Killian Onida

Laurent Vanoye

Anis Tlili

Direct Synthesis of Thiocarbamoyl Fluorides and Trifluoromethylamines Through Fluorinative Desulfurization

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Introduction

Fluorinated compounds have gained widespread interest in the last years. [1] The physicochemical properties gained through the incorporation of fluorine or fluorinating groups is the driving force for this perpetual interest. To be said, high lipophilicity as well as high oxidation resistance are among the most desired properties. [2] On the other hand the presence of nitrogen moieties is even more frequent in bioactive compounds. [3] Thus, the association of fluorine and nitrogen represent a highly covetable synthetic target.

In this context, thiocarbamoyl fluorides constitute an attractive core structure in organic synthesis. [4] The direct synthesis of these compounds has been revisited recently. The group of Schoenebeck [5] demonstrated that amines react with (Me4)NSCF3, a nucleophilic trifluoromethylthiolating reagent, furnishing thiocarbamoyl fluorides within 10 min at room temperature (Scheme 1, I). Herein the stability of the trifluoromethylating reagent is the key to success. Indeed, it has been shown that the in-situ generation of thiocarbonyl fluoride (F2C=S) allows the formation of the desired thiocarbamoyl fluoride. Afterwards, the group of Lin and Xiao [6] demonstrated that thiocarbonyl fluoride can be obtained in-situ by reacting difluorocarbene generated from Ph3P + CF2CO2-(PDFA) and S8 (Scheme 1, II). In both methodologies (I & II), the authors used thiocarbamoyl intermediates for the synthesis of trifluromethylamines. [7] Recently, the group of Jiang [8] demonstrated that thiocarbonyl fluoride can be formed by two distinct ways. On the one hand, the association of the Ruppert-Prakash reagent with elementary sulfur and potassium fluoride allows the in-situ formation of thiocarbonyl fluoride. The latter can also be generated starting from AgSCF3 in conjunction with potassium bromide (Scheme 1, III). More recently, the group of Jiang and Yi demonstrated that the Langlois reagent could also be used in conjunction with triphenylphosphine to form thiocarbamoyl fluorides (Scheme 1, IV). [9] In this context, we thought that the straightforward synthesis of thiocarbamoyl fluoride through fluorinative desulfurization would constitute an attractive option for the access thiocarbamoyl fluorides.

Results and Discusion

We recently initiated a new research program aiming at activating small molecules for the fluorination of organic compounds and presumed that investigating an unprecedented fluorinative desulfurization would be the key to success for our methodology. [10] To do so, we selected amine 1a as model substrate in conjunction with 1 equivalent of CS2 and an excess of (diethyamino)sulphur ). We were pleased to observe the formation of the desired product in 45 % yield (table 1, entry 1) when the reaction was performed in toluene in the presence of an external base (0.5 equiv. of DIPEA). Next, we investigated the impact of the solvent on the reaction outcome. The use of DMSO furnished the desired product in very low yield of 9% (table 1, entry 2). When DMF and THF were used the desired product was observed in similar yield as before with toluene (Table 1, entries 3 &4). Moving on to MeCN, a better yield of 51% (entry 5) was obtained. It turns out that DCM is the most efficient solvent under our reaction conditions since 71% of the desired product was obtained. It should be mentioned that lower reaction outcome was observed when the reaction was performed with 1 equivalent of the base. Also, the presence of DIPEA is fundamental to the formation of the desired product since only traces of the product were obtained in the absence of the base (table 1, entry 7).

Afterwards, we decided to screen several bases. Good reactivity was observed when NEt3 and DBU were used as 62 and 57% of the desired product was formed, respectively. Only low yield was obtained when pyridine derivatives, DMAP and 2,6-Lutidine were employed (Table 1, entries 10 & 11). Finally, the yield dropped to 60% when the quantity of DAST was decreased from 3 to 2 equivalents (Table 1, entry 12). [a] Reactions were performed with 1a (1 mmol, 1 equiv.), DAST (3 mmol, 3 equiv.), CS2 (1 mmol, 1 equiv.), base (0.5 mmol, 0.5 equiv.) and solvent (4 mL). The reaction mixture was stirred at rt for 4 hours. Next, we investigated the use of different deoxyfluorinating reagents. It turns out that the product was formed only in the presence of DAST and no traces were observed with XtalFluor-E ® , XtalFluor-M ® [11] neither with Fluolead ™ [12] (Table 2 Entry DeoxF reagent Yield (%) b

DAST 71

2

XtalFluor-E 0 3 XtalFluor-M 0 4 Fluolead 0

[a] Reactions were performed with 1a (1 mmol, 1 equiv.), DAST (3 mmol, 3 equiv.), CS2 (1 mmol, 1 equiv.), base (0.5 mmol, 0.5 equiv.) and solvent (4 mL).

The reaction mixture was stirred at rt for 4 hours. [b] Determined by 19 F NMR spectroscopy with PhOCF3 as an internal standard.

With the best conditions in hand we turned our attention to study the reaction scope (Scheme 2). Overall, the desired product was obtained in synthetically useful to excellent yields with excellent selectivities. The reaction conditions tolerate the presence of unsaturated compounds. For example, thiocarbamoyl fluoride 2c was obtained in moderate yield of 48%. The presence of chlorine substituent is also tolerated under our reaction conditions and the corresponding product was obtained in a moderate yield of 51% (product 2e, Scheme 2).

Tetrahydropyridine 1i was also converted successfully into the corresponding thiocarbamoyl fluoride in good yield of 69%. Interestingly benzylic amines were tolerated under the developed conditions furnishing the desired products in excellent yield (up to 90%), products 2k and 2l. Finally, we were able to to observe that bioactive amine compounds were also tolerated under these conditions. Indeed Notriptyline, Desipramine as well as Maprotiline derivatives were converted to their corresponding thiocarbamoyl derivatives in very good to excellent yield (up to 99%, products 2m, 2n & 2o). Reactions were performed with 1a (1 mmol, 1 equiv.), DAST (3 mmol, 3 equiv.), CS2 (1 mmol, 1 equiv.), base (0.5 mmol, 0.5 equiv.) and solvent (4 mL). Yields shown are those of isolated products; yields determined by 19F NMR spectroscopy with PhOCF3 as internal standard are shown in parentheses. [c] Starting amine derivative was used as HCl salt in the presence of 1.5 equiv. of DIPEA. [c] 1a (0.5 mmol, 1 equiv.), DAST (1 mmol, 2 equiv.), CS2 (1 mmol, 1 equiv.), base (0.5 mmol, 0.5 equiv.) and solvent (4 mL).

As demonstrated earlier by the group of Schoenebeck, we confirmed that trifluoromethyl amines can be obtained starting with thiocarbamoyl fluorides through sample reaction with silver fluoride in CH3CN at 50 °C for 4h (Scheme 3). The desired trifluromethylamine derivatives are obtained in general with excellent isolated yields through sample filtration of the crude. Scheme 4. Reaction scope, direct trifluoromethylation of amines : [a] Reactions were performed with 1a (1 mmol, 1 equiv.), CS2 (0.5 mmol, 1 equiv.), DAST (1.5 mmol, 3 equiv.), and DCM (2 mL).; yields determined by 19 F NMR spectroscopy with PhOCF3 as internal standard are shown in parentheses. [b] Starting amine derivative was used as HCl salt in the presence of 1 equiv. of NEt3.

It should be mentioned that the direct access to trifluoromethylated amines could be established in the absence of the base. Indeed, excellent yield (up to 99%, 3a, 3n & 3o, scheme 4) of the corresponding trifluoromethyl amines was obtained starting from amines (Scheme 4). Unfortunately, we observed the degradation of these compounds upon purification on column chromatography with silica but also by using alumina of Florisil ® . The main by product obtained is the corresponding carbamoyl fluoride.

Regarding the mechanism, we assume that amines reacts with carbon disulphide to form intermediate A. this later could be transformed to compounds B in the presence of DAST and the starting amine is regenerated in the presence of the base (DIPEA). Finally, the desired thiocarbamoyl fluoride is obtained through inter/intra molecular fluorine attack and diethylsulfuramidothious fluoride C (Scheme 5).

Conclusion

In conclusion we demonstrated herein that thiocarbamoyl fluoride could be easily obtained starting with amines through unprecedented fluorinative desulfurization of carbon disulfide with DAST. The desired products are obtained in moderate to very good isolated yields. The title product can easily be converted to trifluoromethyl amines.

Experimental Section

Synthesis of Piperidine-1-carbothioyl fluoride: To a flame dried Schlenk flask were added the amine (1.0 mmol, 1.0 equiv.), if a solid. The flask was evacuated and backfilled with N2 3 times, then dry DCM (4 mL) was added by syringe. Then, the amine (1.0 mmol, 1.0 equiv), if a liquid, and the CS2 (1.0 mmol, 1.0 equiv.) were successively added to the Schlenk flask equipped with a balloon of N2. Then DAST is added (3.0 mmol, 3 equiv.) dropwise the reaction has been observed to be exothermic. The reaction mixture was stirred at 25°C for 4h (conversion is checked by 19 F NMR with PhOCF3 as internal standard). The crude reaction mixture was diluted with DCM (8 mL) and water (4 mL). Following phase separation, the aqueous layer was extracted with DCM (3 x 5 mL). The resulting organic layer was washed with brine (1 x 2 mL), The organic layer was dried over anhydrous MgSO4 and evaporated under reduced pressure (rotary evaporator). The residue was purified by column chromatography to give the desired thiocarbamoyl fluoride product.

Scheme 1 .

 1 Scheme 1. State of the art: Synthesis of thiocarbamoyl fluoride using amines sstarting with amines

  [b] Determined by19 F NMR spectroscopy with PhOCF3 as an internal standard. [c] Reaction performed with 1 equiv. of DIPEA. [d] Reaction performed with 2 equiv. of DAST. DIPEA= N,N-Diisopropylethylamine. DMAP = 4

  Scheme 2. Reaction scope, Thiocarbamoyl Fluoride: [a] Reactions were performed with 1a (1 mmol, 1 equiv.), DAST (3 mmol, 3 equiv.), CS2 (1 mmol, 1 equiv.), base (0.5 mmol, 0.5 equiv.) and solvent (4 mL). Yields shown are those of isolated products; yields determined by 19F NMR spectroscopy with PhOCF3 as internal standard are shown in parentheses.[c] Starting amine derivative was used as HCl salt in the presence of 1.5 equiv. of DIPEA.[c] 1a (0.5 mmol, 1 equiv.), DAST (1 mmol, 2 equiv.), CS2 (1 mmol, 1 equiv.), base (0.5 mmol, 0.5 equiv.) and solvent (4 mL).

Scheme 3 .

 3 Scheme 3. Reaction scope, Trifluoromethylation of amines: [a] Reactions were performed with 1a (0.2 mmol, 1 equiv.), AgF (0.6 mmol, 3 equiv.) and MeCN (2 mL). Yields shown are those of isolated products; yields determined by 19 F NMR spectroscopy with PhOCF3 as internal standard are shown in parentheses.

  Scheme 5. Proposed mechanism

  Fluorine in Life Sciences: Pharmaceuticals, Medicinal Diagnostics, and Agrochemicals. (Eds.: G. Haufe, F. Leroux), Elsevier Science, London, United Kingdom, 2018, pp. 459-518.e) P. Kirsch in Modern Fluoroorganic Chemistry, Wiley-VCH, Wheineim, 2013; f) D. O'Hagan, Chem. Soc. Rev. 2008, 37, 308-319. [2] a) A. Leo, C. Hansch, D. Elkins, Chem. Rev. 1971, 71, 525-616; b) L. Bruno, W. Zhong, C. Guillaume, P. Vincent, F. C. Q., W. Neil, W. W. Alex, Angew. Chem. Int. Ed. 2016, 55, 674-678; c) W. K. Hagmann, J. Med. Chem. 2008, 51, 4359-4369. [3] E. Vitaku, D. T. Smith, J. T. Njardarson, J. Med. Chem. 2014, 57,

Table 1 .

 1 Reaction optimisation

	Entry	Solvent	Base	Yield (%) b
	1	PhMe	DIPEA	45
	2	DMSO	DIPEA	9
	3	DMF	DIPEA	42
	4	THF	DIPEA	45
	5	ACN	DIPEA	51
	6	DCM	DIPEA	71, 50 c
	7	DCM	_	2
	8	DCM	Et3N	62
	9	DCM	DBU	57
	10	DCM	DMAP	30
	11	DCM	2,6-Lutidine	6
	12	DCM	DIPEA	60 d

Acknowledgements

This work was supported by the CNRS, COLUMN, ICL (Institut de Chimie de Lyon).


Irène Amblard

Edmond Dupont

Isabel Alves

Julie Miralvès

Isabelle Queguiner

Alain Joliot
email: alain.joliot@college-de-france.fr

Bidirectional transfer of Engrailed homeoprotein across the plasma membrane requires PIP 2

Keywords: homeoprotein, unconventional secretion, membrane translocation, PIP2, internalization

Abstract

Homeoproteins are a class of transcription factors sharing the unexpected property of intercellular trafficking that confers to homeoproteins a paracrine mode of action. Homeoprotein paracrine action participates to the control of patterning processes, including axonal guidance, brain plasticity and boundary formation. Internalization and secretion, the two steps of intercellular transfer, rely on unconventional mechanisms but the cellular mechanisms at stake still need to be fully characterized.

Thanks to the design of new quantitative and sensitive assays dedicated to the study of homeoprotein transfer in HeLa cell culture, we demonstrate a core role of the phosphatidylinositol-4,5-biphosphate (PIP 2 ) together with cholesterol in the translocation of Engrailed2 (EN2) homeoprotein across the plasma membrane. Using drug and enzymatic treatments, we show that both secretion and internalization are regulated according to PIP 2 levels. The requirement of PIP 2 and cholesterol in EN2 trafficking correlates with their selective affinity for this protein in artificial bilayers, which is drastically decreased with a paracrine-deficient mutant of EN2. We propose that the bi-directional plasma membrane translocation events occurring during homeoprotein secretion and internalization respectively are parts of a common process.

Introduction

The plasma membrane is a pleiotropic structure that preserves cell homeostasis while allowing the exchange of various molecules, including large biological molecules endowed with signaling properties such as proteins. Along this view, the transfer across the plasma membrane constitutes the landmark that characterizes protein internalization and secretion, in opposite directions. In both cases, membrane fusion is the prevalent mechanism, endocytosis for entry and conventional secretion for exit but alternative mechanisms have been proposed.

Journal of Cell Science • Accepted manuscript

Intercellular trafficking of homeoproteins constitute a fascinating example of protein transfer across the plasma membrane. These proteins were originally identified thanks to their role in transcriptional regulation and are defined by the nature of their DNA-binding domain, the homeodomain [START_REF] Gehring | Homeodomain proteins[END_REF]. Some years ago, we have observed that homeoproteins present the unexpected property of transfer between cells [START_REF] Joliot | Identification of a signal sequence necessary for the unconventional secretion of Engrailed homeoprotein[END_REF]. Indeed, it was recently reported that most if not all homeoproteins are able to transfer [START_REF] Lee | Global Analysis of Intercellular Homeodomain Protein Transfer[END_REF], also emphasizing the pivotal role of the conserved homeodomain in this process [START_REF] Sagan | Homeoproteins and homeoprotein-derived peptides: going in and out[END_REF]. Since the original ex vivo observations, the physiological significance of homeoprotein intercellular trafficking was revealed in several contexts (reviewed in Di Nardo et al., 2018), as it provides to homeoproteins a paracrine mode of action that superimposes on their transcriptional activities. In particular, the paracrine action of Engrailed subclass has been reported in diverse species, including flies, fishes and mice. Targeted inhibition of Engrailed paracrine action, thanks to the use of neutralizing extracellular antibodies, induces local and specific patterning defects in Drosophila [START_REF] Layalle | Engrailed homeoprotein acts as a signaling molecule in the developing fly[END_REF]and zebrafish [START_REF] Rampon | Control of brain patterning by Engrailed paracrine transfer: a new function of the Pbx interaction domain[END_REF], or axonal guidance defects [START_REF] Wizenmann | Extracellular Engrailed participates in the topographic guidance of retinal axons in vivo[END_REF], which in the latter case involved the regulation of local translation by the transferred protein [START_REF] Stettler | Engrailed homeoprotein recruits the adenosine A1 receptor to potentiate ephrin A5 function in retinal growth cones[END_REF].

Understanding the mechanism of homeoprotein intercellular trafficking remains challenging as both secretion and internalization rely on unconventional mechanisms. Homeoproteins are devoid of secretion signal and predominantly reside in the nucleus. However, homeoproteins such as the chick Engrailed2 homeoprotein (EN2) used as a model homeoprotein, shuttles between the nucleus and the cytosol and around 10 % of the intracellular protein pool is secreted in the soluble fraction of the conditioned medium, thus not entrapped within extracellular vesicles [START_REF] Maizel | A short region of its homeodomain is necessary for engrailed nuclear export and secretion[END_REF]. In the cytosol, EN2 associates with the membrane fractions enriched in cholesterol and glycosphingolipids [START_REF] Joliot | Association of Engrailed homeoproteins with vesicles presenting caveolae-like properties[END_REF]. In addition to secretion, homeoproteins are internalized when added in the extracellular medium and accumulate in cytosolic and nuclear compartments, even when endocytosis is Journal of Cell Science • Accepted manuscript inhibited [START_REF] Joliot | alpha-2,8-Polysialic Journal of Cell Science • Accepted manuscript acid is the neuronal surface receptor of antennapedia homeobox peptide[END_REF]. The aim of our study was to identify at the cellular and molecular levels the mechanism of homeoprotein transfer across the plasma membrane, which occurs in opposite directions during secretion and internalization.

Conventional protein secretion is initiated by the recognition of a short N-terminal secretion sequence through a co-translational process, which drives the translocation of the nascent polypeptide chain into the Endoplasmic Reticulum. In the last decade, unconventional protein secretion (UPS)

pathways have been identified, involving distinct mechanisms [START_REF] Nickel | Unconventional protein secretion: Diversity and consensus[END_REF][START_REF] Zhang | Cell biology. Unconventional secretion, unconventional solutions[END_REF]. Four distinct UPS types are now defined [START_REF] Rabouille | Pathways of Unconventional Protein Secretion[END_REF], which differ on the nature (soluble, transmembrane…) and the mode of secretion (plasma membrane translocation, vesicular-based…) of the secreted substrate. Although different UPS can drive the secretion of leaderless cytosolic/nuclear substrate in a free state in the medium, only UPS Type I seems to be constitutive and independent of any specific stimuli, as observed for homeoprotein secretion. UPS Type I refers to soluble proteins that are secreted through direct translocation across the plasma membrane and among them, FGF2 [START_REF] Schäfer | Unconventional secretion of fibroblast growth factor 2 is mediated by direct translocation across the plasma membrane of mammalian cells[END_REF] and HIV Tat [START_REF] Rayne | Phosphatidylinositol-(4,5)-bisphosphate enables efficient secretion of HIV-1 Tat by infected T-cells[END_REF] are prototypical examples. FGF2 is synthesized as a cytosolic protein but needs to be secreted to activate its extracellular receptors. It is devoid of secretion signal but creates pores within the plasma membrane, driving its secretion [START_REF] Müller | Formation of Disulfide Bridges Drives Oligomerization, Membrane Pore Formation and Translocation of Fibroblast Growth Factor 2 to Cell Surfaces[END_REF]. A critical step in FGF2 secretion is its accumulation at the plasma membrane, which requires its specific interaction with PIP 2 [START_REF] Temmerman | A direct role for phosphatidylinositol-4,5-bisphosphate in unconventional secretion of fibroblast growth factor 2[END_REF], a quantitatively minor but essential component of the plasma membrane inner leaflet [START_REF] Hammond | PI4P and PI(4,5)P2 Are Essential But Independent Lipid Determinants of Membrane Identity[END_REF]. A similar requirement for PIP 2 was demonstrated for HIV Tat secretion [START_REF] Rayne | Phosphatidylinositol-(4,5)-bisphosphate enables efficient secretion of HIV-1 Tat by infected T-cells[END_REF]. Although functionally and structurally unrelated, Tat and FGF2 proteins share a high content in basic amino acids (pI of 9.8 and 9.5 respectively).

Protein internalization mostly relies on endocytosis, a vesicular-based pathway initiated by invagination from the plasma membrane [START_REF] Doherty | Mechanisms of endocytosis[END_REF]. Consequently, internalized Journal of Cell Science • Accepted manuscript proteins remain entrapped within the vesicles and never reach the cytosol. Among exceptions, some bacterial toxins are able to translocate through biological membranes and accumulate in the cytosol [START_REF] Williams | Intracellular trafficking of bacterial toxins[END_REF]. We also learned from the study of Cell Penetrating Peptides that extracellularly loaded protein or polypeptides could have access to the cytosol, although the efficacy and the mechanism(s) involved remain debated [START_REF] Kauffman | Mechanism Matters: A Taxonomy of Cell Penetrating Peptides[END_REF].

In HeLa cells, we have focused our study on the events taking place at the plasma membrane, the actual barrier that separates the intra and extracellular spaces. We demonstrate that, similarly to FGF2

and Tat, EN2 translocates across the plasma membrane during secretion thanks to its interaction with PIP 2 . Most importantly, reverse translocation during EN2 internalization that also occurs at the plasma membrane exhibits a similar requirement for PIP 2 , thus revealing striking similarities in their mechanisms.

Results

PIP2 is required for EN2 secretion

Homeoprotein intercellular trafficking have been best studied with EN2, a polypeptide of 28 kD with a pI of 9.2, it was thus kept for this study. To accurately address the mechanism of protein secretion, we generated stable cell clones in the engineered FlpIn-TREX HeLa line [START_REF] Tighe | Mps1 kinase activity restrains anaphase during an unperturbed mitosis and targets Mad2 to kinetochores[END_REF] that permits the targeted insertion of the transgene at an invariant site and its inducible expression upon doxycycline addition. Once secreted by EN2 cell clone, EN2 accumulates at the cell surface and can be detected by flow cytometry [START_REF] Layalle | Engrailed homeoprotein acts as a signaling molecule in the developing fly[END_REF]. We verified that EN2 secretion was insensitive to the classical inhibitor of conventional secretion Brefeldin A but became sensitive upon EN2 fusion to an ectopic secretion signal sequence (ssEN2) (Fig. 1A). FGF2 secretion was also insensitive to Brefeldin

A to the same extent as EN2 (Fig. S1A). Once secreted, both proteins interact with cell surface carbohydrates and were released from the cell surface by heparin wash treatment (Fig. 1B).

Journal of Cell Science • Accepted manuscript

The PIP 2 -interacting drug neomycin efficiently decreases FGF2 secretion [START_REF] Temmerman | A direct role for phosphatidylinositol-4,5-bisphosphate in unconventional secretion of fibroblast growth factor 2[END_REF]. We asked whether it also impaired EN2 secretion. Overnight neomycin treatment inhibited EN2 and FGF2 secretion to similar extent (Fig. 1C), indicating the involvement of PIP 2 in EN2 secretion.

We then evaluated whether EN2 directly interacts with PIP 2 as demonstrated for HIV Tat [START_REF] Rayne | Phosphatidylinositol-(4,5)-bisphosphate enables efficient secretion of HIV-1 Tat by infected T-cells[END_REF] and FGF2 [START_REF] Temmerman | A direct role for phosphatidylinositol-4,5-bisphosphate in unconventional secretion of fibroblast growth factor 2[END_REF]. The interaction of EN2 recombinant protein with lipid bilayers of controlled composition was analyzed by Plasmon-Waveguide Resonance (PWR) [START_REF] Harté | Probing the kinetics of lipid membrane formation and the interaction of a nontoxic and a toxic amyloid with plasmon waveguide resonance[END_REF], a spectroscopic technique allowing to directly measure the interaction and affinity of molecules with membrane lipids organized in the form of a planar lipid membrane. Significant interaction was observed (Fig. 1D) with pure phosphatidylcholine (PC) bilayer (KD:6.3 nM) but incorporation of PIP 2 (10%) dramatically lowered the KD to 75 pM, compared to the mild effect elicited by addition of phosphatidylserine (PS) (KD:2.8 nM) (Fig. 1E). As expected, the specific interaction between FGF2 and PIP 2 was confirmed using the same experimental set-up, although with slightly lower affinity compared to EN2 (Fig. S1B).

We have previously shown that a pool of EN2 associates with the membrane fraction, both in vivo and ex vivo [START_REF] Joliot | Association of Engrailed homeoproteins with vesicles presenting caveolae-like properties[END_REF]. We thus asked whether this association was depending on PIP 2 interaction. The membrane fraction of HeLa EN2 or ssEN2 cells clones was incubated with neomycin and the protein released in the medium was separated from membranes by ultracentrifugation. More than 50 % of the EN2 protein was retrieved in the supernatant following neomycin treatment (Fig. 1F), highlighting the involvement of PIP 2 in this association. The distribution of FGF2 (Fig. S1C), but neither the ones of ssEN2 nor the endoplasmic reticulum marker calregulin distributions were affected by this treatment, attesting vesicle integrity and consequently, that the EN2 pool sensitive to neomycin was not entrapped within vesicles.

Journal of Cell Science • Accepted manuscript

A new assay for the analysis of unconventional secretion

As PIP 2 mostly resides in the inner leaflet of the plasma membrane, we decided to focus on this compartment. Flow cytometry, although quantitative at a single cell level, is poorly informative on the mechanism of secretion. To address this question, we took advantage of the Rush strategy developed by [START_REF] Boncompain | Synchronization of secretory protein traffic in populations of cells[END_REF], an inducible secretion assay. A secreted substrate fused to a streptavidin binding peptide (SBP) [START_REF] Wilson | The use of mRNA display to select highaffinity protein-binding peptides[END_REF] is sequestered by the co-expression of a "hook" protein fused to core streptavidin (Strep). Biotin addition induces the synchronized release of the substrate from the hook and its eventual secretion. Importantly, the subcellular localization of the hook determines the site of release along the secretion pathway of the substrate upon biotin addition.

We chose a hook that localizes at the inner side of the plasma membrane, by Strep fusion either with the acylation sequence (aa1-10) of the tyrosine kinase LCK or with the cytosolic tail of a transmembrane protein (TM1). Flow cytometry was not sensitive enough to monitor the secretion burst induced by biotin addition (not shown). We thus implemented our assay with a highly sensitive detection tool (HiBit assay, Promega), based on light production upon spontaneous complementation of two fragments of an engineered Nanoluciferase [START_REF] Dixon | NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells[END_REF].

To monitor protein secretion, the small 11 aa fragment (HiBiT) was fused to the secreted substrate and the large one (LgBiT) was expressed on the extracellular side plasma membrane upon fusion to the Extracellular domain of the TM1Strep trans-membrane protein. By combining the two strategies (Fig. 2A), secretion of the pool of EN2 sequestered at the cytosolic side of the plasma membrane was measured by the specific increase in light production induced by biotin addition compared to control conditions (without biotin). To minimize non-specific release, stable cell clones were generated for all secreted substrates in FlpIn-TREX HeLa cells. LCK-StrepCherry (hook) was transiently expressed in SBP-Myc-EN2-Hibit (named EN2* for convenience) cell clone. By immunofluorescence we verified on permeabilized cells the retention of EN2* at the plasma membrane Journal of Cell Science • Accepted manuscript in transfected cells (white arrows) and its release by biotin addition (Fig. 2B). Concomitantly, EN2* cell surface staining (visualized in non-permeabilized cells) was increased by biotin treatment (Fig. 2C). A similar behavior was observed with the FGF2* cell clone (Fig. S2).

The secretion of three different proteins was then analyzed by luminescence measurements, GFP* and FGF2* being used respectively as negative and positive controls. The sensor/hook (LgBitTM1Strep) was expressed in each clone and following biotin addition together with the luciferase substrate furimazin, the kinetics of secretion was monitored for one hour. Cells were then lysed and the intracellular content for each protein was measured by luminescence for normalization.

Subtracting biotin-negative from biotin-positive measurement allowed us to remove the contribution of non-sequestered proteins (e.g. expressed in non-transfected cells). Secretion-induced luminescence started right after biotin addition with FGF2* and EN2*clones, supporting a direct translocation of the intracellular hooked proteins. In the same conditions, no GFP* secretion could be observed (Fig. 2D), thus validating our secretion assay which was named TransRush assay (Rush targeting translocation).

We also noticed a higher efficiency of FGF2* secretion compared to EN2*. An additional benefit of our assay is to allow the measurement of secretion on a short time scale, more suitable to analyze the impact of pharmacological treatments while limiting indirect or toxic effects. We first tested the effect of short-time neomycin treatment (added 30 min before biotin addition) in the TransRush assay. One hour after biotin addition, both EN2* and FGF2* secretions were drastically reduced by neomycin treatment to similar extents, further supporting a direct action of the drug at the site of the plasma membrane (Fig. 2E).

PIP2 levels modulate EN2 secretion

We next asked whether EN2 secretion was sensitive to PIP 2 levels, using an enzymatic strategy.

OCRL is a 5-phosphatase that dephosphorylates PIP 2 into PI4P and its ectopic expression drastically Journal of Cell Science • Accepted manuscript reduced PIP 2 levels, as revealed with anti-PIP 2 antibodies (Fig. 3A). The Cherry-OCRL coding sequence was inserted in a bi-expressor plasmid together with LgBiTM1Strep sequence to maximize co-expression. Cherry-OCRL protein, but not Cherry co-expression, reduced EN2* and FGF2* secretion after biotin addition (Fig. 3B). Conversely, we expressed the PI4P5K1 enzyme to increase PIP 2 levels at the plasma membrane. As shown in Fig. 3C, cells expressing Cherry-PI4P5K showed a consistent concomitant increase of PIP 2 levels and EN2* or FGF2* secretion (Fig. 3D). The effect of the enzymatic treatments on EN2 trafficking suggested that PI4P could not substitute for PIP 2 in the process. We thus asked whether the nature of the phospholipid could modulate EN2 interaction.

Incorporation of PI4P into PC bilayer increased the affinity for EN2 but to a significantly lower extend (3 folds) compared to PIP 2 (Fig. 3E). In comparison, the affinity for FGF2 was drastically reduced as previously reported [START_REF] Temmerman | A direct role for phosphatidylinositol-4,5-bisphosphate in unconventional secretion of fibroblast growth factor 2[END_REF], suggesting different modes of interaction for the two proteins. Taken together, these results demonstrate the pivotal and highly specific role of PIP 2 levels in EN2 unconventional secretion.

PIP2 modulates EN2 internalization

Homeoproteins are not only secreted but are also internalized and reach cytosolic and nuclear compartments. After a one-hour incubation in the culture medium, FITC-labelled recombinant EN2 (F-EN2) was mainly detected at the cell surface (Fig. 4A, Ctrl). Addition in the medium of Trypan blue (TB), an efficient quencher of extracellular fluorescence [START_REF] Vranic | Deciphering the mechanisms of cellular uptake of engineered nanoparticles by accurate evaluation of internalization using imaging flow cytometry[END_REF], revealed the specific accumulation of intracellular F-EN2 (Fig. 4A, +TB), although impacting signal detection (Fig. S3A).

Both punctuate and diffuse distributions were observed, the latter highlighting the cytosolic and nuclear accumulation of the protein. As PIP 2 are required for EN2 secretion, we asked whether they would also be involved in the reverse process of internalization. In cells treated with neomycin, F-EN2 intracellular accumulation was drastically reduced (Fig. 4B). Importantly, the extracellular association of F-EN2 with the plasma membrane, detected before trypan blue addition, was not affected by Journal of Cell Science • Accepted manuscript neomycin treatment (Fig. S3), supporting an intracellular action of the drug. Next, we tested the effect of PIP 2 -modulating enzymatic treatments. Hela cells were transfected with Cherry fusion constructs and F-EN2 internalization was compared between transfected (Cherry positive) and non-transfected cells. Cherry-OCRL expression significantly impaired F-EN2 internalization (Fig. 4C). Using the same protocol, Cherry-PI5P4K expression increased F-EN2 internalization (Fig. 4D).

A distinctive feature of plasma membrane translocation-driven processes is the direct access of the internalized protein to the cytosol. We have thus adapted the HiBiT assay to selectively target this mode of internalization. We first added a HiBiT tag to the recombinant EN2 protein (Hb-EN2) and secondly generated a reporter cell clone that expressed the LgBiT protein in the cytosol. Light production only occurs if the Hb-EN2 protein added in the medium reaches the cytosol and can be measured in live cells thanks to the quick diffusion of the luciferase substrate into live cells [START_REF] Song | A convenient luminescence assay of ferroportin internalization to study its interaction with hepcidin[END_REF]. One hour after its addition in the extracellular medium, Hb-EN2 efficiently stimulated light production together with the cytosolic reporter (Fig. 4E). As expected, neither Hb-GST nor Hb-FGF2 could activate the reporter. Both neomycin treatment (Fig. 4E) and OCRL ectopic expression (Fig. 4F) lowered the cytosolic accumulation of Hb-EN2, whereas PI4P5K ectopic expression had an opposite enhancing effect (Fig. 4G).

Journal of Cell Science • Accepted manuscript

Selective role of raft associated PIP2 in EN2 trafficking

In a previous study, we have shown that EN2 selectively associates with cholesterol-enriched membranes [START_REF] Joliot | Association of Engrailed homeoproteins with vesicles presenting caveolae-like properties[END_REF]. Using the PWR assay, we demonstrated that cholesterol incorporation in the membrane increased 10 fold EN2 affinity for a PIP 2 containing lipid bilayer (Fig. 5A). We then asked whether cholesterol levels directly modulate EN2 intercellular trafficking. Extracting cholesterol from the plasma membrane by methyl-ß-Cyclodextrin (MBCB) treatment reduced F-EN2 internalization (Fig. S4A) and EN2* secretion (Fig. S4B). Due to the reported side-effects of MBCB, alternative strategies were used to deplete plasma membrane cholesterol. Overnight treatment of HeLa cells with U18886A (U18), an inhibitor of cholesterol transport and synthesis [START_REF] Cenedella | Cholesterol Synthesis Inhibitor U18666A and the Role of Sterol Metabolism and Trafficking in Numerous Pathophysiological Processes[END_REF], provoked a drastic depletion of plasma membrane cholesterol, visualized by the removal of the fluorescent D4H cholesterol sensor (Maekawa and Fairn, 2015) from the plasma membrane (Fig. S4C), and strongly impaired both Hb-EN2 internalization (Fig. 5B) and EN2* secretion (Fig. 5C). In conclusion, the two membrane components, PIP 2 and cholesterol, are essential to EN2 bi-directional trafficking across the plasma membrane.

PIP 2 are known to associate with rafts, the cholesterol enriched membrane domains [START_REF] Pike | Localization and turnover of phosphatidylinositol 4,5-bisphosphate in caveolin-enriched membrane domains[END_REF]. We verified that PIP 2 co-purified with the raft fraction isolated by density gradient separation of detergent-treated membranes (Fig. S5A). We next asked whether this PIP 2 pool was specifically involved in EN2 trafficking. To selectively deplete raft-associated PIP 2 , we used an assay developed by Varnai et al. [START_REF] Varnai | Rapidly inducible changes in phosphatidylinositol 4,5-bisphosphate levels influence multiple regulatory functions of the lipid in intact living cells[END_REF]. It consists in the selective and conditional membrane targeting of a cytosolic TypeIV-5Phosphatase fragment (5Dom) that has retained its catalytic activity thanks to the ability of LCK and SRC acylation motifs to target proteins towards raft and non-raft domains respectively (Fig. S5B and [START_REF] Varnai | Rapidly inducible changes in phosphatidylinositol 4,5-bisphosphate levels influence multiple regulatory functions of the lipid in intact living cells[END_REF]. Rapamycin is known to promote dimerization of two protein modules, FRB (FKBP binding domain of mTOR) and FKBP [START_REF] Stockwell | Probing the role of homomeric and heteromeric receptor interactions in TGF-β signaling using small molecule dimerizers[END_REF]. A 5Dom-FRB-Cherry fusion was co-expressed with a FKBP-CFP protein fused to either LCK Journal of Cell Science • Accepted manuscript or SRC acylation motif and rapamycin addition induced its relocation according to the localization of the co-expressed acylation motif (Fig. 5D). Both LCK and SRC motifs promoted plasma membrane targeting of 5Dom and PIP 2 level decrease (Fig. 5D), but only raft-targeted 5Dom (with LCK motif) significantly impaired F-EN2 internalization (Fig. 5E). By contrast, non-raft PIP 2 depletion induced a small increase in EN2 internalization (Fig. 5F). Such depletion was previously shown to increase the raft-associated PIP 2 pool [START_REF] Johnson | Compartmentalization of phosphatidylinositol 4,5-bisphosphate signaling evidenced using targeted phosphatases[END_REF]. We concluded that cholesterol and the raft associated PIP 2 pool cooperate to regulate EN2 intercellular trafficking.

Competing for PIP 2 interaction in live cells

Several protein domains are known to selectively interact with PIP 2 and therefore, they have been used to probe PIP 2 in live cells. The most widely used PIP2 biding-domain is the Pleckstrin homology domain of Phospholipase C ∂1 (PHPLC) [START_REF] Várnai | Visualization of phosphoinositides that bind pleckstrin homology domains: calcium-and agonist-induced dynamic changes and relationship to myo-[3H]inositol-labeled phosphoinositide pools[END_REF]. HIV Tat, whose secretion also depends on PIP 2 , efficiently delocalizes a GFP-PHPLC sensor from the plasma membrane [START_REF] Rayne | Phosphatidylinositol-(4,5)-bisphosphate enables efficient secretion of HIV-1 Tat by infected T-cells[END_REF]. We thus decided to follow the behavior of GFP-PHPLC during EN2 trafficking by measuring the plasma membrane/cytosol index of GFP-PHPLC (see method section). EN2 uptake quickly induced a delocalization of GFP-PHPLC probe toward the cytosol (Fig. 6A), but not that of a Cherry protein fused to the LCK acylation sequence (LCK-Cherry) which remained at the plasma membrane (Fig. S6A). Redistribution started only few minutes after EN2 addition and increased with time (Fig. 6B) We then asked if EN2 secretion also promotes PHPLC delocalization. GFP-PHPLC was expressed in the TransRush assay. Promoting EN2* secretion by biotin addition induced a delocalization of GFP-PHPLC towards the cytosol similarly to EN2 internalization (Fig. 6C). The distribution of LCK-Cherry remained unmodified in the same conditions (Fig. S6B). These results further confirm that PIP 2 is an essential relay in EN2 translocation across the plasma membrane in either direction. Using the same experimental protocol, FGF2* secretion also delocalized GFP-PHPLC Journal of Cell Science • Accepted manuscript upon biotin addition (Fig. 6D) but had little effect when applied in the extracellular medium, in agreement with its inability to enter cells by direct translocation (Fig. 4E).

A paracrine-deficient mutant of Engrailed has a reduced affinity for PIP2

Intercellular transfer confers to homeoproteins a paracrine mode of action which contribute to homeoprotein physiological functions [START_REF] Di Nardo | The Physiology of Homeoprotein Transduction[END_REF]. In zebrafish embryo, we have shown recently that the paracrine activity of ectopically expressed EN2 protein induces eye defects [START_REF] Rampon | Control of brain patterning by Engrailed paracrine transfer: a new function of the Pbx interaction domain[END_REF]. We have characterized a paracrine-deficient mutant of EN2 (EN2 ww>KK ), unable to induce such phenotype and strongly impaired in its intercellular trafficking. We first confirmed the altered behavior of the EN2 ww>KK mutant with our assays. Both internalization (Fig. 7A) and secretion (Fig. 7B) were significantly impaired. Additionally, the EN2 ww>KK mutant was unable to delocalize PHPLC in either situation (Fig. 7C,D). We thus asked whether the interaction of EN2 ww>KK with PIP 2 was concomitantly affected by the mutation. Compared to the wild type EN2 protein, the affinity of EN2 ww>KK mutant for a pure PC-containing membrane (KD:20 nM) was not significantly reduced but a high impact was observed with membrane containing PIP 2 with an almost 30 fold decrease in affinity (KD:2.5 nM) compared to the wild-type protein(Fig. 7E). These results further support the pivotal role of PIP 2 interaction not only in EN2 trafficking but also in the paracrine activity of the protein.

Journal of Cell Science • Accepted manuscript

Discussion

In the present study, we have investigated the molecular and cellular mechanisms of homeoprotein intercellular transfer, focusing on the mechanism of plasma membrane crossing. The two events of crossing, occurring during secretion and internalization respectively, have been analyzed

separately in this study. Combining Rush and HibiT assays in a new TransRush assay, we demonstrate that a pool of EN2 that has reached the inner side of the plasma membrane is secreted by direct translocation towards the extracellular medium. We further demonstrate the specific interaction of EN2 with PIP 2 and its pivotal role in secretion. First, the extent of secretion strictly correlates with PIP 2 levels, modulated either by enzymatic treatments or drugs. Secondly, EN2 competes for PIP 2 binding with PHPLC∂ when secreted. EN2 is a new example of PIP 2 -dependent secreted substrate along with FGF2 and HIV Tat, both synthesized in the cytosol and highly basic. EN2 secretion would thus relies on a UPS Type I pathway, although the mechanism of translocation across the acyl part of the bilayer remains to be clarified. Using a dedicated assay that selectively targets the cytosolic accumulation of the internalized protein, we show here that this mode of internalization shares with secretion a very same requirement for PIP 2 and cholesterol, strongly suggesting that these two events are part of a same process. Both involve the translocation of the protein across the plasma membrane but in two opposite directions. EN2 secretion and internalization would thus result in a dynamic equilibrium between the two sides of the plasma membrane, which depends, at least in part, on the local micro-environment at the two membrane interfaces.

Soluble proteins that translocate across the plasma membrane encounter distinct environments, the hydrophilic cytosol and the hydrophobic core of the bilayer. At the plasma membrane, FGF2 and Tat proteins assemble into oligomers which enable the formation of pores [START_REF] Müller | Formation of Disulfide Bridges Drives Oligomerization, Membrane Pore Formation and Translocation of Fibroblast Growth Factor 2 to Cell Surfaces[END_REF][START_REF] Zeitler | HIV-Tat Protein Forms Phosphoinositide-dependent Membrane Pores Implicated in Unconventional Protein Secretion[END_REF]. We have no evidence of multimer formation in the case of EN2 which in addition, possesses only one cysteine residue. Importantly, in the presence of negatively charged lipid bicelles, Journal of Cell Science • Accepted manuscript the conformation of EN2 protein analyzed by NMR is shifted in a way that promotes the exposure of the hydrophobic core of the homeodomain and its insertion into the acyl part of the bilayer [START_REF] Carlier | Investigation of homeodomain membrane translocation properties: insights from the structure determination of engrailed-2 homeodomain in aqueous and membrane-mimetic environments[END_REF]. Indeed, bilayer interaction with EN2 not only relies on electrostatic interactions because it is increased by cholesterol addition and reduced with the EN2 ww>KK mutant that has two less aromatic residues and two more basic ones. The bilayer organization might also be required as PHPLC, which interact almost exclusively with the polar head of PIP 2 [START_REF] Watt | Subcellular localization of phosphatidylinositol 4,5-bisphosphate using the pleckstrin homology domain of phospholipase C delta1[END_REF], efficiently binds to PIP 2 beads whereas neither EN2 (or FGF2) could do so (Fig. S7). The interplay between electrostatic and hydrophobic interactions with PIP 2 , shown to promotes oligomer formation in the case of FGF2 [START_REF] Steringer | Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-dependent oligomerization of fibroblast growth factor 2 (FGF2) triggers the formation of a lipidic membrane pore implicated in unconventional secretion[END_REF], might act as a hub for EN2 to exchange between the cytosol and the bilayer. At this stage, we cannot ruled-out the participation of additional components in the translocation process.

For instance, ABC transporters (UPS Type II) mediate the secretion of acylated peptides in yeast [START_REF] Kuchler | The a-factor transporter (STE6 gene product) and cell polarity in the yeast Saccharomyces cerevisiae[END_REF] and recently, Bidirectional translocation of EN2 across the plasma membrane occurs despite the asymmetrical composition of this structure. PIP 2 mostly resides in the cytosolic leaflet of the membrane but its translocation to the exofacial leaflet has been reported through an ABCA1-dependent mechanism [START_REF] Gulshan | PI(4,5)P2 Is Translocated by ABCA1 to the Cell Surface Where It Mediates Apolipoprotein A1 Binding and Nascent HDL Assembly[END_REF] and might participate to EN2 trafficking. Interestingly, unconventional secretion and translocation of annexin 2 and 5 depend on the phospholipid scramblase TMEM 16F, even in basal conditions [START_REF] Stewart | Transbilayer phospholipid movement facilitates the translocation of annexin across membranes[END_REF]. From the extracellular side, mirroring the "hub" function of PIP 2 inside the cell, other components might initiate EN2 translocation and in particular, glycosaminoglycans (GAGs). First, they are essential determinants of homeoprotein uptake in vivo [START_REF] Beurdeley | Otx2 binding to perineuronal nets persistently regulates plasticity in the mature visual cortex[END_REF], and of homeoprotein-derived peptide uptake ex vivo [START_REF] Jiao | Translocation and endocytosis for cell-penetrating peptide internalization[END_REF]. They are also indispensable to FGF2 secretion [START_REF] Zehe | Cell-surface heparan sulfate proteoglycans are essential components of the unconventional export machinery of FGF-2[END_REF]. Due to their flexibility, these long chains of negatively charged sugars might not only concentrate EN2 at the cell surface, playing the role of Journal of Cell Science • Accepted manuscript recruiting platforms, but also bring the protein in close proximity of the exofacial leaflet and possibly favor its insertion in the bilayer.

Although critical to understand the whole process, plasma membrane translocation of EN2 constitutes only one step along EN2 intercellular transfer. Within the cell, EN2 has to be addressed to the plasma membrane in order to be secreted and conversly, internalized EN2 should be able to escape the plasma membrane to exert its intracellular functions. For the first point, we [START_REF] Maizel | A short region of its homeodomain is necessary for engrailed nuclear export and secretion[END_REF] and others [START_REF] Zhou | Nkx3.1 functions as para-transcription factor to regulate gene expression and cell proliferation in noncell autonomous manner[END_REF] have shown in previous works that secreted homeoprotein originates from the nucleus because secretion requires the nuclear export of the protein. Intriguingly, nuclear accumulation and secretion are strictly correlated in an ex-vivo model of intercellular transfer [START_REF] Tassetto | Plant and animal homeodomains use convergent mechanisms for intercellular transfer[END_REF], suggesting an instructive role of the nucleus in the process. A similar correlation was reported for Galectin-3 [START_REF] Gong | The NH2 terminus of galectin-3 governs cellular compartmentalization and functions in cancer cells[END_REF]. One can note that polyphosphoinositides, including PIP 2 , are also found within the nucleoplasm and their interaction with nuclear proteins can regulate nucleocytoplasmic translocation events [START_REF] Jacobsen | Polyphosphoinositides in the nucleus: Roadmap of their effectors and mechanisms of interaction[END_REF]. Once in the cytosol, EN2 addressing to the plasma membrane might involves specific association with intracellular vesicles and/or posttranslational modifications as reported for EN2 [START_REF] Maizel | Engrailed homeoprotein secretion is a regulated process[END_REF].

How internalized EN2 could spread into the cytosol despite its strong interaction with PIP2. At this stage, we can only speculate on the mechanism at stake, but two hypotheses are favored. The first hypothesis involves the participation of cytosolic partners that would compete with PIP2 for EN2

interaction. The second one is based on the study of lipid transfer proteins that convey various lipids between two membrane compartments. Binding/release cycles rely on intramolecular conformational switches that regulate the accessibility of the phospholipid binding motif within the protein [START_REF] Wong | Advances on the Transfer of Lipids by Lipid Transfer Proteins[END_REF].

The paracrine mode of action of homeoprotein is the functional outcome of their trafficking property. First demonstrated with selected homeoproteins [START_REF] Di Nardo | The Physiology of Homeoprotein Transduction[END_REF], a recent study now Journal of Cell Science • Accepted manuscript

shows that intercellular trafficking is common to most if not all homeoproteins [START_REF] Lee | Global Analysis of Intercellular Homeodomain Protein Transfer[END_REF] and consequently, that paracrine functions might be a shared property of the entire protein family. The reduced affinity of the paracrine-deficient mutant EN2 ww>KK for PIP 2 might reveal a new physiological role of PIP 2 as a generic regulator of homeoprotein trafficking. Because PIP 2 levels regulate multiple cellular processes, from cell morphology and cytoskeleton dynamics to second messenger production, their specific requirement in UPS Type I might create a bridge between this pathway and the physiological state of the cell.

Methods

Cell culture

Cell culture experiments were performed on HeLa cells grown in DMEM supplemented with 10% fetal bovine serum. Transient transfections were performed with Lipofectamine 2000 (Life Technologies) according to the manufacturer's instructions. Cells were cultured for an additional 24 h before being processed for analysis. Stable clones were generated for each protein used in the secretion assays. All cell clones were produced in a modified HeLa cell line (HeLa FlpIn-TREX) kindly provided by S. Taylor [START_REF] Tighe | Mps1 kinase activity restrains anaphase during an unperturbed mitosis and targets Mad2 to kinetochores[END_REF] according to the instructions of the FlpIn-TREX Core Kit (Invitrogen) and are listed in Table S3. Briefly, the cell line allows the targeted insertion of the transgene by co-expression of the FlpIn recombinase as well as its inducible expression by doxycycline when using appropriate promoter thanks to the constitutive expression of the tetracycline repressor.

Cells were checked routinely for the absence of mycoplasma contamination (MycoAlert, Lonza).

Chemicals and Pharmacological treatments

All drugs and Chemicals were purchased at Sigma unless specified. To inhibit conventional protein secretion, protein expression was induced for 4h with doxycycline before addition of 10 µg/ml Journal of Cell Science • Accepted manuscript

Brefeldin A for 2 additional hours. Wash-out of cell-surface associated proteins was performed by incubation with 10 mg/ml Heparin for 5 min followed by 2 washes with PBS. To reduce PIP 2 accessibility, cells were pre-treated with 10 mM neomycin. For cholesterol extraction experiments, cells were treated for 16h with 2 µg/ml U1866A in delipidated FCS or alternatively, for 30 minutes with 10 mM methyl-B-cyclodextrin. Membrane targeting of 5Dom using FKBP/FRB conditional heterodimerization was performed by treatment with 100 nM rapamycin for 30 minutes.

Immunocytochemistry

Cells grown on coverslips were fixed with paraformaldehyde (4%, 10 min, room temperature) in PBS, permeabilized or not with Triton X-100 (0.3%, 5 min, room temperature) and saturated with PBS containing 10% FCS before incubation with primary antibodies (1 h, room temperature) and Alexalabeled secondary antibodies (Thermo 1:500, 1 h, room temperature). Signal levels were adjusted independently of permeabilized and unpermeabilized conditions because of significant differences in signal intensity (higher under permeabilized conditions).

EN2* and FGF2* were revealed with a polyclonal anti-myc antibody (Sigma C3956, 5 µg/ml) followed by secondary antibody staining (AlexaFluor488 donkey anti rabbit IgG 1/400).

PIP2 was performed according to (Hammond et al., 2009). Cells grown on coverslips were washed in PBS and fixed (PBS, 4% PFA, 0.25% glutaraldehyde) for 15 minutes at RT and then on ice for 1h. All subsequent steps were performed on ice. After 3 washes (PBS, NH 4 Cl 50mM, 15 minutes each), saturation was proceeded in Buffer A (20mM Pipes, 137mM sodium chloride, 2.8mM potassium chloride. pH 6.8) supplemented with 0.5% Saponin and 5% FCS for 2h. Primary antibody (anti-PIP2, 2C11, Echelon, 5 µg/ml in Buffer A, 0.1% Saponin, 2% FCS) was added for 2h or overnight. After three washes (Buffer A, 0.1% Saponin), secondary antibody (AlexaFluor488 donkey anti mouse IgM 1/400, Life Technologies in Buffer A, 0.1% Saponin 2% FCS) was performed. Coadded for 1h.

Journal of Cell Science • Accepted manuscript

Coverslips were then washed three times (Buffer A, 0.1% Saponin) before post-fixation with PFA 2%

in Buffer A (15 minutes on ice and then 10 minutes at RT). After rinsing with water, coverslips were dried and mounted.

Internalization

Cells (30,000 per well) were plated on μ-slide six-well plates (Ibidi). After 24 h, the medium was removed and cells were incubated with the fluorescent protein (1 µM) diluted in DMEM for 30 minutes at 37°C before visualization. Cells were analyzed either directly to visualize the cell-surface staining or following addition of Trypan Blue (0.1% final concentration), an efficient quencher of all extracellular fluorescence and a marker of permeabilized cells, to visualize only the intracellular staining in live cells.

DNA constructs and recombinant proteins

All DNA constructs used in this study are listed in Table S2-3 and available upon request.

His6-tagged recombinant proteins were produced in BL21 (DE3) grown in MagicMedia (Invitrogen) 24h, 28°C and purified on HisTrap columns (GE Healthcare) by Imidazole gradient elution on AKTA Prime according to manufacturer instructions. Following tag removal by incubation with PreScisson protease (6h, 4°C), the protein was purified on Heparin column (GE Healthcare), eluted by NaCl gradient elution and dialyzed for 2 days (20 mM phosphate buffer, 100 mM NaCl, pH 7.5). For protein labelling, 100 μM of dialyzed purified protein was incubated with a twofold molar excess of fluorescein isothiocyanate in labeling buffer ( Carbonate buffer 50 mM pH 9.5, 100 mM NaCl) overnight at 4°C and free FITC was removed by dialysis (48 h, 4°C, 20 mM phosphate buffer, 100 mM NaCl, pH 7.5). The efficacy of FITC incorporation was controlled by SDS-PAGE and spectral analysis. The FITC:protein molecular ratio was estimated between 1.5 and 2 for all proteins.

Journal of Cell Science • Accepted manuscript

Quantitative Secretion assay

Cells (13,000 per well) were plated on 96-well plates (Greiner Bio-one) coated with 1.5 µg/ml PolyDL-ornithine and induced for protein expression with doxycycline (1µg/ml). After 10 h, cells were transfected with the indicated plasmids and cultured for 24h. The medium was then replaced with fresh medium in the incubator and protein release was induced by biotin addition (100 µM final) and following Furmazin (Promega, 1/500) addition, luciferase activity was measured 1h later on a plate reader (Tristar, Berthold). Cells were then lysed in the presence of recombinant LgBiT (0.7 µM final concentration) and Furimazin to measure intracellular protein expression. Rush-specific secretion was calculated by subtracting biotin-treated with biotin-untreated (control) wells for each condition and normalization with intracellular protein expression.

Quantitative Internalization assay

Cells (90,000 per well) were plated on 24-well culture dishes. After 24h, the protein was added to the medium and incubated for 30 minutes at 37°C. Following to washes with PBS, cells were collected by centrifugation after Trypsin treatment (0.25% final concentration) and resuspended in PBS 2% serum. Furimazin (1/500) was added to live cells and luciferase activity of the internalized protein was measured on a plate reader (Tristar, Berthold).

Journal of Cell Science • Accepted manuscript

PHPLC delocalization assay

HeLa cells were co-transfected with GFP-PHPLC and LCK-Cherry expression plasmids, PHPLC delocalization was visualized in live cells, following either biotin addition in the Trans-Rush assay or direct addition of the indicated protein in the medium. The kinetic of PHPLC delocalization was more accurate on time lapse image acquisition from a same cell, but equivalent results were obtained with images acquired at different time points on different cells. Delocalization index was calculated as followed.

Index is set at 100% at t=0 into the silica surface of the PWR sensor (prism). PWR assays were performed by using a homemade PWR instrument [START_REF] Harté | Probing the kinetics of lipid membrane formation and the interaction of a nontoxic and a toxic amyloid with plasmon waveguide resonance[END_REF]. Light was generated from a polarized CW laser (He-Ne;

wavelength of 632.8 nm) incident on the back surface of a thin metal film (Ag) deposited on a glass prism and coated with a layer of SiO2. The spectral angular resolution was  1 mdeg. PWR spectra corresponding to plots of reflected light intensity versus incident angle was excited with light whose electric vector was either parallel (s-polarized) or perpendicular (p-polarized) to the plane of the resonator surface. After formation and stabilization of the lipid bilayer (no further spectral changes with time), the protein was incrementally added to the PWR cell sample. Spectral changes were acquired for both polarizations. The system was let to equilibrate before each peptide addition. PWR being sensitive to the optical properties of material deposited on the resonator surface (so peptide bound to the lipid bilayer), interference from the material present in the bulk solution (non-bound) is unlikely. Apparent dissociation constants (KD) were obtained by plotting the resonance minimum position as a function of the peptide concentration and by fitting the plot through a hyperbolic binding function using GraphPad Prism™ version 5.0a (GraphPad Software, San Diego, California, US).

Binding data for both pand s-polarized light are presented, that characterize binding affinity along the two membrane axes (perpendicular and parallel to the membrane, respectively). The binding affinities obtained from the two axes are usually quite comparable, which is the case also here.

Journal of Cell Science • Accepted manuscript

FiguresFigure 1 :

 1 Figures

Figure 2 :

 2 Figure 2: Assessing EN2 unconventional secretion

Fig

  Fig S3 : F-EN2 staining in cell culture

Fig

  Fig S4: Role of Cholesterol in EN2 trafficking

Journal of Cell Science • Supplementary information

J.Cell Sci.: doi:10.1242/jcs.244327: Supplementary information Journal of Cell Science • Supplementary information

permeabilized or unpermeabilized conditions respectively together with Cherry staining (red). (D) Kinetics of biotin-induced secretion of EN2* (black), FGF2* (grey) and GFP* (green) proteins. (E) Quantification of EN2*, FGF2* and GFP* protein secretion in cells treated (Neo) or not (Ctrl) with neomycin (10 mM) for 1 hour (n=12; p<0,0001 (FGF2 and EN2) by one-way ANOVA followed by Tukey's post-tests). Bar: 20 µm Journal of Cell Science • Accepted manuscript

Funding: This work supported by CNRS, INSERM, Collège de France, Université de Paris, Labex PSL Memolife and Agence Nationale de la Recherche, (ANR-17-CE11-0050).

Journal of Cell Science • Accepted manuscript

Statistical Analysis

Data were analyzed with GraphPad Prism 6 and expressed as the mean +/-SEM. Two groups were compared with student's t-test. For multiple group comparison, one-way analysis of variance (ANOVA) followed by Tukey's post-tests was performed. P-values < 0.05 were considered to be statistically significant.