixture at T lip . In the case of methane, the transition from Ia to Ib was shown to be close to 10 [START_REF] Lamige | Burner lip temperature and stabilization of a non-premixed jet flame[END_REF].

The introduction of ammonia in the jet leads to several changes in terms of thermal properties, diffusive properties and fundamental combustion characteristics as introduced in Table 1, where ammonia mixing ratio, E, and ammonia mole fraction in fuel mixture, X NH3 , are first recalled. It could be observed in previous work that, though the flame is considered as mostly diffusive, partial premixing is assumed to occur in the dark region close to the burner, enabling the leading edge of the flame to develop in the vicinity of the burner, with higher heat release rates and supporting the diffusion trail [START_REF] Takahashi | Attachment mechanisms of diffusion flames[END_REF][START_REF] Otakeyama | Stability of CH4-N2/air jet diffusion flame for various burner rim thicknesses[END_REF][START_REF] Marin | Experimental study of the lifting characteristics of the leading-edge of an attached non-premixed jet-flame: Air-side or fuel-side dilution[END_REF]. As a first approach, the laminar burning velocity of a stoichiometric ammonia/methane/air premixed flame, S L0 , is reported in Table 1. As ammonia is added S L0 decreases, reflecting the lower reactivity of the mixture observed for larger ammonia content. This decrease is expected to play an important role in flame destabilization [START_REF] Marin | Experimental study of the lifting characteristics of the leading-edge of an attached non-premixed jet-flame: Air-side or fuel-side dilution[END_REF] and the diminution of the liftoff velocity, U LO , as developed in previous work [START_REF] Colson | Study of the Combined Effect of Ammonia Addition and Air Coflow Velocity on a Non-premixed Methane Jet Flame Stabilization[END_REF]. Other parameters are also expected to affect the local flame stabilization. The flame adiabatic temperature decreases by close to 70 K when ammonia is introduced up to E = 0.3, while the maximum heat released by the flame is divided by 3. This might drive changes in the thermal interaction with the burner, affecting in turn flame stabilization. The thermo/diffusive balance is also introduced in Table 1 with the effective Lewis number, Le eff = (Le F + A f • Le O2 ) / (1+A f ) , calculated as in [START_REF] Park | Effects of Lewis number and preferential diffusion on flame characteristics in 80%H2/20%CO syngas counterflow diffusion flames diluted with He and Ar[END_REF], To investigate how those changes will affect the flame-burner interaction, the evolution of the local stabilization dynamic of the flame (flame position, burner temperature and heat transfer to the burner) was observed with progressive addition of ammonia, taking the regimes detailed earlier for pure methane flame as a reference.

taking

Effect of ammonia addition on flame position

The effect of ammonia addition on flame stabilization is first observed looking at the attached flame position evolution with ammonia addition. The attachment radius, R a , and the attachment height, H a , are represented in Fig. 5 for ammonia methane mixing ratio from E = 0 to E = 0.3 as a function of MR, the momentum ratio of fuel and air defined as criteria for the transition from the regime I to regime II as previously developed in the case of a methane flame. From Figure 5, it can be observed that the global evolution of the attached flame as described in the introduction of this work is still valid for this type of mixture, with the simultaneous observation of maximum of R a and minimum of H a corresponding to the transition from I to II and the position (R E , H E ). For low ammonia content (E < 0.2), the transition II to III is still

H a [mm] MR [-] E = 0 E = 0.1 E = 0.2 E = 0.3 b E = 0 E = 0.1 E = 0.2 E = 0.3 E = 0 E = 0.1 E = 0.2 E = 0.3 Regime I Regime II Regime I Regime II
observed with a change in the variation of H a and a larger decreasing slope for R a is observed for jet velocity such as Re J > 2300. For larger ammonia content though, the liftoff occurred before the jet laminar to turbulence transition occurred at Re J = 2300.

In addition, the introduction of ammonia lead to a decrease in R a , and an increase in H a for all the jet velocity condition U J explored (respectively MR, Re J ).

Looking further in the evolution of R a and H a , it is worth noticing in Fig. 5 that, though the 3 regimes are still observed, the transition between regime I and II, corresponding to the flame extremum position (R E , H E ), appears for a gradually higher MR E as ammonia is introduced, from

MR E = 5 (U JE = 0.61 m/s) for E = 0 to MR E = 20 (U JE = 1.2 m/s) for E = 0.3.
The evolution of the flame position and the transitions between the identified stabilization regime with MR (as well as U J or R eJ ) is varying with E.

To understand this increase in MR E , it is necessary to go back to the physical change corresponding to this transition. The transition from regime I to II corresponds to the motion of the stoichiometric line toward the jet due to the air dragged in the wake of the lip by larger jet velocity. This transition has been associated with a momentum ratio of 3 -7 in a previous study on methane flame [START_REF] Lamige | Burner lip temperature and stabilization of a non-premixed jet flame[END_REF]. Nonetheless, this parameter remains global and does not take into account the eventual local variations of the stoichiometric mixture line due to air drag. One explanation for the increase of MR E when ammonia is introduced is to relate to the higher flame position, as represented in the sketch in Fig 6 . The impact of the air dragged on the Z contour is expected to gradually become more important as the velocity is increased, with at first small impact relatively close to the burner, and then gradually more pronounced, with streamlines gradually going further toward the jet axis. The difference in MR E observed for the point of transition might be attributed to the fact that for greater ammonia content, larger jet velocity is necessary to drag sufficient air to change the stoichiometric line Z st up to the position at which is stabilized the flame. Taking this extremum position as a reference, as represented in Fig. 7, it can be seen that the flame positions for different mixtures are overlapping, except for some particularly low-velocity

Burner axis

Fuel Air

Burner lip

Burner axis

Fuel Air

Burner lip

(a) MR = MR E (E = 0) < MR E (E = 0.3) (b) MR = MR E (E = 0.3) > MR E (E = 0)
conditions. The complete evolution of the flame position for increasing U J can then be described referring to the methane flame case, and the evolution on R a and H a with ammonia addition limited to the observation of the evolution of the extremum (R E , H E ). The decrease of R E can be attributed to the change in the Z st with ammonia addition. Indeed, when ammonia is added Z st is increased as recalled in Table 1. Thus, for a similar jet/coflow momentum ratio MR, the location of the mixture contour, Z = Z st , called in the following stoichiometric line, is moving toward the jet. For the same MR value, an ammonia/methane flame is thus closer to the jet centerline and R E is reduced as ammonia is added. A strong correlation between R E and Z st is thus observed in the present data as shown in Fig. 8

H a -H E [mm] R a -R E [mm] E = 0 E = 0.1 E = 0.2 E = 0.3 E = 0 E = 0.1 E = 0.2 E = 0.2
stoichiometric mixing fraction Z st . Extrapolation of this relation for other jet configurations is however subject to caution, as the geometry of the burner outlet, particularly the lip, might greatly affect the local velocity flow field and the distribution of the Z-contours. Generalization of such relation would require the actual measurement of mixing fraction Z in the wake of the burner lip and its direct surrounding, to derive a more accurate relationship. Such type of measurement is however extremely complex in practice, considering the fuel involved and the burner dimension. Several interpretations of the evolution of H E with ammonia addition can be done. Following the leading-edge theory and considering partial premixing at the flame tip, the reduction of the local flame speed, due to the lower reactivity of ammonia as observed from its lower laminar burning velocity in Table 1, is expected to be one of the reasons of the higher attachment

E = 0 E = 0.1 E = 0.2 E = 0.3
position. For a constant MR, the flame is thus expected to move toward a region of lower local velocities where its speed can balance the local velocity. Thus, it moves further away from the jet, toward the oxidizer side, in the upper direction.

In addition, the higher Z st with ammonia addition, will also lead the flame to stabilize higher above the burner lip. Indeed, with ammonia addition, the flame will move toward a new stoichiometric contour Z st, E>0 > Z st, E=0 , located closer to the jet, in a higher-velocity region. It will thus need to move higher up along this line, in a lower-velocity region, allowing sufficient time for the diffusion of radicals and reactions to occur. Similar observations were done in the case of the fuel-side dilution of a methane jet with inert gas (CO 2 , Ar, N 2 ) for which attached flame stabilization was observed closer to the jet centre and higher above the burner lip [START_REF] Marin | Experimental study of the lifting characteristics of the leading-edge of an attached non-premixed jet-flame: Air-side or fuel-side dilution[END_REF].

The present interpretation relies essentially on the aero-chemical couplings and their evolution with ammonia addition, which are expected to be dominant in the flame stabilization mechanism. This analysis should be however further developed, considering the evolution of thermo-diffusive properties of the gas in addition to their reactive properties. More specifically, the thermal and chemical interactions between the flame and burner and their evolution with ammonia addition should be considered.

The thermal interaction with the burner and their evolution with ammonia addition are thus further detailed in the next section 3.3. Chemical quenching to the burner lip, though not investigated in the present study, might also contribute to some minor extent to the changes observed with ammonia addition. Indeed, the changes in the combustion chemistry induced by ammonia addition lead to a greater dependency in the presence of OH radicals relative to the larger dependency in H radicals in the methane flame [START_REF] Kobayashi | Science and technology of ammonia combustion[END_REF][START_REF] Colson | Extinction characteristics of ammonia/air counterflow premixed flames at various pressures[END_REF]. This difference might trigger a different flame response in terms of chemical quenching to the burner lip. This effect is however expected to remain neglectable compared to the aero-chemical coupling or thermal interactions, particularly when considering that the mixtures investigated remains under E = 0.3 (X NH3 = 0.52), that the burner lip material employed is merely reactive (Al 2 O 3 ) and the burner surface temperature is relatively low [START_REF] Lamige | Burner lip temperature and stabilization of a non-premixed jet flame[END_REF][START_REF] Prakash | OH concentration profiles over alumina, quartz, and platinum surfaces using laser-induced fluorescence spectroscopy in lowpressure hydrogen/oxygen flames[END_REF][START_REF] Kim | Effects of thermal and chemical surface-flame interaction on flame quenching[END_REF].

Effect of ammonia addition to heat transfer to the burner

It was observed that flame stabilization is particularly influenced by the modification in the aero-chemical coupling with ammonia addition. With the attached flame being in close contact with the burner, the thermal interactions of the flame with the burner should also be carefully investigated. More specifically, and as recalled in section 3.1, regime I can be subdivided into two considering the evolution of the temperature at the burner lip, T lip , and the conductive heat flux in the burner lip from the surface, Φ, as shown in Fig. 9.

Figure. 9. T lip and Q as a function of Re δ for gradual ammonia addition.

A clear decrease of both T lip and Φ is observed as ammonia is introduced whatever the jet velocity. However, the trends observed in the present work when increasing the jet velocity for the different E are similar to those observed in previous work on methane [START_REF] Lamige | Burner lip temperature and stabilization of a non-premixed jet flame[END_REF]. The large decrease of temperature observed at the burner lip can be explained by the decrease in the flame temperature with ammonia addition, as recalled in Table 1, and the increase in the flame-burner distance, d a .

As a first approach, and to understand the relative importance of those parameters on the burner lip temperature variations, the variations with E of the adiabatic flame temperature, T ad , obtained from 1D calculations [START_REF] Ansys | Chemistry Simulation Software[END_REF], the maximum flame temperature, T max , observed from experimental temperature mapping, and the burner lip temperature, T lip , are introduced in Fig. 10. It can be seen that the adiabatic flame temperature variation, ΔT ad , remains under 5 % in Re lip

E = 0 E = 0.1 E = 0.2 E = 0.3 E = 0 E = 0.1 E = 0.2 E = 0.3 Ia III II Ib T lip Q E = 0 E = 0.1 E = 0.2 E = 0.3 E = 0 E = 0.1 E = 0.2 E = 0.3
the range E = 0 -0.3. Similarly, the temperature mapping of the flame in the four different regimes (Ia, Ib, II, III) gives similar variation order (3 -4 %). However, the variation of the maximum burner temperature, ΔT max, lip , is more than 20 % and the flame temperature variations alone seem insufficient to explain this drop. As observed in the previous section, the flame tip position varies with ammonia addition and the changes in the local aero-chemical coupling. This change in position is expected to affect the thermal interaction with the burner. The relationship between the burner lip temperature, T lip , and the distance flame-burner, d a , is introduced in Fig. 11a for regime I and Fig. 11b for regime II and III, for various E and U J . It can be observed in Fig. 11a that in regime I, T lip is wellcorrelated to d a , with a temperature decreasing gradually as the flame gets more distant. In regime II and III, as shown in Fig. 11b, the relationship is more complex. For each E, a large decrease of the temperature is observed as U J is increased whereas d a remains close to constant.

This important decrease in temperature corresponds to the larger transfer by convection as jet velocity is increased. Nonetheless, for a constant U J , a decreasing trend is observed with an increasing d a , also corresponding to the increase E. Flame-burner distance plays thus an important role in those regimes too.

Effect of ammonia on liftoff dynamics

After investigating the evolution of the stabilization dynamics of a methane attached flame with ammonia addition, we will now focus more specifically on the case of the liftoff, corresponding to the rupture of stabilization balance as described in the previous section 3.1 to 3. Re lip

E = 0 E = 0.1 E = 0.2 E = 0.3 E = 0 E = 0.1 E = 0.2 E = 0.3
Regime I Regime II Regime III normalized 1 x 1 domain above the burner lip in Fig. 13.

Figure 13. Evolution of the extremum position (red) and the position just before liftoff (black)

for various E.

As developed in section 3.2, when ammonia is introduced in the mixture, the extremum position moves toward the jet, due to changes in the stoichiometric mixture fraction Z st . For each E, when the jet velocity is increased from (R E , H E ), the Z-contours are pushed toward the jet side where local velocities and strain rates are higher, up to local extinction and liftoff.

When E increases, this region becomes eventually more restricted, as observed from the convergence of the extremum position (R E , H E ) and the position just before liftoff (R LO , H LO ) in Fig. 13. The fact that the region becomes gradually more restricted and flame position constrained is also observable from the temperature gradients across the flame as represented in

0 1 0 1 H a /e [R a -(D i /2)]/e ■ = 0 ◆ E = 0.1 ▲ E = 0.2 • E = 0.3

Fuel Air

The extremum and liftoff line eventually reach each other close to E = 0.3, at the corner of the normalized 1 x 1 region, corresponding to a height close to the burner lip size (2 mm) and a radius corresponding to the outer edge of the burner lip. This mixing ratio particularly close to the blowoff limit observed on this burner (E = 0.32) [START_REF] Colson | Study of the Combined Effect of Ammonia Addition and Air Coflow Velocity on a Non-premixed Methane Jet Flame Stabilization[END_REF]. When getting closer to this fuel mixing condition, if the jet velocity, U J , is increased from the extremum position, the region in which flame can stabilize close to the burner is limited to a cornered point due to the higher velocities on the fuel side. The flame cannot stabilize anymore in the close-burner-region and it lifts from the burner. Besides, when the flame is in the extremum position, (R E , H E ), the jet is laminar, and no air is yet dragged in the wake of the burner lip. The fuel-oxidizer mixing is thus expected to be particularly low in the shear layer above the burner lip. Those conditions will thus lead the flame to blowoff directly, as its base cannot stabilize, even at a higher position in the jet, with no region favorable in terms of both sufficiently low velocities and flammable mixtures.

Conclusions

The stabilization mechanisms of an ammonia/methane non-premixed jet flame were investigated experimentally in the present work using a local approach.

 When compared to the methane non-premixed jet flame, it could be observed that the flame position moves toward the jet and downstream as ammonia is added. This phenomenon can be understood as the effect of the increase in stoichiometric fraction, Z st , leading the flame toward the fuel side, where mixing fraction is more favorable to its propagation; and the reduction of the mixture reactivity due to ammonia introduction, leading the flame to stabilizes further downstream where the local velocities are sufficiently low.

 The transition from regimes I to II was shown to be shifted to larger MR E as ammonia is introduced into the mixture. This later transition could be associated with the higher fuel jet velocities that are necessary to drag sufficient air in the wake of the burner lip and cause a change in the position of the stoichiometric line Z st up to the higher flame position. The momentum ratio MR, though representative of the overall dynamics at stake, cannot fully apprehend the local dynamics.

 The thermal interactions with the burner were shown to follow the same dynamics as defined in previous work [START_REF] Lamige | Burner lip temperature and stabilization of a non-premixed jet flame[END_REF], but the temperature at the burner decreased drastically as ammonia was introduced in the fuel jet, under the combined effect of flame temperature decrease and higher attachment height. Moreover, similarly to the transitions between regimes I and II, the transitions between regimes Ia and Ib were observed for slightly larger local Reynolds numbers Re δ . This could be associated with the evolution of the flame position with ammonia addition.

 Finally, it could be observed that as ammonia is introduced, the domain, in terms of attached flame position, on which the attached flame can stabilize get gradually reduced under the effects of the lower flame reactivity (and lower burning velocity) and the modification of the local stabilization dynamics evolution (particularly the position of the Z st contours).

λ

  Nomenclature r radial direction (r = 0 at the burner center axis) [mm] z axial direction (z = 0 at the burner lip surface) [mm] (R a , H a ) Flame base attachment position (radius, height) [mm] (R E , H E ) Flame base extremum position (radius, height) [mm] (R L-T , H L-T ) Flame base position at laminar to turbulent transition (radius, height) [mm] (R LO, H LO ) Flame base position just before liftoff (radius, height) [mm] d a Distance flame-burner lip [mm] L Length of the fuel injection pipe [mm] D i Inner diameter of the fuel injection pipe [mm] e Burner lip thickness [mm] U J Jet velocity [ms -1 ] U co Coflow velocity [ms -1 ] U LO Jet velocity at liftoff [ms -1 ] E Ammonia mixing ratio X i Mole fraction of specie i in fuel mixture LHV i Lower heating value of specie i [kJmol -1 ] Φ Conduction heat flux in the burner lip, assuming unidirectional conduction [ k W m -2 ] Thermal conductivity of the burner, for temperature corresponding to the mean temperature between T lip and T 6mm [Wm -1 K -1 ] T lip Burner lip temperature, at the lip surface (z = 0 mm) [K] T 6mm Burner temperature at 6 mm under the lip surface (z = -6 mm) [K] S L0 Unstretched laminar burning velocity at T = 298 K, 1 atm and ϕ = 1.0 [ms -1 ] T ad Adiabatic flame temperature [K]

Figure 1 .

 1 Figure 1. Experimental setup overview.

  R a and H a were determined for each image of the set of 300 images, and the value reported in the following corresponds to the maximum of the statistical distribution observed. The distribution span is represented by the mean of error bar in the following, including both flame position fluctuations due to physical flame instability and uncertainty due to the measurement (particularly a smaller signal to noise ratio might contribute to a larger distribution span). Further details on the post-processing can be found in the supplementary material section.

Figure 2 .

 2 Figure 2. Chemiluminescence imaging and post-processing: (a) raw image; (b) post-processed image.

Figure 3 .

 3 Figure 3. Burner lip and thermocouple positions.

  change. Radiative heat losses and heat losses by conduction to the thermocouple were not considered in the present work as only relative evolution was considered for qualitative analysis and not for absolute measurement. Details on the thermocouple and its interactions with the flame are available in the supplementary material section. The direct, uncorrected, temperature measurement gave maximum temperature for the velocity range investigated between 1850 K and 1950 K for E = 0, and 1840 K and 1870 K for E = 0.3, and are in agreement with previous work[START_REF] Lamige | Analysis of the influence of thermal boundary conditions on nonpremixed flame stabilization[END_REF][START_REF] Lamige | Burner lip temperature and stabilization of a non-premixed jet flame[END_REF]

  into account both the effective Lewis number of the fuel, Le F , and oxidizer, Le O2 , and the parameter A f = aY F /Y O with a the stoichiometric oxidizer-to-fuel mass ratio, and Y F and Y O the fuel and oxidizer mass fractions. The variation of Le eff in the range of mixture investigated remains moderated and close to unity. Finally, the change in the fuel also leads to a change in the stoichiometric mixing fraction, Z st , which is expected to influence the flame position and the local stabilization balance.

Figure. 5 .

 5 Figure. 5. Evolution of flame tip height and radius with gradual ammonia addition: (a) Attachment radius, R a ; (b) Attachment height, H a . The position just before liftoff is highlighted by black symbol.

Figure. 6 .

 6 Figure. 6. Schematics explaining the evolution of the I to II transition (extremum position): (a) transition for E = 0; (b) Transition for E = 0.3.

Figure 7 .

 7 Figure 7. Flame position for various U J relative to the extremum position (R E , H E ) for E = 0, 0.1, 0.2 and 0.3.

  , relating the radius of the flame normalized by the burner lip thickness, [R E -(D i /2)]/e, with the

Figure 8 .

 8 Figure 8. Correlation flame radius -stoichiometric mixture fraction Z st for various E.

Figure 10 .

 10 Figure 10. Flame and burner lip temperature variations with ammonia addition. Maximum flame temperatures from temperature mapping were taken at constant velocity in regimes Ia, Ib, II and III.

Figure 11 .

 11 Figure 11. Burner lip temperature variations with the burner-flame distance for various E and U J : (a) in regime I; (b) in regimes II and III.

Figure 12 .

 12 Figure 12. Flame-burner distance evolution along with the three aerodynamic regimes. The position just before liftoff is highlighted by black symbol.

  3. The extremum position, (R E , H E ), and the position of the flame tip before liftoff, (R LO , H LO ), are shown in the

Fig. 14 .

 14 Fig. 14. For U J = 2 ms -1 , in regime II, between the extremum position and the position just

Figure 14 .

 14 Figure 14. Radial temperature gradients across the flame at z = 2 mm, for various E in the regime II for U J = 2 ms -1 . The burner lip is denoted by a thick black line on the abscissa.

25 cm 6 mm 2 mm 2 m

	ammonia/methane non-premixed flames, shown in the supplementary material section of this
	work, it can be seen that CH radicals remain a good marker for the heat release zone for
	ammonia/methane mixtures. The spatial variation between the CH distribution peak and the heat
	release rate peak being smaller than the spatial resolution achievable in the present work, CH*
			Perforated plates
	was selected as a marker of the HRR region. Other markers, such as NH*, presented lower
			Mixing
	CH 4 intensity in the range of ammonia mixing ratio, E, investigated and were thus not considered in Fuel MFC
		NH 3	MFC
	the present study.	Air	MFC

Table 1 .

 1 Some fundamental combustion parameters for methane/ammonia mixtures.

	E Ammonia mixing ratio	0	0.1	0.2	0.3
	X NH3 Ammonia fuel mixing mole fraction	0	0.22	0.39	0.52
	S L0 [m.s -1 ]
	Laminar burning velocity at stoichiometric condition	0.35	0.27	0.22	0.19
	from experiment [11]
	T ad [K]
	Adiabatic temperature	2229	2194	2174	2157
	Stoichiometric, calculation from PREMIX code [9,29]
	HRR max [J.m -3 .s -1 ]
	Maximum heat release rate	4.31E9 2.71E9 1.94E9 1.43E9
	Stoichiometric, calculation from PREMIX code [9,29]
	Le eff
	Effective Lewis number for diffusion flame	1.113	1.107	1.101	1.096
	From [1]
	Z st Stoechiometric mixture fraction	0.0552 0.0642 0.0731 0.082

Acknowledgment

Funding:

A part of the work was carried out under the Collaborative Research Project of the Institute of Fluid Science, Tohoku University and in the framework of the Japan-France International Associated Laboratory (LIA) ELyT Global.

CRediT authorship contribution statement: