Bash-Imam Zeina

Thérizols Gabriel

Vincent Anne

Lafôrets Florian

Polay Espinoza

Pion Nathalie

Macari Françoise

Pannequin Julie

David Alexandre

Saurin Jean-Christophe

Mertani Hichem

Textoris Julien

Auboeuf Didier

Catez Frédéric

Dalla Venezia

Dutertre Martin

Marcel Virginie

Diaz Jean- Jacques

Dr Jean-Jacques Diaz

Translational reprogramming of colorectal cancer cells induced by 5-fluorouracil through a miRNA-dependent mechanism

Keywords: 5-fluorouracil, translation, translatome profiling, miRNA, colorectal cancer

5-Fluorouracil (5-FU) is a widely used chemotherapeutic drug in colorectal cancer. Previous studies showed that 5-FU modulates RNA metabolism and mRNA expression. In addition, it has been reported that 5-FU incorporates into the RNAs constituting the translational machinery and that 5-FU affects the amount of some mRNAs associated with ribosomes.

However, the impact of 5-FU on translational regulation remains unclear. Using translatome profiling, we report that a clinically relevant dose of 5-FU induces a translational reprogramming in colorectal cancer cell lines. Comparison of mRNA distribution between polysomal and non-polysomal fractions in response to 5-FU treatment using microarray quantification identified 313 genes whose translation was selectively regulated. These regulations were mostly stimulatory (91%). Among these genes, we showed that 5-FU increases the mRNA translation of HIVEP2, which encodes a transcription factor whose translation in normal condition is known to be inhibited by mir-155. In response to 5-FU, the expression of mir-155 decreases thus stimulating the translation of HIVEP2 mRNA.

Interestingly, the 5-FU-induced increase in specific mRNA translation was associated with reduction of global protein synthesis. Altogether, these findings indicate that 5-FU promotes a translational reprogramming leading to the increased translation of a subset of mRNAs that involves at least for some of them, miRNA-dependent mechanisms. This study supports a still poorly evaluated role of translational control in drug response.

Introduction

Translational control regulating one of the last steps of gene expression, plays a key role in tumor development [START_REF] Marcel | p53 Acts as a Safeguard of Translational Control by Regulating Fibrillarin and rRNA Methylation in Cancer[END_REF][START_REF] Truitt | New frontiers in translational control of the cancer genome[END_REF]. By finely regulating the synthesis of a specific subset of proteins, translational control contributes to tumor initiation, invasion and metastasis. In contrast, the role of translational control in anti-cancer drug response is just starting to emerge and largescale analysis of the translatome has been carried out for only a few anti-cancer drugs [START_REF] Wiita | Global cellular response to chemotherapy-induced apoptosis[END_REF][START_REF] Zaccara | p53-directed translational control can shape and expand the universe of p53 target genes[END_REF][START_REF] Boussemart | eIF4F is a nexus of resistance to anti-BRAF and anti-MEK cancer therapies[END_REF][START_REF] Larsson | Distinct perturbation of the translatome by the antidiabetic drug metformin[END_REF].

Among the drugs commonly used in chemotherapy, 5-Fluorouracil (5-FU) is an antimetabolite widely given in first-line of treatment in many types of solid cancers. For a long time, the 5-FU-induced cytotoxic effects were thought to result exclusively from its impact on DNA metabolism, in particular from 5-FU-induced inhibition of DNA synthesis and induction of DNA damage [START_REF] Longley | 5-Fluorouracil: mechanisms of action and clinical strategies[END_REF]. However, several evidences indicate that the cytotoxic effect of 5-FU also results from its capacity to alter RNA metabolism and mRNA expression [START_REF] Longley | 5-Fluorouracil: mechanisms of action and clinical strategies[END_REF]. 5-FU can be incorporated into all species of RNAs after its conversion into fluorouridine triphosphate (FUTP) and media complementation with uridine, which allowed recovery of normal RNA metabolism, compensate most of the 5-FU-induced cytotoxic effects [START_REF] Kufe | 5-Fluorouracil incorporation into human breast carcinoma RNA correlates with cytotoxicity[END_REF][START_REF] Pritchard | Inhibition by uridine but not thymidine of p53-dependent intestinal apoptosis initiated by 5-fluorouracil: evidence for the involvement of RNA perturbation[END_REF][START_REF] Hoskins | Evidence for distinct DNA-and RNA-based mechanisms of 5fluorouracil cytotoxicity in Saccharomyces cerevisiae[END_REF][START_REF] Pettersen | UNGinitiated base excision repair is the major repair route for 5-fluorouracil in DNA, but 5fluorouracil cytotoxicity depends mainly on RNA incorporation[END_REF]. Furthermore, exposure to 5-FU promotes a profound transcriptional reprogramming leading to modification of mRNA and miRNAs expression profiles that contributes in modifying cell fate [START_REF] Rossi | Modification of miR gene expression pattern in human colon cancer cells following exposure to 5-fluorouracil in vitro[END_REF][START_REF] Hernández-Vargas | Transcriptional profiling of MCF7 breast cancer cells in response to 5-Fluorouracil: relationship with cell cycle changes and apoptosis, and identification of novel targets of p53[END_REF][START_REF] Shah | 5-Fluorouracil drug alters the microRNA expression profiles in MCF-7 breast cancer cells[END_REF].

Although 5-FU directly alters RNA metabolism and mRNA expression, the impact of 5-FU on translation has yet been poorly characterized. Several evidences indicate that 5-FU could alter translation. It has been shown that 5-FU affects processing and functions of two components of the translational machinery, ribosomal RNA (rRNA) and transfer RNA (tRNA). [START_REF] Mojardã N | New Insights into the RNA-Based Mechanism of Action of the Anticancer Drug 5′-Fluorouracil in Eukaryotic Cells[END_REF][START_REF] Kaiser | Studies on 5-fluorouracil-containing ribonucleic acid. I. Separation and partial characterization of fluorouracil-containing transfer ribonucleic acids from Escherichia coli[END_REF][START_REF] Tseng | Specific inhibition of transfer RNA methylation and modification in tissues of mice treated with 5-fluorouracil[END_REF][START_REF] Burger | Chemotherapeutic Drugs Inhibit Ribosome Biogenesis at Various Levels[END_REF]. Moreover, genome-wide screening in yeast revealed that rRNA and tRNA processing factors mediate a part of 5-FU cytotoxicity [START_REF] Giaever | Chemogenomic profiling: identifying the functional interactions of small molecules in yeast[END_REF][START_REF] Fang | 5-Fluorouracil Enhances Exosome-Dependent Accumulation of Polyadenylated rRNAs[END_REF][START_REF] Lum | Discovering modes of action for therapeutic compounds using a genome-wide screen of yeast heterozygotes[END_REF][START_REF] Gustavsson | Evidence that tRNA modifying enzymes are important in vivo targets for 5-fluorouracil in yeast[END_REF]. Finally, two large-scale studies showed that 5-FU could regulate the translational output of a set of mRNAs [START_REF] Xi | Multi-level gene expression profiles affected by thymidylate synthase and 5-fluorouracil in colon cancer[END_REF][START_REF] Kudo | Translational control analysis by translationally active RNA capture/microarray analysis (TrIP-Chip)[END_REF].

For some of these mRNAs, their global expression levels were not strongly affected by 5-FU, while their abundance in the polysomal fractions (i.e. ribosome-bound mRNAs) was modulated. Although these data raised the possibility that their translational efficiency might be regulated by 5-FU, it was not clearly addressed in these studies. Therefore, the regulation of translational efficiency by 5-FU has not been determined on a large-scale, and the molecular mechanisms mediating such regulations were not determined.

In the present study, to investigate whether treatment with a clinically relevant dose of 5-FU affects the translational efficiency of specific mRNAs, we performed a genome-wide analysis of the translatome by comparing the distribution of mRNAs within polysomes (actively translated RNAs) and non-polysomes (free non-translated and poorly translated mRNA) in a panel of colorectal cancer cells treated by 5-FU. This approach showed that 5-FU induces a translational reprogramming, characterized by an increased translational efficiency of specific genes that was mediated, at least in part, through 5-FU-modulation of miRNA expression.

Results

Cells remain viable and metabolically active in response to 10 µM of 5-FU

We first optimized 5-FU treatment condition to harvest HCT-116 colorectal cancer cells that retain cell viability and metabolic activity. Cell response to increasing concentrations of 5-FU was monitored in real-time for 72 hrs using electrical impedance-derived technology (Figure 1A) [START_REF] Urcan | Real-time xCELLigence impedance analysis of the cytotoxicity of dental composite components on human gingival fibroblasts[END_REF]. Compared to non-treated cells, two different kinetics of 5-FU response were observed depending on the dose. At 100 µM and 500 µM, 5-FU induced a decrease of impedance signal from 24 hrs post-treatment. At these doses, a significant reduction of total cell number and an increase in the percentage of dead cells were also observed (Figures 1B-1C). In response to 10 µM of 5-FU, the decrease of impedance signal was delayed by 24 hrs (Figure 1A). In this condition, the total cell number remained stable between 24 and 48 hrs post-treatment and no significant change in the percentage of dead cell was observed (Figures 1B-1C), while the 5-FU treatment was effective as soon as after 4 hrs of exposure as shown by the increased expression of the stress sensor p53 (Figure S1A). These data showed that, compared to 100 µM and 500 µM, cytotoxicity in response to 10 µM of 5-FU was delayed, as cells were still viable at 24 and 48 hrs post-treatment.

To determine whether HCT-116 cells treated with different doses of 5-FU retained a metabolic activity at 24 hrs and 48 hrs, we performed MTS assays (Figure 1D). Cells treated with 100 µM and 500 µM of 5-FU showed a strong decrease of their metabolic activity. In contrast, a slight but significant increase in metabolic activity 24 hrs post-treatment was observed in 10 µM 5-FU treated cells, followed by a decrease back to their initial level by 48 hrs of treatment. Altogether, these data indicated that cells exposed to 10 µM of 5-FU remained viable and retained metabolic activity even at 48 hrs post-treatment.

5-FU reduces global protein synthesis

Before analyzing specific changes in translation that occur in response to 5-FU in viable cells retaining metabolic activity, we determined whether HCT-116 cells treated with 10 µM of 5-FU maintained their capacity to synthesize proteins. Using 35 S pulse-labeling experiments, we compared levels of global protein synthesis between non-treated cells (NT) and cells exposed to 10 µM of 5-FU for 24 hrs (Figure 2A). Cycloheximide (CHX) treatment was used as a positive control of complete inhibition of global protein synthesis. 35 S quantification revealed a decrease of about 20% of the global level of protein synthesis in treated cells compared to non-treated cells (Figures 2A-2B).

We then compared polysome profiles through sucrose gradients from non-treated cells and cells treated with 10 µM of 5-FU (Figures 2C-2D). Typical polysome profiles were obtained for non-treated and treated HCT-116 cells using both real-time absorbance detection and RNA visualization on agarose gel. The total quantity of 40S, 60S subunits, 80S monosomes and polysomes for a given amount of cytosolic extract was decreased in 5-FU treated cells.

In particular, the 60S subunits and the monosomes 80S were drastically decreased. Using 32 P pulse labeling, we confirmed that levels of ribosome decreased in 5-FU-treated cells compared to non-treated ones, and that 60S and 80S are more prone to reduction than 40S (data not shown). This decrease in ribosomes quantity is concordant with the reduction of ribosome production previously described in response to 5-FU [START_REF] Mojardã N | New Insights into the RNA-Based Mechanism of Action of the Anticancer Drug 5′-Fluorouracil in Eukaryotic Cells[END_REF][START_REF] Burger | Chemotherapeutic Drugs Inhibit Ribosome Biogenesis at Various Levels[END_REF][START_REF] Lum | Discovering modes of action for therapeutic compounds using a genome-wide screen of yeast heterozygotes[END_REF]. Reduction in ribosome production in response to 5-FU was probably partly responsible for the decrease of global protein synthesis (Figures 2A-2B). These data showed that HCT-116 cells exposed to 10 µM of 5-FU for 24 hrs retained the capability to synthesize proteins although the global protein synthesis rate was reduced.

5-FU promotes association of a subset of mRNAs with polysomes

Because global protein synthesis was slightly decreased but still highly effective in response to 10 µM of 5-FU for 24 hrs in HCT-116 cells, we investigated whether this treatment induced a modulation of the translational efficiency of some specific mRNAs using translatome profiling, a widely used method [START_REF] Zong | Messenger RNA translation state: the second dimension of high-throughput expression screening[END_REF]. We used a three-step process that allows determining the variation of distribution of each cytosolic mRNA in non-polysomal fractions (NP, including free non-translated and poorly translated RNAs) and polysomal fractions (P, including actively translated RNAs) (Figures 2C-2D and Figure S2A). First, for every gene, the detection of mRNA in non-polysomal (NP) and polysomal (P) fractions was assessed in nontreated and 5-FU treated cells using DNA microarray. Second, in each condition, the relative distribution of a given mRNA between NP and P fractions was estimated by calculating the ratio of the probe signal obtained for the P fraction to the NP fraction. Third, comparison of mRNA distribution between non-treated and treated cells was performed by calculating the translational index (TI), which represents the variation of the distribution of a particular mRNA between NP and P in response to 5-FU treatment. Thus, for each mRNA, the TI reflects the fold-change of its translation efficiency in response to 5-FU.

Two independent experiments were performed using this approach to determine change in TI in 10 µM 5-FU treated cells compared to non-treated cells. The probe intensities issued from the two biological replicates were significantly correlated supporting the robustness of this translatome profiling (Figures S2B-S2E). Globally, 12,131 genes were commonly detected in both non-treated and treated conditions (Figure S2F). These common genes were used to analyze changes in translation efficiency in response to 5-FU by calculating the translational index (TI). By applying cut-off values of 1.5 and P-value < 0.05 to the TI of the 12,131 common genes [START_REF] Dacheux | BRCA1-Dependent Translational Regulation in Breast Cancer Cells[END_REF], we identified 313 genes (2.6%) whose Ti was significantly changed in response to 10 µM of 5-FU for 24 hrs in HCT-116 cells (Figure 3A and Table S1).

These data indicated that 5-FU treatment altered translation efficiency of some mRNAs.

Among them, 29 were translationally down-regulated (9%) while 284 were up-regulated (91%). The range of TI varied from -1.5 to -2.5 and from 1.5 to 2.9 (Figure 3B and Table S2).

In particular, 25 genes displayed a TI above 2, indicating that 5-FU treatment induces a 2fold increase in polysome association of some mRNAs (Table 1). Using lower or higher cutoff values (TI: 1.3 or 2.0), we observed that up-regulation of translation efficiency was always much more frequent than down-regulation (Table S1). Thus, gene-specific modulation of translational efficiency in response to 10 µM of 5-FU for 24 hrs in HCT-116 cells corresponded mainly to stimulation (for genes list see Table 1 and Table S2).

Validation was performed by calculating the TI of 11 genes, which were selected on the basis of the significance and the absolute values of their TI measured using microarray. RT-qPCR were performed on HCT-116 NP and P fractions that were used for microarrays analyses (two independent replicates) and from two additional independent experiments. Compared to microarray data, similar up-and down-regulation of TI were observed in the panel of 11 genes analyzed by RT-qPCR (Figure 4A). A difference in the range of TI was observed between the two methods that can be explained by a difference in sensibility of these methods. However, a significant and positive correlation was observed between TI obtained from RT-qPCR and microarray analyses (Figure 4B). Similar analyses using RT-qPCR quantification of mRNA distribution within polysome profiles were performed in two additional colorectal cancer cells (Figure 4C). We used the p53-null HCT-116 cells and the mutant p53.R273H HT-29 cells (Figure S1, IARC TP53 Database), to determine whether this translational regulation occurs in different colorectal cancer cell lines and whether it is dependent of p53 since several data support a role of p53 as a regulator of translational reprogramming [START_REF] Marcel | onc201525a[END_REF]. Like in HCT-116 cells, TI of a panel of 7 genes were either reduced or increased in response to 5-FU in these two cell lines, similarly to what observed in HCT-116 cells. Of note only 3 genes in HT-29 did not show variation in their TI. Interestingly, since similar pattern of TI was observed in wild-type p53 HCT-116 cells, p53-null HCT-116 cells and mutant p53.R273H HT-29 cells, it appeared that changes in TI of each mRNA revealed by the translatome profiling are independent of p53. Altogether, the validation procedure performed on different genes in several cell lines confirmed that 5-FU treatment at 10 µM for 24 hrs reduced or increased the association of a subset of mRNAs with polysomes, indicating respectively either a reduction or an increase in translational efficiency of some mRNAs.

To decipher whether alteration in translational efficiency directly resulted from 5-FU treatment or from 5-FU-induced change in transcriptional and post-transcriptional regulation, we quantified the levels of total and cytosolic mRNAs in response to 24 hrs of 10 µM 5-FU treatment in HCT-116 p53-null and HT-29 cells (Figure S3). No concordance was observed between 5-FU-induced transcriptional, post-transcriptional and translational variation, as confirmed by correlation analyses. These data suggest that 5-FU alters translational efficiency independently of any change at mRNA levels. Finally, no change in TI analyzed in cytosolic lystates was observed in HCT-116 cells treated for 4 hrs by 10 µM 5-FU while increased in cytosolic mRNA levels were observed (Figure S4). Once again, no concordance between transcriptional and translational events was observed at early time point although 5-FU affects more rapidly transcriptional regulatory events than the translational ones.

Altogether, our data showed that 5-FU treatment at 10 µM for 24 hrs alters the translatome independently of any transcriptional and post-transcriptional regulation of the targeted mRNA.

In addition, 5-FU mainly increases the translation efficiency of a subset of mRNAs.

Genes involved in DNA replication and gene expression regulation are selectively regulated by 5-FU at the translation level

To determine the main functions of genes whose recruitment whithin polysomes was altered in response to 5-FU in HCT-116 cells, we performed Gene Ontology analysis using the Functional annotation clustering analytic modules of DAVID bioinformatics resources that provides a rank classification of enriched functions based on determination of P-values and enrichment scores [START_REF] Huang | Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources[END_REF]. Statistical enrichment analyses were performed separately on genes translationally up-and down-regulated, using the lists of 313 (TI cut-off of 1.5, P < 0.05) and 798 (TI cut-off of 1.3, P < 0.05) translationally dysregulated genes. Clusters of functional annotation common to both lists are presented in Figure 5 (for a complete list of functional annotation clustering, see Table S3).

Using this approach, only one functional cluster was identified for the genes whose translation efficiency was down-regulated in response to 5-FU treatment. This cluster contained 5 of the 29 translationally down-regulated genes (17%). It was composed of genes involved in DNA replication, such as ASF1B or TBCD (Figure 5 and Table S3). These data suggested that 5-FU reduced the translation efficiency of mRNAs involved in DNA replication.

Genes whose translation efficiency was up-regulated by 5-FU clustered into five groups

showing enrichment scores ranging from 1.7 to 4.9 (Figure 5). The group which displayed the most significant P-value and the highest enrichment score, contained genes encoding zinc finger proteins, including several ZNF family members and HIVEP2 (Figure 5 and Table S3).

These proteins are mainly involved in transcription regulation. A second cluster closely related to the transcription process (cluster 3) was enriched in genes coding proteins with Bbox domains. These data suggest that 5-FU treatment selectively increases the translation efficiency of a subset of mRNAs encoding transcriptional regulators. Translationally upregulated genes were also enriched in genes involved in translation and translational regulation. Indeed, clusters 2 and 4 contained genes involved in translation initiation and elongation (EIF3F, EIF3E…) and in production of the translational machinery, including ribosome components (RPL13…) and tRNA maturation factors. Altogether, these data showed that genes whose translation efficiency was up-regulated in response to 5-FU treatment for 24 hrs were mainly involved in regulation of gene expression, including transcription and translation.

5-FU induces translational up-regulation of HIVEP2 mRNA through down-regulation of mir-155

To investigate the molecular mechanisms contributing to the modulation of translational efficiency of specific mRNA in response to 5-FU treatment, we focused our attention on HIVEP2 mRNA one of the most translationally deregulated genes (Figure 3B). Indeed, our translatome profiling and validation process showed that the distribution of HIVEP2 mRNA was significantly increased in polysomes compared to non-polysomes in response to 5-FU in a panel of three colorectal cancer cell lines (Figures 3B, 4A, 4C and6A). We determined whether this change in HIVEP2 mRNA translation was paralleled with a change in mRNA and protein levels in HCT-116 cells. RT-qPCR analysis showed a significant 40% decrease of HIVEP2 mRNA levels in both total and cytosolic extracts in response to 5-FU treatment (Figure 6B). In parallel, a significant 1.5-fold increase in HIVEP2 protein levels was observed in response to 5-FU treatment (Figure 6C and Figure S5A). These data demonstrated that despite the decrease of HIVEP2 mRNA levels in response to 5-FU, the selective increase in its translational efficiency led to an increase in HIVEP2 protein levels. miRNAs reduce mRNA translation by direct interaction with specific mRNAs that is a prerequisite for subsequent mRNA degradation [START_REF] Meijer | Translational Repression and eIF4A2 Activity Are Critical for MicroRNA-Mediated Gene Regulation[END_REF][START_REF] Wilczynska | The complexity of miRNA-mediated repression[END_REF]. HIVEP2 mRNA was previously identified as a direct target of mir-155 [START_REF] Yin | MicroRNA-155 is an Epstein-Barr virus-induced gene that modulates Epstein-Barr virus-regulated gene expression pathways[END_REF]. We thus wondered whether mir-155 expression may be regulated by 5-FU, thereby participating in the stimulation of HIVEP2 mRNA translational efficiency. In HCT-116 cells, quantification of mir-155 expression by RT-qPCR showed a significant reduction of 50% in response to 5-FU treatment for 24 hrs (Figure 6D). Thus, by decreasing mir-155 expression, 5-FU could counteract mir-155-mediated inhibition of HIVEP2 translation and thus promote HIVEP2 mRNA translation. Interestingly, reduction in mir-155 levels was observed from 4 hrs (Figure S5B), suggesting a direct effect of 5-FU on mir-155 expression. To determine the effect of mir-155 on HIVEP2 mRNA translation, we performed 3'UTR luciferase reporter assays using HIVEP2 3'UTR reporter. Over-expression of mir-155 in the absence of 5-FU significantly reduced the Firefly/Renilla luciferase activity ratio of the HIVEP2 3'UTR reporter and of the positive control BACH1 3'UTR reporter while it had no impact on a 3'UTR-less negative control (Figure S5C) [START_REF] Yin | MicroRNA-155 is an Epstein-Barr virus-induced gene that modulates Epstein-Barr virus-regulated gene expression pathways[END_REF]. Using this system, we assessed the role of HIVEP2 3'UTR and mir-155 expression in the increase in HIVEP2 mRNA translation in response to 5-FU treatment (Figure 6E). In the absence of transient mir-155 over-expression (mir-CTL condition), 5-FU treatment significantly increased the Firefly/Renilla luciferase activity ratio of the HIVEP2 3'UTR reporter. Importantly, this increase in luciferase activity was not paralleled with a variation in luciferase mRNA levels (Figure 6F). These data suggested that increase in luciferase activity results from change in translation rather than change in transcription of the chimeric Firefly Luciferase -HIVEP2 3'UTR. Thus, HIVEP2 mRNA translation in response to 5-FU treatment was, at least in part, related to the 3'UTR of HIVEP2 mRNA. In 5-FU treated cells, mir-155 expression was then restored using an expression vector (Figure 6E). Restoration of mir-155 expression reduces the induction of Firefly/Renilla luciferase activity ratio in 5-FU treated cells. In addition, no significant change in the luciferase mRNA levels was observed, indicating that restoration of mir-155 in 5-FU treated cells inhibits translation without affecting transcriptional expression of the luciferase reporter assay (Figure 6F). Interestingly, similar reduction in mir-155 expression from 4 hrs post-treatment and in mir-155-dependent HIVEP2 translation in response to 5-FU was observed in p53-null HCT-116 cells, indicating a p53-independent effect (Figure S6). Thus, over-expression of mir-155 was sufficient to inhibit the increase in HIVEP2 mRNA translation that was induced by 5-FU. Altogether, these data showed that the alteration of mir-155 in response to 5-FU mediated a translational regulation of the HIVEP2 mRNA.

To determine whether mir-155 may have a broader role in translational regulation in response to 5-FU treatment, we first analyzed the enrichment of mir-155 target genes among the genes whose translation efficiency was altered by 5-FU treatment in HCT-116 cells, using a list of 719 experimentally validated mir-155 target genes (miRTarBase ( 33)) (Figure 6G and Table S4). Interestingly, a significant enrichment in mir-155 target genes was observed among the translationally up-regulated genes (Up) when compared to the whole genome (Up vs Microarray, P = 0.0389), while no enrichment of mir-155 target genes was found among the genes translationally down-regulated by 5-FU (Down vs Microarray, P = 0.3088). As shown in Figure 6G, 19 of the genes that were translationally up-regulated by 5-FU corresponded to mir-155 target genes (6.7%). We validated the translationnally upregulation of a panel of mir-155 target genes in response to 5-FU in HCT-116 p53-null cells (Figure S7). These data suggest that the down-regulation of mir-155 in response to 5-FU treatment may contribute to the increased translation of a subset of mRNAs. Interestingly, Gene Ontology analysis of these mir-155 target genes whose translation was up-regulated in response to 5-FU mainly corresponded to proteins involved in transcription (Table S5).

Discussion

The anti-metabolite 5-FU has been shown to alter gene expression at transcriptional and splicing levels and to alter the translation machinery, mainly by affecting the processing and functions of rRNA and tRNA [START_REF] Mojardã N | New Insights into the RNA-Based Mechanism of Action of the Anticancer Drug 5′-Fluorouracil in Eukaryotic Cells[END_REF][START_REF] Burger | Chemotherapeutic Drugs Inhibit Ribosome Biogenesis at Various Levels[END_REF][START_REF] Giaever | Chemogenomic profiling: identifying the functional interactions of small molecules in yeast[END_REF][START_REF] Lum | Discovering modes of action for therapeutic compounds using a genome-wide screen of yeast heterozygotes[END_REF][START_REF] Gustavsson | Evidence that tRNA modifying enzymes are important in vivo targets for 5-fluorouracil in yeast[END_REF]. However, the impact of 5-FU on translational control remained unclear. Using translatome profiling, we show here for the first time that 5-FU induces a translational reprogramming since, while reducing the global protein synthesis, 5-FU increases translation of a subset of mRNAs, at least in part through miRNA-based mechanisms.

As already reported, we identify two kinetics of cellular response to 5-FU [START_REF] Longley | 5-Fluorouracil: mechanisms of action and clinical strategies[END_REF]. While high doses of 5-FU exposure result in drastic and rapid cytotoxic effects, low doses, which correspond to doses used in the clinic [START_REF] Casale | Plasma concentrations of 5-fluorouracil and its metabolites in colon cancer patients[END_REF][START_REF] Trump | Pharmacokinetic and pharmacodynamic analysis of fluorouracil during 72-hour continuous infusion with and without dipyridamole[END_REF], promote delay in cytotoxicity. In particular, cells exposed to 10 µM of 5-FU for 24 hrs remain viable and metabolically active, and keep their capacity to synthesize proteins. By comparing mRNA levels in polysomal (translated mRNAs) and non-polysomal (free and poorly translated mRNAs) fractions using DNA microarray [START_REF] Zong | Messenger RNA translation state: the second dimension of high-throughput expression screening[END_REF], we demonstrate that low doses of 5-FU specifically alter the translation efficiency of about 300 mRNAs. Importantly, the validation procedure performed on different genes in a panel of three colorectal cancer cell lines confirmed that 5-FU treatment alters the association of some mRNAs with polysomes. By analyzing the abundance of mRNAs in polysomal fractions using genome-wide approaches two previous studies have suggested that 5-FU affects translation [START_REF] Xi | Multi-level gene expression profiles affected by thymidylate synthase and 5-fluorouracil in colon cancer[END_REF][START_REF] Kudo | Translational control analysis by translationally active RNA capture/microarray analysis (TrIP-Chip)[END_REF]. However, since no normalization to non-polysomal fraction or total RNA was done in these studies, alteration of translation efficiency was not directly addressed in these studies. In addition, both studies had potential bias. Indeed, one of these studies used colorectal cancer cells with decreased or increased expression of thymidylate synthase, a key mediator of 5-FU cytotoxicity, thus complicating the analysis of the direct impact of 5-FU on translation [START_REF] Longley | 5-Fluorouracil: mechanisms of action and clinical strategies[END_REF][START_REF] Xi | Multi-level gene expression profiles affected by thymidylate synthase and 5-fluorouracil in colon cancer[END_REF]. The second study used Hsp70 immunoprecipitation to purify ribosomes presenting newly synthesized proteins [START_REF] Kudo | Translational control analysis by translationally active RNA capture/microarray analysis (TrIP-Chip)[END_REF]. This approach may suffer from several biases since Hsp70 was recently reported to bind RNA, Hsp70 is not required for newly synthesized small proteins, and Hsp70 levels are reduced in response to 5-FU [START_REF] Wong | Identification of 5-fluorouracil response proteins in colorectal carcinoma cell line SW480 by two-dimensional electrophoresis and MALDI-TOF mass spectrometry[END_REF][START_REF] Willmund | The cotranslational function of ribosome-associated Hsp70 in eukaryotic protein homeostasis[END_REF][START_REF] Hentze | Making sense of nonsense[END_REF][START_REF] Kwon | The RNA-binding protein repertoire of embryonic stem cells[END_REF].

Our data indicate that exposure to 5-FU promotes increase in translational efficiency of only a subset of mRNAs. It appeared that the 5-FU-induced change in translation efficiency obtained in our experimental conditions is independent of any transcriptional and posttranscriptional modulation induced by 5-FU treatment in a panel of three colorectal cancer cell lines. Indeed, we showed that, although change in translation efficiency is not an early event, modulation of translation does not correlate with modulation of transcription, as shown for several genes including HIVEP2. Change in transcription can thus not explain the observed change in translation. Several mechanisms can explain the change in translational control of only a subset of transcripts in response to 5-FU. Such modulation in translational efficiency can result for example from 5-FU-induced alteration of expression of factors involved in translational control such as proteins or miRNAs [START_REF] Truitt | New frontiers in translational control of the cancer genome[END_REF]. Our data showed that 5-FUinduced change in translational efficiently occurred independently of the p53 protein, a stress sensor known to regulate translation through numerous mechanisms [START_REF] Marcel | onc201525a[END_REF]. Our data rather identified mir-155 as a common regulator of 5-FU-mediated alteration of translational efficiency for several target genes.

We indeed showed that reduction of mir-155 in response to 5-FU can promote HIVEP2 mRNA translation in HCT-116 colorectal cancer cells. In addition, determination of the molecular mechanism by which 5-FU regulates HIVEP2 also supports that 5-FU regulates translation efficiency. Indeed, cell exposure to low doses of 5-FU decreases HIVEP2 mRNA levels in both total and cytosolic fractions while in the same time, HIVEP2 mRNA is concentrated in the actively translated polysomal fractions, and HIVEP2 protein levels are increased in 5-FU treated cells. Moreover, our data reveal that increased translation of HIVEP2 mRNA in response to 5-FU results from abolition of mir-155-mediated inhibition of HIVEP2 mRNA translation. By reducing mir-155 expression, 5-FU promotes translation of HIVEP2 mRNA. This observation is in accordance with the repressing role of miRNAs, which have been shown to inhibit translational regulation before inducing mRNA degradationinhibition, which can be removed by reducing miRNA expression [START_REF] Meijer | Translational Repression and eIF4A2 Activity Are Critical for MicroRNA-Mediated Gene Regulation[END_REF][START_REF] Wilczynska | The complexity of miRNA-mediated repression[END_REF]. Furthermore, we showed and validated that mir-155 target genes are enriched in genes translationally upregulated, establishing that reduction in mir-155 levels in response to 5-FU could explain about 7% of the translationally up-regulated gene. Interestingly, plasmatic mir-155 expression levels were found to be decreased after 5-FU-based chemotherapy exposure of colorectal cancer cells' patients [START_REF] Chen | Predicting distant metastasis and chemoresistance using plasma miRNAs[END_REF]. miRNA regulation is likely a more general mechanism leading to alteration of translation in response to 5-FU, because miRNA profiling identified several miRNAs, whose expression is either up-or down-regulated in response to 5-FU [START_REF] Rossi | Modification of miR gene expression pattern in human colon cancer cells following exposure to 5-fluorouracil in vitro[END_REF][START_REF] Shah | 5-Fluorouracil drug alters the microRNA expression profiles in MCF-7 breast cancer cells[END_REF][START_REF] Hou | MicroRNA profiling in human colon cancer cells during 5-fluorouracil-induced autophagy[END_REF]. However, until now, these changes in miRNA expression levels have not been linked to alteration of translation. Additional mechanisms remain to be explored to explain the translational control of a specific subset of mRNAs in response to 5-FU treatment. In particular, the impact of 5-FU incorporation into RNAs of the translational machinery on translational control remains to be investigated.

We show that the selective translation of mRNAs in response to 5-FU is accompanied by a decrease in global protein synthesis. This reduction in protein synthesis could result from the previously described reduction in pre-rRNA processing in response to 5-FU since a strict correlation occurs between the rate of ribosome biogenesis and protein synthesis [START_REF] Burger | Chemotherapeutic Drugs Inhibit Ribosome Biogenesis at Various Levels[END_REF][START_REF] Brooks | Continuous protein synthesis is required to maintain the probability of entry into S phase[END_REF][START_REF] Pederson | The Nucleolus[END_REF].

Therefore, the concomitant decrease in global protein synthesis and increase in translation of a selected subset of mRNAs indicate that 5-FU promotes translational reprogramming in colorectal cancer cells.

In conclusion, in the present study we provide a novel mechanism, which supports the importance of translational control in 5-FU-induced cellular response. Overall, our data indicate that low doses of 5-FU promote translational reprogramming of colorectal cancer cells. The gene-specific stimulation of translation induced by 5-FU involves at least in part miRNA-dependent translational regulation. However, additional mechanisms remain to be investigated in the future. Altogether, our data add to the growing body of evidences that support the direct contribution of translation in establishing anti-cancer drug response as well as potential treatment failures.

Materials and Methods

Cell culture. Human colorectal cancer HCT-116 and HT-29 cells were obtained from ATCC (CCL-247, HTB-38). Cells were maintained in DMEM-glutaMAX supplemented with 10% FBS and 1% penicillin/streptomycin (Life technologies) at 37°C under 5% CO 2 atmosphere. 5-Fluorouracil (5-FU) was kindly provided by Centre Léon Bérard (purchased at Sanofi-Aventis), stored at room temperature at 384 mM in sterile water and diluted in cell culture medium.

Cell proliferation and viability. Real-time cell behavior was monitored using xCELLigence RTCA system (Roche), which allows label-free and dynamic monitoring of cells by measuring electrical impedance. 10 5 cells were seeded in 96 E-plates (Roche) 24 hrs prior 5-FU treatments. RTCA system displays the measurements of impedance signal as Cell Index (CI) values, providing quantitative information about the different biological status of the cells including number, viability, proliferation and mobility. CI values curves were normalized to the time point of 5-FU administration. MTS were performed at different time points on 10 5 cells seeded into 96 well-plate 24 hrs prior 5-FU treatments using Cell Titer Aqueous One Solution Cell Proliferation assays as described by the manufacturer (Promega). Cell viability and total cell numbers were quantified in response to 5-FU treatment by trypan blue staining method using Cedex XS analyzer (Roche) from 150.10 5 cells seeded in 24 well-plates 24 hrs prior 5-FU treatment.

Global protein synthesis. Cells were plated 72 hrs before labeling in normal medium and treated with 10 µM 5-FU for 24 hrs before labeling. Normal medium was replaced with methionine-cysteine free DMEM for 30 min before labeling. [ 35 S]-methionine-cysteine was added in the medium at 75 µCi/mL and cells were incubated for 1 hr at 37°C. Labeling was stopped by washing cells with ice cold 1X PBS. Cells were scraped in SDS-PAGE laemmli sample buffer, and incubated 10 min at 95°C. Fifteen µg of protein was loaded onto a 4-20% gradient SDS-PAGE. [ 35 S]-met-cys incorporation was quantified using PhosphorImaging on a FLA-9500 apparatus (GE). For each labeling experiment, protein quantification and SDS-PAGE were performed in duplicate.

Preparation of mRNA-associated polysomes. Cells were seeded at 10 7 cell/15 cm dish and treated for 24 hrs with 5-FU. Cells were then incubated for 15 min with 100 µg/ml cycloheximide (CHX) and washed twice with cold 1X PBS-100 µg/ml CHX before harvesting.

Cytosolic lysates were prepared in lysis buffer (20 mM KCl, 5 mM MgCl 2 , 50 mM Tris-HCl pH 7.4, 4.5% sucrose, 0.5 mM DTE and 100 µg/ml CHX) using dounce homogenizer and two successive 10 min centrifugations at 4°C at 1,000 and 12,000 x g. Ten-40% sucrose gradients were prepared using four solutions (10%, 20%, 30% and 40% of sucrose dissolved in lysis buffer) that were introduced one by one in increased concentration order after 30min at -80°C in Ultra-Clear Tube 9/16 x 3 ½ (Beckman). One mg of cytosolic proteins was loaded onto a sucrose gradient defrozen overnight at 4°C and sedimented by ultra-centrifugation at 40,000 rpm for 2 hrs at 4°C using SW41 rotor on L7-55 ultracentrifuge (Beckman). The gradients were collected in 18 fractions and absorbance profiles were generated at 254 nm using an ISCO UA-6 detector. RNA quality of the different fractions was checked on agarose gel. Non-polysomal or polysomal fractions were pooled for further RNA extraction using Trizol LS Reagent as described by the manufacturer (Life technologies).

Affymetrix exon-array. Quality of RNA purified from polysome fractionation was verified using BioAnalyzer (Agilent) (RIN ranging from 6.6 to 9.3). 250 ng of total RNA was processed with the GeneChip WT Sense Target Labeling kit and hybridized to GeneChip Human Exon 1.0 ST arrays. Affymetrix exon-array data were normalized with quantile normalization. Antigenomic probes were used to perform the background correction. Only probes targeting exons annotated from full-length cDNA were retained for analysis. Cross hybridizing probes and probes with lower signals intensity than anti-genomic background probes showing the same GC content were removed. Only probes with a DABG P-value ≤ 0.05 in at least half of the arrays were considered for further statistical analysis. Arrays were performed in two independent replicates. The median intensity of all constitutive exonic probes was calculated for each gene in each sample, and the experimental samples and control groups were compared using a Student's paired t-test. The adopted strategy to identify the translationally regulated genes depends on calculating the translational index (TI) (Figure S2A). First the ratios of active mRNAs (polysomal fraction P) to inactive mRNAs (non-polysomal fraction NP) was calculated for both 5-FU treated and non-treated cells.

Then the translational index (5-FU P/NP)/(Control P/NP) presents the translational changes for each individual transcript. List of genes significantly deregulated at translational levels was determined using Student's paired t-test (P < 0.05) and TI cut-off (1.3, 1.5 or 2) that are classical values used in translatome analyses [START_REF] Dacheux | BRCA1-Dependent Translational Regulation in Breast Cancer Cells[END_REF]. Gene ontology was performed on gene lists using the online DAVID tools using Functional annotation clustering analytic modules that performed dedicated statistical analysis [START_REF] Huang | Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources[END_REF]. The Affymetrix exon-array data reported in this article have been deposited in NCBI's Gene Expression Omnibus (GEO) database (accession number GSE77180).

Real-time quantitative RT-PCR. The Affymetrix exon-array was validated by RT-qPCR using RNA purified from the two polysomal profiles used for the exon-array analyses and from four additional and independent polysomal profiles. Three criteria have been used to select genes for validation: a significant difference in mRNA distribution within polysomal and non-polysomal fractions between non-treated and 5-FU-treated cells; a Translational Index (TI) corresponding to the highest TI variations; and a mix of translationnally up-and downregulated genes. 250 ng of total RNA were reverse transcribed using the M-MLV RT kit and random primers (Invitrogen), according to the manufacturer's instructions. Quantitative realtime PCR (qPCR) was carried out using the Light cycler 480 II real-time PCR thermocycler (Roche). Expression of mRNAs was quantified using LightCycler 480 SYBR Green I Master Mix (Roche) (Table S6) and normalized using GAPDH expression according to the 2 -∆∆Ct method. Reverse transcription of miRNAs was performed using TaqMan MicroRNA RT kit (no. 4366596, Life Technologies). miRNA RT-qPCR was carried out using 7900HT Fast Real-Time PCR System (Applied Biosystems). The expression of mature miR-155 and RNU6B was assessed using qPCR primer sets (Life Technologies, cat.no. 4427975-002623 and 4427975-001093, respectively).

Western blot. Cells were washed in 1X PBS, harvested and total proteins were directly extracted in Laemmli buffer (62.5 mM Tris-HCl pH 6.8, 1% SDS, 0.1 M DTE). Twenty µg of total proteins were separated on a 12 % SDS-PAGE and transferred on a nitrocellulose membrane using a semi-dry transfer apparatus. Membranes were saturated with 5% milk and incubated with mouse monoclonoal antibodies against HIVEP2 (ab70599, AbCam), p53 (DO-1, ab1101, AbCam) and Ku80 (ab87860, AbCam). Detection was performed with the secondary anti-mouse antibody (A4416, Sigma) with Clarity Western ECL substrat kit using ChemiDoc Imager (BioRad).

Luciferase reporter assays. 500 ng of either miRNA control plasmid (pMSCV-puro-GFPmir-CTL) or miR-155 expression vector (pMSCV-puro-GFP-mir-155) was co-transfected with 200 ng of either HIVEP2 reporter plasmid (pMIR-REPORT-dCMV 3' UTR HIVEP2) or control reporter plasmid (pMIR-REPORT-dCMV) into HCT-116 or HCT-116 p53-null cells using Lipofectamine reagent (Invitrogen) following the manufacturer's procedure. Firefly luciferase activity was used to monitor 3'UTR activity while Renilla luciferase activity was used as a control of transfection efficiency. 10 5 cells were seeded 24 hrs prior transfection and were treated with 5-FU 24 hrs post-transfection for additional 24 hrs before analyzing luciferase activity using Dual Reporter Luciferase Assays (Promega). The plasmids are a kind gift of Dr. Erik K. Flemington laboratory.

Statistical analyses

Statistical analyses were performed using GraphPad Prism 5.0a (GraphPad Software, Inc).

Mean comparison was performed using Student t-test. Correlation between translational index (TI) issued from DNA microarrays and RT-qPCR was tested using the non-parametric Spearman r test. Enrichment of mir-155 target genes was investigated by Chi-square test. All translationally deregulated mRNAs in response to 5-FU. Among the 12,131 genes commonly detected on the eight Affymetrix exon-arrays (NP-CTL, P-CTL, NP-5-FU, P-5-FU -each in duplicates), 2.6% (n=313) were significantly deregulated at translational levels (TI cut-off = 1.5, P < 0.05). Among these translationally deregulated genes, 9% were down-regulated and 91% up-regulated. These data were issued from two independent experiments. (B) Distribution of the Translational Index (TI). Among the 313 genes significantly deregulated at translational levels, TI varies from -2.5 to 2.9. TI of HIVEP2 mRNA is indicated. For list of genes translationally deregulated see Table 1 and Tables S1 andS2. S3.

PFigure 1 .

 1 Figures legends

Figure 2 .

 2 Figure 2. Impact of 5-FU treatment on protein synthesis in HCT-116 cells. (A-B) Global protein synthesis in response to 5-FU. Protein synthesis was quantified by 35 S labeling pulsechase assays in non-treated and 5-FU treated cells. A representative gel is shown in (A) and mean quantification of three independent experiments is shown in (B). Compared to nontreated cells, a reproducible decrease in protein synthesis was observed in response to 10 µM of 5-FU for 24 hrs. Cycloheximide (CHX) was used as a positive control. (C-D) Polysome profiles in response to 5-FU. 40S and 60S ribosomal subunits, 80S monosomes and polysomes were separated by ultracentrifugation on sucrose gradients. One representative polysome profile of non-treated (C) and 10 µM 5-FU treated cells (D) is shown, as well as gel analysis of 18S and 28S rRNA used to verified RNA quality. On top of each profile, the fractions collected for microarray analyses (non-polysomal NP and polysomal P) are indicated. After RNA extraction, RNA quality was checked using bioanlayzer, the RNA Integrity Number (RIN) ranging from 6.6 to 9.3.

Figure 3 .

 3 Figure 3. Effect of 5-FU on translatome of HCT-116 cells. (A) Percentage of

Figure 4 .

 4 Figure 4. Validation of translatome profiling in a panel of colorectal cancer cell lines. (A) Comparison of TI determined by Affymetrix exon-array and RT-qPCR in HCT-116 cell line. Validation in HCT-116 cells was performed by calculating the TI of 11 genes in response to 24 hrs exposure to 10 µM 5-FU using RT-qPCR on polysome profiles used for DNA microarrays analyses (two replicates) and additional, independent fraction preparation (at least two additional replicates). Similar down-and up-regulation were observed. (B) Correlation between TI calculated from Affymetrix exon-array and RT-qPCR in HCT-116 cells. Mean TI obtained by microarrays and RT-qPCR were plotted and correlation was assessed by Spearman correlation. A significant correlation was observed between Affymetrix exonarray and RT-qPCR data. (C) Comparison of TI in a panel of colorectal cancer cell lines in response to 10 µM of 5-FU for 24 hrs. TI of 7 genes determined by Affymetrix exon-array in HCT-116 cells (black bars) and determined by RT-qPCR in HCT-116 p53-null cells (white bars) and in HT-29 cells (striped bars) were compared. All the 7 genes showed similar translational modulation in both HCT-116 and HCT-116 p53-null cells. Only 3 genes out of 7showed no translational dysregulation in HT-29 cells, whereas the 4 other genes showed similar translationnally up-and down-regulation in response to 24 hrs of 5-FU exposure in the three cell lines. Graph presents mean and SD. TI below cut-off used for microarrays analyses were represented by grey area.

Figure 5 .

 5 Figure 5. Gene ontology analysis of translationally deregulated genes in 5-FU treated HCT-116 cells. GO enrichment was determined using the Functional annotation clustering tools from DAVID. Translationally up-regulated genes are mainly involved in transcription and translation, while translationally down-regulated genes are involved in DNA replication. Only the 3 first terms were shown for all clusters identified (up-regulation: 5 clusters; downregulation: 1 cluster). For complete data see TableS3.

Figure 6 .

 6 Figure 6. Translation regulation of HIVEP2 mRNA by mir-155 through its 3'UTR under 5-FU treatment in HCT-116 cells. (A) Distribution of HIVEP2 mRNA in polysomal and nonpolysomal fractions. HIVEP2 mRNA levels was quantified in polysomal (mRNA actively translated) and non-polysomal fractions (free and poorly translated mRNA) by RT-qPCR in two additional and independent polysome separation. Increase in HIVEP2 mRNA levels was observed in polysome in 5-FU treated HCT-116 cells compared to non-treated HCT-116 cells. (B) Variation of HIVEP2 mRNA levels in response to 5-FU in HCT-116 cells. HIVEP2 mRNA levels were quantified by RT-qPCR in both total and cytosolic RNA. 5-FU treatment significantly reduces HIVEP2 mRNA levels. (C) Expression of HIVEP2 protein in response to 5-FU in HCT-116 cells. HIVEP2 protein levels were analyzed by Western blot. 5-FU treatment increases HIVEP2 protein levels. (D) Expression of mature mir-155 under 5-FU treatment. Mature mir-155 expression levels were quantified by RT-qPCR in treated and untreated HCT-116 cells. 5-FU treatment significantly reduces mature mir-155 expression levels. (E-F) Analysis of HIVEP2 translational regulation through its 3'UTR using luciferase reporter assays. Luciferase activities were measured to determine the role of mir-155 on HIVEP2 3'UTR-dependent translation in response to 0 or 10 µM 5-FU for 24 hrs (E). mRNA levels of Firefly and Renilla genes were analysed to verify that variation in luciferase activity shown as F-Luc/R-Luc (RLU) is not due to variation of F-Luc/R-Luc at mRNA levels (F). (G) Enrichment of mir-155 target genes in translationally dysregulated genes. Statistical analyses showed that mir-155 target genes were enriched in translationally up-regulated genes in response to 5-FU. The list of mir-155-target genes translationaly up-regulated is given in the box. Graphs present means and SD of at least two independent experiments. . *: P < 0.05; **: P < 0.01; ***: P < 0.001.

Table 1 . List of the 25 most translationally dysregulated genes in response to 10 µM 5- FU in HCT-116 cells.

 1

	Gene symbol	Translational Index	P-value
	Translationally up regulated mRNAs
	SLC10A5	2.852	8.80E-07
	SERTAD2	2.413	4.60E-09
	FRK	2.325	0.00E+00
	AC005035.1	2.254	0.00E+00
	HIST1H2AM	2.216	4.19E-04
	ANP32B	2.191	1.00E-10
	C4orf14	2.153	0.00E+00
	KLF7	2.142	4.16E-08
	FAM123B	2.128	8.25E-08
	MT-ND1	2.106	1.96E-03
	C12orf5	2.105	0.00E+00
	ZNF502	2.086	2.50E-04
	RIPK2	2.078	0.00E+00
	HIVEP2	2.073	0,00E+00
	GPX2	2.064	9.81E-03
	ZNF385B	2.052	0.00E+00
	TRIM4	2.047	0.00E+00
	MAP3K13	2.042	0.00E+00
	C14orf126	2.035	6.00E-09
	PLEKHM3	2.032	0.00E+00
	MRPS18C	2.032	6.74E-04
	CPEB4	2.024	0.00E+00
	B3GALT1	2.024	8.81E-03
	FAM200A	2.022	3.17E-06
	GNPNAT1	2.018	0.00E+00
	Translationally down regulated mRNAs
	CDKL4	-2.455	5.84E-05

Acknowledgments

The authors thanks Dr. Erik K. Flemington laboratory for providing the luciferase reporter plasmids.

Funding

This work was supported by CNRS, INSERM, Université Claude Bernard Lyon 1 and Centre Léon Bérard. This project was funded by Ligue Contre le Cancer Comité du Puy-de-Dôme (PPE 2016, PME 2017). ZBI was a recipient of PhD program from Syrian government, GT was a recipient of PhD fellowships from Ligue Nationale Contre le Cancer (LNCC) and

Conflict of interests

The authors declare no conflict of interests. Fondation ARC, FL was a recipient of PhD fellowship from French Ministry of Research, MPE was a recipient of PhD fellowship from Association Française contre les Myopathies (AFM).


Dr Stéphane Arbault
email: stephane.arbault@u-bordeaux.fr

Pauline Lefrançois

Bertrand Goudeau

Dynamic monitoring of a bi-enzymatic reaction at a single biomimetic giant vesicle

Giant unilamellar vesicles were used as individual biomimetic micro-reactors wherein a model bi-enzymatic reaction involving a glucose oxidase (GOx) and the horseradish peroxidase (HRP) was monitored by confocal microscopy. These giant vesicles were formed from a natural mix of phospholipids in physiological conditions of pH and osmolarity (phosphate buffer, pH 7.4, 330 mOsm). The so-called Amplex Red assay, which generates the highly fluorescent resorufin species, was performed in individual vesicles and used to report on the progress of the whole reaction. We aimed at controlling kinetically and quantitatively the different steps of the bi-enzymatic reaction in vesicles. To do so, substrates (glucose and Amplex Red) were provided in individual reactors by two ways. Electro-microinjection allowed to control volume variations owing to a reservoir of lipids connected to the vesicle membrane. Alternatively, substrates could passively diffuse from the surrounding medium to the vesicle compartment. The semi-permeability feature of the phospholipidic membrane was characterized for all substrates and products while we demonstrated that enzymes remain sequestrated in the vesicles after their injection. The Amplex Red assay was thus achieved in individual vesicles under steady-state conditions, and could pursue over tens of minutes. Such giant vesicles are stable, fully compatible with media used for bioanalyses and allow out-of-equilibrium reactions at time-scales compatible with living reaction dynamics, making them a good choice for the development of minimal cell-like systems.

Introduction

Liposomes are widely used as bio-compatible carriers , rehydration [12][13][14] , microfluidic 15,16 , etc.). Phospholipid unilamellar vesicles are used to perform diverse biochemical and biological reactions 17 . However, when compared to biological cells, these synthetic systems often lack a transport of reactants and products between the inside of vesicles and their external environment. Consequences are that reaction dynamics cannot be studied due to a fast runout of encapsulated substrates (contained in nanolitre volumes), or when reactions stop when reaching their thermodynamic equilibrium 18 . To overcome this major limitation and reach steady-state activities inside these reactors, a continuous transport of substrate(s) from the outside to the inside of the compartment is required. This can be facilitated by membrane proteins 19 such as pore-forming toxins (α-hemolysin) 15,[START_REF] Noireaux | Proc. Natl. Acad. Sci[END_REF] or peptides (melittin) [START_REF] Li | [END_REF] , which allow passive transport of molecules up to ~5 nm in diameter. Alternatively, semipermeable membranes allow the selective diffusion of certain molecules (typically small molecules below 200 g•mol -1 molecular weight, or 1 nm in diameter) while larger ones (diameter >1 nm, e.g. enzymes or nucleic acids) remain entrapped in the micro-reactor. Giant unilamellar vesicles (GUVs: 10-200 µm diameter) have raised growing interest as biomimetic reactors, owing to their size, compatibility with proteins and with usual biochemical aqueous buffers, a control of passive diffusion across their membrane and ease for in situ analytical monitoring, particularly by optical microscopy. Herein, we report the preparation of GUVs from a natural phospholipid mix, with semi-permeable membrane properties and use them to monitor a bi-enzymatic reaction in real-time and in individual vesicles. GUVs were formed by a dehydrationrehydration method 12 . With this technique, GUVs grow on lipid reservoirs. The connection between the membrane and the reservoir allows the transfer of lipids from one to the other. Consequently, the GUV membrane surface can increase or decrease as a function of internal volume changes during microinjection or subsequent to osmotic changes. Enzymatic reactants were either directly encapsulated during GUVs growth when they were solubilized in the rehydration solution,

ARTICLE Analyst 2 | Analyst, 2020,xx
This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins Please do not adjust margins or microinjected inside vesicles to trigger the reactions. The first approach allows the incorporation of species at a fixed concentration inside all the GUVs whereas the second enables the encapsulation of defined volumes in single vesicles. These were used to achieve a model bi-enzymatic reaction coupling the glucose oxidase (GOx) and horseradish peroxidase (HRP) reactions. Reaction dynamics were monitored by confocal microscopy using the fluorescent Amplex Red assay. The assay was conducted systematically by incorporating each reactant in vesicles using passive diffusion and microinjection. We monitored GOx and HRP activities in single GUVs under steadystate condition for up to 30 min. Our study shows how simple liposomes can be used as bioreactors by combining diffusion and electro-microinjection, paving the way for more complex enzymatic pathways to mimic out-of-equilibrium cell-like activities. . Briefly, a natural extract from soybean (see the Chemicals section) was solubilized in pure chloroform at 10 mg•mL -1 in a 10 mL pear-shaped flask. The solvent was evaporated with a rotary evaporator during 3 to 5 h (80-100 rpm) leading to the formation of a lipid film. KPi buffer (see composition in the Chemicals section) was added in the flask allowing a gentle hydration of the film (~12 h at 4°C). Glycerol was then added to the suspension (10 vol%). Finally, a sonication step allowed the formation of SUV-MLVs (sonication bath, 3 min). GUVs were obtained from this preformed liposome suspension by a second dehydration-rehydration step. 5 µL of the suspension were dropped onto a glass coverslip and dehydrated for 30 min in a vacuum desiccator using a vacuum membrane pump. The dehydrated lipids form a bevelled-edge transparent film with salt crystals in the centre. The presence of glycerol prevents from complete dehydration of the lipids. Final rehydration was obtained by addition of PBS (0.5 mL) onto the coverslip. Osmotic gradient allows the swelling of GUVs on lipid reservoirs. After 10-15 min of equilibrium, the vesicles suspension was transferred onto coated glass slides (see next section). After ~10 min, vesicles had sedimented and were immobilized on the slide by interaction with the coating material (see next section). 12 . The coated film thickness is estimated at ~3 µm according to supplier's tables and spin speed used here. Microscopy observations. All images were obtained on an inverted laser scanning confocal microscope (Leica TCS-SP5 II) with a 20x dry objective (HCX PL Fluotar L, IP1, NA 0.4). We used an argon laser (output power 20%) to excite resorufin at 514 nm (acousto-optic tunable filter, 30%). Emission was collected within the adjusted wavelength window of 550 to 710 nm. Detection was done with a photomultiplier set to a gain of 800 V. Experiments were monitored in time-lapse mode with a 1.47 s time-interval between acquisitions (700 Hz frequency, 1024x1024 pixels window; no averaging nor accumulation). Electro-microinjection. Microinjection experiments were performed with manual micromanipulators using mechanical and piezoelectric control of translation stages (ThorLabs PCS-5200). Vesicles were electro-microinjected with a glass micropipette formed from a glass capillary (WPI, TW100F-4) pulled with a pipette puller (PC-100 model, Narishige, Japan) with parameters preliminary determined to provide pipette tips with a diameter below 1 µm (heat level = 82, force = 2.45 N). A platinum wire (Good Fellow PT005140) was inserted in the pipette and another one was positioned randomly in the solution used as a counter electrode. Both wires were connected to an electric pulse generator (Digitimer DS2A-MkII). When the tip of the pipette was in touch with the vesicle membrane, the application of electric pulses (40 V, 20 ms) allowed the formation of a local pore in the membrane 12,22 (not observable here). The pipette was moved simultaneously to application of pulses leading to its penetration inside the vesicle. Finally, the solution contained in the pipette was

Experimental

Please do not adjust margins Please do not adjust margins injected with an air injector (Eppendorf FemtoJet). One should note that this type of injectors doesn't allow the injection of a pre-set volume, but instead the injection is based on an injection pressure and duration. Pulling pipettes present variability in their tip opening size (Figure S1), thus variations of solution flow during injection for a specific pressure and injection duration were observed. Consequently, the injected volumes and therefore the quantities of injected reactants were systematically calculated based on the GUV diameter and inner volume increases after each injection and for each experiment.

Image analyses. All images were analysed using the software ImageJ

23

. For the cross-section profile analysis, a line-type region of interest (ROI) was drawn with a thickness of 18.3 µm across a GUV. For time lapse experiments, circular ROIs were drawn to analyse the fluorescence at the equatorial plane of the microinjected GUVs. Each experiment was reproduced on at least ten different GUVs (not necessarily from the same sample). The fluorescence intensity displayed on the figures corresponds to the raw data without further processing, whereas time offsets were applied to set the start of the experiment at t = 0. Each vesicle and each micropipette being different from one another (mostly in size and tip opening diameter respectively), the reported fluorescence intensity varied between replicates but the trend remained the same for all replicates of a specific experiment. We therefore show the analysis of only 1 GUV per experiment, representing the general observed behaviour for each condition. In most of the results, fluorescence decays are observed after the injection of resorufin or after the onset of the enzymatic reactions. To assess for the effect on fluorescence variations of resorufin photobleaching versus its diffusion from the vesicle to the outer solution, all decays were fitted with one and two exponential decay curves. Results of treatments for each curve are reported in Supplementary Materials (Figures S7 to S10).

Results and discussion

We studied at the level of single GUVs a bi-enzymatic reaction involving a glucose oxidase (GOx, 160 kDa dimer) and the horseradish peroxidase (HRP, 40 kDa). The first enzyme catalyses the oxidation of β-D-glucose to D-glucono-δ-lactone with a production of hydrogen peroxide (H 2 O 2 ). This is a well characterized reaction 24,25 , used herein as a model. Then, hydrogen peroxide serves as a substrate to HRP, which oxidizes Amplex Red (AR; N-acetyl-3,7-dihydroxyphenoxazine), to form the highly fluorescent species resorufin 26 (Figure 1).

The AR assay is commonly used in bioanalytical techniques to assess hydrogen peroxide released by cells and biochemical activities due to oxidases 27 . Also, it was used to monitor enzymatic reactions in micro and nano-volumes 28 . However, even though the AR assay is considered as a routine technique, it has been demonstrated that precautions should be taken to ensure accurate data interpretations 29,30 .

The main recommendation is to avoid high concentrations of hydrogen peroxide. Above 100 µM H 2 O 2 , enzyme inactivation (HRP) or substrate inhibition (Amplex Red) are described 29 . Thus, we did a preliminary bulk spectroscopic study, which provided us with the optimal conditions to get the fastest kinetics of the bienzymatic system (Figures S2, S3 andS4) and be able to later observe a dynamic fluorescence report of the AR assay in GUVs. The Amplex Red assay was conducted in single vesicles following a step by step method. Each result presented below is the typical trend obtained for each experiment. Every experiment was repeated at least with ten vesicles. Trends were very reproducible at each step although we observe differences in the fluorescence intensity amplitudes between different experiments. This is explained by the differences of size between each GUV and each micropipette, resulting in various reactant final concentrations inside a GUV. As function of these individual parameters, convection and mixing times after injections might therefore vary slightly before reaching steady-state. Such variability is inherent to any single vesicle study with a population poly-dispersity.

The first tested compound was the end product of the assay, resorufin. Preformed vesicles were dehydrated and rehydrated with PBS (10 mM, pH 7.4) resulting in the formation of a GUV connected to a lipid reservoir (bright spots on Figure 2A). A GUV was then microinjected with a resorufin solution (Figure 2). The cross-section fluorescence profile obtained by laser scanning confocal microscopy (LSCM) at the equatorial plane of the vesicle (Figure 2A andB), shows a maximal intensity at its centre, a sharp decay at the membrane, due to the partition coefficient of the membrane for resorufin, and a fluorescent halo surrounding the GUV due to the molecule diffusion toward the outside. This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins Please do not adjust margins Figure 3 depicts the variations of the fluorescence intensity inside a GUV upon sequential micro-injections of a resorufin solution (initial concentration of 2.5 µM in the pipette). The final concentration of resorufin in the GUV is calculated as 0.24 ± 0.08 µM (details in table S1, ESI). We observed a fast increase of the fluorescence right after the injection, followed by its rather fast decrease immediately after the end of injection. Each time, the signal reached back the background value in ~1 min, driven by the concentration gradient between the outside and the inside of the vesicle (inner volume: 2-3 nL; outer volume: 500 µL). This experiment was repeated at least 3 times per vesicle, and for several tens of them. We always obtained the same profiles. The fluorescence decays may be attributed to the simple diffusion of resorufin and/or its photobleaching due to laser exposure (see experimental section). The effect of photobleaching was shown as minimal based on two results: 1. when a solution of resorufin (up to 50 µM in PBS), even in the presence of GUVs (Figure S5), was exposed under the same experimental conditions as the above, the local decrease of fluorescence intensity was weak (below 5 %. over a 1 min timescale); 2. the fluorescence decays were fitted with exponential decay functions (one or two components; see Figure S7) to assess for the effect of photobleaching. In this case, and in further experiments, a very slow decay function could possibly be attributed to the dye photobleaching, though a diffusion kinetic was mainly observed. Moreover, the flux of resorufin provided by the pipette in the GUV led, after each injection, to an ensuing fluorescence in the solution surrounding the GUV (as shown also on Figure 2), which shows that resorufin diffuses within a second time-scale across the membrane of these giant liposomes. Resorufin permeation is indeed reported as dependent on membrane compositions (phospholipids, sterols, proteins) of vesicles, interfaces or cells 28,31 .

At this initial stage of the study, several control experiments were made to check whether the microinjection and electrical pulses may induce damages to the membrane resulting in the formation of pores and an increased permeability. GUVs were prepared in PBS followed by an addition of resorufin in their outer solution. In less than 30 seconds, all GUV lumina were filled with resorufin (Figure S5). The same experiment was also performed with fluorescein and calcein fluorophores; both are anionic at pH 7.4 but calcein is a slightly larger molecule than fluorescein (6.5 Å Stocks' radius for calcein 32 and ~5 Å for fluorescein 33 ) (Figure S5). A similar filling of GUVs was observed within less than a minute for both molecules. This demonstrates that the semi-permeable feature is intrinsic to the GUVs, according to their preparation method and composition, and is not influenced by electric pulses, nor to membrane tension due to microinjection. In order to evaluate if the permeability would be associated with the presence of transient pores, we performed a negative contrast experiment, similar than before, with a much larger molecule, a Dextran-FITC (70 kDa; Stocks' radius 6.0 nm 34 ). A progressive diffusion of Dextran-FITC toward the vesicle interior was also observed, though at a very slow rate (several hours), notably compared to the size of the vesicles. One should note as well that Dextran polymers are coil-shaped (length 44 nm, width 2.5 to 3 nm 35 ) and are known to follow reptation movements 35 , transient unfolding and slow diffusion of Dextran chains through small pores in membranes.

These results indicate that the membrane of GUVs is semipermeable essentially to small hydrophilic molecules, including resorufin, calcein and fluorescein. Moreover, the diffusion of these fluorophores across the membrane is homogeneously distributed, either around GUVs after internal microinjections (Figure 2B), or inside their lumen during negative contrast experiments (Figures S5 andS6). This shows that no specific defects at the interface between the vesicle and the lipid reservoir induce any leakage of the vesicle content during the following experiments. The AR reaction was then performed in single GUVs. Vesicles were grown on lipid reservoirs by rehydration in PBS supplemented with 10 µM H 2 O 2 (the reaction substrate, see Figure 1 bottom part). Hydrogen peroxide was thus present in the inside and outside of the liposomes, and was prone to diffuse passively across the membrane, as previously reported

. We injected a mix of the co-reactant AR (6.8 ± 3.2 µM final) and HRP enzyme (0.03 ± 0.01 U•mL -1 final) in single GUVs. The local fluorescence signal evolution was followed by LSCM (Figure 4). We observed a fast fluorescence increase subsequent to the injection. After injection was stopped, the signal decreased progressively to reach the background level. Five successive injections were performed in the same vesicle, all showing the same response profile and thus the reproducibility of the response. Similar to Figure 3, the fluorescence decays observed here were analysed kinetically (Figure S8) and could be attributed mainly to the passive diffusion of resorufin across the membrane. Thus, we may conclude that a short injection (5 s) provides only a little amount of AR that is immediately consumed by the reaction (table S2, ESI). AR may also progressively diffuse outside of the GUV. When missing its substrate, the reaction stops very fast after the injection and since resorufin is not sequestrated, spikes of fluorescence are observed. In this first experimental condition; despite the AR enzymatic reaction lasts at most one minute, it can be performed in individual vesicles with a fine temporal and quantitative control of the injected compounds.

Nevertheless, the diffusion of AR, and possibly of HRP, across the membrane need to be better understood (see below). A third experiment was designed to explore this latter point. Vesicles were formed by rehydration in PBS (10 mM, pH 7.4) containing 50 µM H 2 O 2 and 50 µM AR. Higher concentrations of both substrates were used in this experiment to maximize their in situ concentrations in vesicles and the observed This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins Please do not adjust margins fluorescence response. The conversion reaction started upon a single injection of HRP in a vesicle (Figure 5). Resorufin fluorescence intensity inside the GUV increased, indicating its effective enzymatic production. After 40 s of injection (corresponding to 270 pL volume; table S3, ESI), the fluorescence kept increasing for about 30 s and reaches a high intensity level. Then it slowly decreased, over more than 20 min in the representative example displayed here. The whole duration of resorufin production and fluorescence emission was dependent on the GUV dimensions and substrate quantities injected inside. Overall, this advanced set of experiments demonstrates the achievement of the AR assay in individual GUVs and its timeresolved monitoring. Several interpretations can be considered at this point to explain these results by comparison with Figures 3 and4. The decay rate in Figure 5 is 50-100-fold slower than on Figures 3 and4. Although diffusion of resorufin toward the external solution continuously occurs as well as a slight photobleaching (kinetic analyses in Figure S9), this result indicates that the production of resorufin inside the GUV is less limited by a substrate runout than previously. Since AR is encapsulated during GUV growth, the concentration inside the vesicle is assumed to be the one of the hydration solution (50 µM); whereas when the AR is injected together with the enzyme (Figure 4), the average concentration after injection is lower, estimated to ~7 µM. This 10-fold difference in concentration changes the reaction rate and allows the reaction to last longer in the case of Figure 5. Moreover, H 2 O 2 diffuses quickly across the phospholipid membrane and is then continuously provided to the inside of the vesicle. Therefore, if the reaction rate decreases over time due to a substrate runout, it would be most likely because of AR. However, previous studies have shown that AR is able to permeate across phospholipid membranes 36 , while HRP should not. In addition, one may argue that confinement in a vesicle affects the AR assay kinetics because of AR inhibition and/or HRP inactivation, although we optimized the reaction conditions in bulk (Figure S3) and limited hydrogen peroxide concentration to 50 µM. Therefore, the slow decrease of fluorescence observed on Figure 5, which represents the kinetic slowdown of the AR assay, is due either to a slow diffusion of AR across the membrane, or to a progressive inactivation or inhibition of HRP or AR, respectively. The next study will enable to address this point. The bi-enzymatic reaction, involving both GOx and HRP, was then performed in GUVs. A glucose solution (100 µM in PBS) was used to rehydrate the lipid film and grow the giant liposomes. The two enzymes (GOx and HRP) were mixed with AR and the solution was subsequently injected inside a vesicle (Figure 6). All concentrations were previously optimized in bulk by spectrophotometry to get a fast resorufin production (Figure S4) and to avoid side perturbations of the AR reaction. We set the GOx reaction condition as the rate limiting step so Please do not adjust margins Please do not adjust margins that any produced H 2 O 2 would be immediately consumed by the AR reaction, avoiding a steady-state oxidative environment. As shown in Figure 6, successive injections of the -GOX+HRP+AR-cocktail could be performed (details in table S4, ESI), leading each time to a fast increase of the resorufin fluorescence signal inside the GUV, followed by a decrease. After resorufin diffusion to the outside of the vesicle, the final fluorescence intensity reaches the background level (in 1 to 2 min, depending on the vesicle size). As previously, the fluorescence increase is associated to the resorufin production and demonstrates the proper working of the bienzymatic reaction inside the vesicles, which is here limited by AR runout.

The fluorescence decrease corresponds to passive diffusion of resorufin across the membrane, as photobleaching was not observed in this case (Figure S10). To complete this systematic study, a final set of experiments was performed. Here, the initial substrates (50 µM glucose and 50 µM AR) were encapsulated during the liposome growing step. The enzymes (GOx and HRP) were injected only once per vesicle (table S5, ESI) and repeated on at least ten vesicles and for various concentrations of substrates (see Figure S11). As shown with the typical obtained signal in Figure 7, we observed a regular rise of the resorufin signal inside the vesicle over the first minute. Then, the intensity remained stable for a long time, at least 15 min and up to 1 hour when monitored (long time experiments were not performed systematically). This plateau corresponds to a balance between the production of resorufin inside the liposome and its diffusion to the outside of the GUV. The steady-state concentration of resorufin was estimated at ~0.5 µM based on results from Figure 3. This mean concentration of produced resorufin, reported by the plateau intensity, was also shown to depend on the AR concentration in the solution (Figure S11). This journal is © The Royal Society of Chemistry 20xx

Please do not adjust margins Please do not adjust margins Finally, we demonstrated that we could manage a long-lasting generation of a compound (resorufin) produced by a bienzymatic reaction inside a single vesicle. Moreover, the observed steady-state response also provides clues on the diffusion of the substrates, glucose and AR across the membrane. We may expect a drop of fluorescence intensity after few min, caused by a substrate runout. However, the signal in the reactor was very stable for long durations (tens of minutes). This proves that glucose and AR substrates can diffuse across the membrane while the enzymes remain sequestrated. Our results confirm previous reports on the diffusion of AR [START_REF] Piwonski | Proc. Natl. Acad. Sci[END_REF] across lipid membranes but also show a sufficient permeability of our vesicles to glucose, which allows to perform stably the bienzymatic reaction inside. Usually, phospholipid bilayers are considered as weakly permeable to glucose (permeation coefficient of 10 -10 cm•s -1 ) 37 . However, in the vast majority of reported studies, liposomes are composed of ideal mixtures of one or two types of pure synthetic phospholipids (DPPC, POPC …). Herein, GUVs were formed from a natural mix (soybean polar extract) composed of phospholipids with a wide variety of polar heads (PC, PE, PI and PA) and fatty acid chains (DO, DP, PO…) (see the Chemicals section), to mimic the complexity of living cell membrane composition. With such a composition, formation of lipid rafts is possible 38 . A mix of fluidic areas where phospholipids have a low transition temperature T m and more rigid phases where T m is higher is expected. These spatial differences on the membrane might favour permeation to small molecules. Moreover, all present experiments were achieved in solutions of physiological osmolarity (with respect to eukaryotic cells) and at low sugar concentrations (10-100 µM) whereas usual GUV experiments are performed either in pure water or buffers supplemented with high sugar concentrations (200-300 mM sucrose). This last condition increases the solution viscosity and therefore slows down diffusion and reaction kinetics. As well, it affects the lipid diffusion in membranes and consequently hampers their permeation to small molecules 39,40 . This additionally explains the semi-permeable behaviour of the current GUVs as compared to other purely synthetic GUVs in other experimental conditions. Overall, this feature is used in the present study as an advantage to let small molecules, i.e. the enzymatic substrates, diffuse from the outer solution towards the internal volume and activate the enzymatic reactions. Additionally, one may argue that phospholipids could be oxidized and henceforth increase membrane permeability 41 . As we showed in control experiments, neither microinjection nor electric pulses are responsible for the semi-permeability of GUVs (Figures S5 andS6). Additionally, the presence of hydrogen peroxide in some of the experiments may lead to phospholipid peroxidation and consequently could modify the permeability of the liposomes 42 . However, the membrane semi-permeability was similar in presence or absence of hydrogen peroxide (Figures S5 andS6). The formation of transient nanopores is more likely to be the source of the observed semi-permeable feature, although these could not be observed directly in our microscopy experiments. The result from Figure 7 provides information allowing to clarify the previous results of Figure 5. First, we can conclude that the slow decay observed on Figure 5 is not likely due to the diffusion of HRP to the outside of the vesicle since we did not observe any fluorescence decay on Figure 7, for a similar experimental time-range. We showed that AR as a cosubstrate of HRP can permeate from the outside to the inside of the vesicle, however certainly slower than H 2 O 2 . Thus, the slow activity decay of Figure 5 Please do not adjust margins Please do not adjust margins 7, the Amplex Red assay can effectively be performed with micro-units of enzymes inside a stable nanolitre-volume reactor. Kinetic and quantitative accuracies are similar to bulk experiments but at the scale of an individual liposome.

Conclusions

The GUVs formed by swelling on a lipid reservoir allow single biomimetic compartment manipulation, injection and monitoring in usual conditions of biochemical analyses (buffers, reactants, concentrations, pH etc.). We characterized their peculiar permeability features (permeation of small neutral molecules, typically < 300 g•mol -1 ) and took advantage of these to probe a bi-enzymatic reaction inside single vesicles.

We believe that such compartments allowing out-ofequilibrium reactions may be useful to develop artificial celllike systems while avoiding the use of membrane proteins. In addition, the system is fully suitable to implement other analytical methods, such as in situ electrochemical detection by a 43 . The direct detection of hydrogen peroxide flux shall provide complementary quantitative information for the analysis of enzymatic activities.

3 . 4 ,

 34 Their membrane can be composed of various synthetic or natural phospholipids and fatty acids mimicking the basic structure of living cell membranes. The size of vesicles (small, large, giant) and the number of their bilayers can be adjusted depending on the preparation technique

Figure 1 .

 1 Figure 1. Scheme of the bienzymatic reaction used in this study. β-D-glucose is catalytically oxidized via glucose oxidase (GOx) to D-glucono-δ-lactone and hydrogen peroxide. Hydrogen peroxide then oxidizes the fluorogenic compound Amplex Red, catalysed by the horseradish peroxidase (HRP), to produce the fluorescent final product resorufin.

Figure 2 .

 2 Figure 2. Micro-injection of resorufin in a single GUV monitored by LSCM. A. Microscopy images of a GUV (connected to its lipid reservoir) before injection. B. Same GUV during injection of resorufin (injection duration = 30 s). Left: differential interference contrast image; Right: emitted fluorescence. Scale bar: 50 µm. Pseudocolour, lookup table (LUT): fire. The dashed zone corresponds to the region of interest used to plot the cross-section profile in C. C. Fluorescence profile across the GUV micro-injected with resorufin. The black trace is the 50-point moving average of the grey trace.

Figure 3 .

 3 Figure 3. A. Fluorescence intensity variations over time inside a GUV (corresponding to the indicated region of interest) injected 4 times with resorufin B. Microscopy images of a GUV connected to its lipid reservoir during injection of resorufin. Left: differential interference contrast image; Right: emitted fluorescence. Pseudo-colour, LUT: fire. Scale bar: 50 µm. The region of interest (dashed circle) used for the analysis of the radial fluorescence inside the GUV is delimited by a white dashed line. Injected volumes were estimated by size changes of the GUV to ~240 ± 50 pL per injection. Injection duration: 5 s. Initial concentration in the pipette: 2.5 µM; final concentration in GUV = 0.24 ± 0.08 µM. Initial GUV diameter: 155 µm; GUV diameter after 4 injections: 177 µm.

Figure 4 .

 4 Figure 4. A. Schematic representation of the Amplex Red assay performed in a single GUV. A vesicle containing H2O2 (10 µM) was injected with a mix of HRP (0.4 U•mL -1 initial concentration) and AR (50 µM initial concentration). The passive diffusion of H2O2 is depicted with a dashed double arrow. B. The resorufin production is reported by the mean fluorescence intensity inside the GUV over time detected by LSCM. C. Microscopy time-lapse images obtained by LSCM of a GUV during and after injection, showing the evolution of fluorescence intensity. Scale bar: 50 µm. Pseudo-colour, LUT: fire.5 successive injections were performed. The HRP and AR concentrations in the vesicle were calculated for each injection according to the injected volume and averaged for the 5 injections: 0.03 ± 0.01 U•mL -1 HRP and 6.8 ± 3.2 µM AR. Average injected volume for each injection = 130 ± 70 pL. Injection duration: 5 s. Initial GUV diameter: 145 µm; GUV diameter after 5 injections: 160 µm.

Figure 5 .

 5 Figure 5. A. Schematic representation of the Amplex Red assay in a single GUV. A hydrogen peroxide (50 µM) and AR (50 µM)-containing GUV was injected once with HRP (0.28 U•mL -1 final). B. The resorufin production is reported by the mean fluorescence intensity inside the GUV over time monitored by LSCM. C. Microscopy time-lapse images obtained by LSCM of a GUV during and after injection, showing the fluorescence intensity increase followed by a slow decay. Scale bar: 50 µm. Pseudocolour, LUT: fire. Injected volume: 270 pL. Initial GUV diameter: 60 µm; GUV diameter after injection: 90 µm.

Figure 6 .

 6 Figure 6. A. Schematic representation of the coupled bi-enzymatic assay in GUVs. A glucose (100 µM)-containing GUV is injected with a mix of GOx, HRP and AR; in the typical experiment displayed here, their respective concentrations in the vesicle were 0.9 U•mL -1 , 0.20 U•mL -1 and 46 µM after the first injection, and 1.3 U•mL -1 , 0.26 U•mL -1 and 35 µM after the second injection. Concentrations of enzymes are given assuming

Figure 7 .

 7 Figure 7. A. Schematic representation of the Amplex Red assay performed in a GUV. A GUV containing glucose (10 µM) and AR (50 µM) was injected once with a mix of GOx (4.76 U•mL -1 in situ concentration) and HRP (0.38 U•mL -1 in situ concentration). B. The resorufin production is reported by the mean fluorescence intensity inside the GUV over time detected by LSCM. C. Microscopy time-lapse images obtained by LSCM of a GUV during and after injection, showing the fluorescence intensity increase until reaching a steady-state. Scale bar: 50 µm. Pseudo-colour, LUT: fire. Injected volume: 630 pL. Initial GUV diameter: 39 µm; GUV diameter after injection: 108 µm.

  may result from a limited diffusion of AR across the membrane, reducing the production rate of resorufin over time. However, under well-chosen conditions of reactant concentrations such as shown in Figure Analyst ARTICLE This journal is © The Royal Society of Chemistry 20xx Analyst, 2020,xx 9

Preparation of microscopy slides. Glass

			slides were thoroughly
	cleaned with ethanol and milliQ water and dried under
	pressurized air flow. Surface activation was conducted with an
	oxygen plasma (PDC-002, Harrick Plasma, USA) for 10 min.
	SU8-2002 epoxy photoresist (Micro-chem) was then spin
	coated on the slides (100 rpm•s	-1 ramp up to 1000 rpm, then
	kept at this speed during 1 min). The photoresist was pre-
	baked at 95°C for 5 min on a hotplate, UV exposed at 40
	mW•cm	-2 during 8 s (Kloe, UV Kub) and post-baked at 95°C
	during 5 min on a hotplate. The SU8 substrate was chosen
	because it had already been used in similar experiments. It was
	shown to present a slight interaction with phospholipids which
	is sufficient to immobilize GUVs, without deforming or
	damaging the membrane

This journal is © The Royal Society of Chemistry 20xxAnalyst, 2020,xx 3

Acknowledgements

This work was financially supported by the Agence Nationale de la Recherche (ANR, NOSYNTHCELL project, ANR-RF-2013-01). The authors wish to thank Raphael Harismendy (Univ. Bordeaux) for preliminary experiments, Dr Adeline Perro (Bordeaux INP, ISM, UMR 5255, Pessac, France) for her help to prepare the microscopy slides and Dr Ann-Sofie Cans (Department of Chemistry and Chemical Engineering, Chalmers University of Technology, Gothenburg, Sweden) and her collaborators for discussions and experimental advices at the onset of this project.

Conflicts of interest

There are no conflicts to declare.