Maelle Bellec

Ruoyu Chen

Jana Dhayni

Cyril Favard

Antonello Trullo

Helene Lenden-Hasse

Ruth Lehmann

Edouard Bertrand

Mounia Lagha

Jeremy Dufourt
email: jeremy.dufourt@irim.cnrs.fr

Affiliations

Boosting the toolbox for live imaging of translation

Live imaging of translation based on a tag recognition by a single chain antibody is a powerful technique to assess the fine tuning of translation regulation in living cells. However, especially in a multicellular organism, this approach is challenging and requires optimization in terms of expression level and detection sensitivity of the system. Here, we improved existing fluorescent tools and developed new ones to image and quantify nascent translation in the living Drosophila embryo and in mammalian cells. We tested and characterized five different Green Fluorescent Protein variants fused to the single chain fragment variable (scFv) and uncover photobleaching, aggregation and intensity disparities. Using different strengths of germline and somatic drivers, we determined that the availability of the scFv is critical in order to detect translation throughout development. Finally, we introduced a new translation imaging method, based on a nanobody/tag system, named ALFA_array, allowing the sensitive and simultaneous detection of the translation of several distinct mRNA species.

Introduction:

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The discovery of the Aequorea victoria green-fluorescent protein [START_REF] Chalfie | Green fluorescent protein as a marker for gene expression[END_REF][START_REF] Prasher | Primary structure of the Aequorea victoria green-fluorescent protein[END_REF] and especially its optimized version mEGFP [START_REF] Zacharias | Partitioning of lipidmodified monomeric GFPs into membrane microdomains of live cells[END_REF] have revolutionized imaging of biological processes [START_REF] Rodriguez | The Growing and Glowing Toolbox of Fluorescent and Photoactive Proteins[END_REF] . Since then, several new FPs have been and continue to be discovered and engineered [START_REF] Lambert | Aequorea's secrets revealed: New fluorescent proteins with unique properties for bioimaging and biosensing[END_REF][START_REF] Bindels | mScarlet: a bright monomeric red fluorescent protein for cellular imaging[END_REF] . The constant race for better FPs generally implies the optimization for fast folding, pH resistance as well as improvement of photo-physics [START_REF] Leake | A guide to small fluorescent probes for single-molecule biophysics[END_REF] .

Introduction of the bacteriophage-derived MS2 RNA binding sites, which are recognized by MS2 Coat Protein (MCP) fused to FP, provided a technique to visualize transcription in living cell. This technological advance has been subjected to numerous improvements from its initial development in 1998 [START_REF] Bertrand | Localization of ASH1 mRNA particles in living yeast[END_REF] . These improvements include increased sensitivity down to single molecules [START_REF] Fusco | Single mRNA Molecules Demonstrate Probabilistic Movement in Living Mammalian Cells HHS Public Access Author manuscript[END_REF] , implementation in a living organism [START_REF] Forrest | Live imaging of endogenous RNA reveals a diffusion and entrapment mechanism for nanos mRNA localization in Drosophila[END_REF] , the generation of new stem loops to visualize single mRNAs at a high temporal resolution [START_REF] Tantale | A single-molecule view of transcription reveals convoys of RNA polymerases and multi-scale bursting[END_REF][START_REF] Dufourt | Imaging translation dynamics in live embryos reveals spatial heterogeneities[END_REF] , artefact-free RNA [START_REF] Tutucci | An improved MS2 system for accurate reporting of the mRNA life cycle[END_REF] and double mRNA species imaging with an orthologous system [START_REF] Hocine | Single-molecule analysis of gene expression using two-color RNA labeling in live yeast[END_REF] . However, several limitations exist with the present tools that hinder quantitative imaging. For example, protein aggregation has been observed for some systems derived from MS2 Coat Protein fused to GFP [START_REF] Weil | Making the message clear: visualizing mRNA localization[END_REF][START_REF] Tutucci | An improved MS2 system for accurate reporting of the mRNA life cycle[END_REF][START_REF] Pichon | New Generations of MS2 Variants and MCP Fusions to Detect Single mRNAs in Living Eukaryotic Cells[END_REF] and for scFv fused to specific GFP variants [START_REF] Tanenbaum | A proteintagging system for signal amplification in gene expression and fluorescence imaging[END_REF] . Furthermore, the expression level of the detector is critical to ensure accurate detection, particularly for translation, as most of the time one mRNA undergoes multiple rounds of translation. However, no such optimization has been made for the SunTag system in the context of a developing organism, although photo-physical properties of molecules can be variable between different systems (i.e. in vitro experiments, cell culture, tissues, organisms…) [START_REF] Leake | A guide to small fluorescent probes for single-molecule biophysics[END_REF][START_REF] Wörn | Correlation between in vitro stability and in vivo performance of anti-GCN4 intrabodies as cytoplasmic inhibitors[END_REF][START_REF] Donahue | An Optogenetic Tool to Raise Intracellular pH in Single Cells and Drive Localized Membrane Dynamics[END_REF] . We tested five different GFP variants for their brightness, photobleaching and propensity to aggregation in fixed and live embryos. We also generated inducible scFv-FP constructs that, when combined with different drivers to extend the use of the SunTag system to a wider range of genes and biological questions. Furthermore, we developed an orthologous method, called ALFA-array, to visualize nascent translation in Drosophila embryo and mammalian cells. This system is based on a small and soluble nanobody and we show its usefulness by combining it with the SunTag to visualize translation of two different mRNA species in Drosophila embryos. Results: New fluorescent protein fused to scFv to detect nascent translation. The SunTag method is a bipartite system based on an antibody-derived single chain variable fragment (scFv) detector fused to a fluorescent protein (FP; the detector) which binds a peptide named Supernova Tag (suntag) derived from the yeast GCN4 protein and are experimentally fused in multiple copies at the N-terminus of the gene of interest [START_REF] Tanenbaum | A proteintagging system for signal amplification in gene expression and fluorescence imaging[END_REF] (Figure 1A). To improve the system, we created new scFvs with different green fluorescent proteins under the control of nanos (nos) enhancer-promoter (EPr). This EPr drives expression in the Drosophila germline 38- 41 allowing the maternal deposition of mature scFv-FP into the laid eggs (Figure S1A). We generated a set of four different constructs, in addition to the previously published scFv fused to the superfolder GFP (sfGFP) [START_REF] Pédelacq | Engineering and characterization of a superfolder green fluorescent protein[END_REF] , named scFv-sfGFP [START_REF] Dufourt | Imaging translation dynamics in live embryos reveals spatial heterogeneities[END_REF] . The new generation of green fluorescent protein variants includes: i) the monomeric superfolder GFP2 (msGFP2) [START_REF] Valbuena | A photostable monomeric superfolder green fluorescent protein[END_REF] , ii) the mGreenLantern (GL) [START_REF] Campbell | mGreenLantern: a bright monomeric fluorescent protein with rapid expression and cell filling properties for neuronal imaging[END_REF] , iii) mNeonGreen [START_REF] Shaner | A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum[END_REF] and iv) the monomeric A. victoria fluorescent protein 1 (mAvic1) [START_REF] Lambert | Aequorea's secrets revealed: New fluorescent proteins with unique properties for bioimaging and biosensing[END_REF] (Figure 1B, upper panel). Live imaging of these five scFv-FPs during embryonic development until the end of nuclear cycle 14 (n.c. 14) showed that all the scFv-FPs are well expressed, localize mainly in the nucleus, due to the presence of a nuclear localization signal, and do not form aggregates (Figure S1B and Movie S1-5). Quantification of the fluorescent intensity during the first forty minutes of n.c.14, showed that the apparent brightness of these four new scFv was similar to the scFv-sfGFP, while scFv-mAvic1 harbors a brighter signal (Figure S1C). Although, we observed that scFv-GL is prone to photobleaching (Figure S1C). We confirmed that the distribution of scFv-FP was homogenous in the embryo and (A) Schematic of the SunTag system. Once the mRNA (dark grey) is translated by the ribosomes (grey), the suntag epitopes (light blue) will be bound by the single chain variable fragment (scFv) (dark blue) fused to a fluorescent protein (FP) (green). Therefore, the nascent peptide will be detected by the accumulation of fluorescent signal. (B) (top) Schematic of the different constructs of scFv-FP generated in this study. scFv-msGFP2, scFv-GreenLantern, scFv-mAvic1 and scFv-NeonGreen were created for this study and scFv-sfGFP in our previous study [START_REF] Dufourt | Imaging translation dynamics in live embryos reveals spatial heterogeneities[END_REF] . (bottom) Schematic of the CRISPR/Cas9 targeted insertion strategy to obtain endogenous twist gene tagged with suntag and MS2 [START_REF] Dufourt | Imaging translation dynamics in live embryos reveals spatial heterogeneities[END_REF] . (C) (top) Schematic of a sagittal view of a Drosophila embryo with twi expression pattern in purple.

Anterior side is on the left, posterior on the right, dorsal on the top and ventral side on the bottom. Black square represents the imaged area of the bottom panels. (bottom) One Z-plane of confocal images from smFISH with direct fluorescent protein signal in green (scFv-msGFP2, scFv-GreenLantern, scFv-mAvic1, scFv-NeonGreen and scFv-sfGFP) and MS2 probes (red) on scFv-FP x twi_suntag_MS2_CRISPR embryos in early-mid n.c. 14

New scFv-FP prevents aggregates formation

To show whether scFv-FPs expressed under the maternal nos EPr could detect translation during embryogenesis, at least until gastrulation, we imaged fixed embryos at the gastrulation stage. We observed tagged Twist protein localized to nuclei in the ventral gastrulation furrow region, consistent with Twist being a mesodermal transcription factor (Figure S2B, upper panel). To test, whether the presence of a NLS in the scFv-FP construct might be responsible for this nuclear localization, we imaged the localization of the Insulin-like peptide 4 (Ilp4), an excreted protein expressed in the mesoderm [START_REF] Brogiolo | An evolutionarily conserved function of the drosophila insulin receptor and insulin-like peptides in growth control[END_REF] . We used the tagged Ilp4 with suntag and observed scFv containing the NLS in the cytoplasm (Figure S2B, lower panel). Thus, the presence of a NLS in the scFv-FP construct appears not to significantly interfere with the native localization of tagged proteins. Strikingly, at the gastrulation stage, bright GFP spots were observed only when using scFv-sfGFP in twi_suntag_MS2_CRISPR embryos but not with the other scFv-FPs (Figure S3A). As only the FP sequences is different between these constructs, this discrepancy might be due to a difference in protein stability, scFv-sfGFP being more stable than the other variants, or that scFv-sfGFP being more prone to aggregation. To decipher between these hypotheses, we used smFISH to label twi_suntag_MS2_CRISPR mRNA in the presence of the different scFv-FPs to determine whether the bright GFP spots observed during gastrulation co-localize with mRNA and represent translation spots. With scFv-sfGFP we saw large green spots that were not colocalized with mRNA (Figure S3B upper left panels, white arrowhead). We did not observe these large spots with the any of the four new scFv-FPs (Figure S3B) suggesting an aggregation of the scFv-sfGFP in the Drosophila embryo at the gastrulation stage in presence of twi_suntag_MS2_CRISPR.

We next asked whether aggregation formation is specific to the tagged twi in developing embryos or could also occur in cultured cells with a different suntag construct. We expressed scFv-sfGFP and scFv-msGFP2 in Drosophila S2R+ cells along with a SunTag-luciferase reporter (SunTagRLuc-MS2) (see methods, Figure S3C). The SunTagRLuc-MS2 plasmid (see methods) was co-transfected with an scFv-FP plasmid as well as an MCP-Halotag plasmid (see methods) to track single mRNA molecules and their translation during live-cell imaging. As expected, scFv-FPs were able to label polysomes by forming GFP foci co-localizing with mRNA (Figure S3D). With the scFv-sfGFP, however, we frequently observed GFP aggregates which were not associated with mRNA. These aggregates were morphologically distinct from translating foci and were more abundant and bigger than the GFP aggregates observed in gastrulating embryos (Figure S3D-E). This result suggests an aggregation of the SunTag peptides bound by the scFv-sfGFP after being translated and released from polysomes. Although few aggregates were also present in cells expressing scFv-msGFP2 with the SunTagRLuc-MS2 reporter (Figure S3D) this occurred with a significantly lower frequency (~55% of cells without aggregates compared to ~15%) and smaller aggregate size, suggesting that msGFP2 is less prone to aggregate formation. These results support that the scFv-FPs containing newer generation FPs are more suitable for translation analysis, as they are less likely to form aggregates.

Monitoring translational arrest during Drosophila embryogenesis

The low amount of translation spots observed during gastrulation suggested that twi translation maybe have ceased at this stage of development (Figure S3A-B). However, a twist mRNA translational arrest had not been described previously. This unexpected observation led us to investigate whether twi transcripts were undergoing a translational arrest during n.c.14 (Figure 2A) using the four new scFv-FPs. Interestingly, we found at mid-late n.c. 14 (Figure 2A) that the twi_suntag_MS2_CRISPR RNA was mostly translated at the border of the mesoderm (Figure 2B and S4A-C). In comparison, polysomes dots in the center of the mesoderm were less intense suggesting reduced translation (Figure 2B and S4A-C). A translational arrest was recently described for Hunchback (Hb) using the SunTag system in the Drosophila embryo [START_REF] Vinter | Dynamics of hunchback translation in real-time and at single-mRNA resolution in the Drosophila embryo[END_REF] . In this study the authors showed that after being translated in the entire Hb domain (anterior half of the embryo), hb translation became restricted to the border of its expression pattern, similarly to what we observed for twist. At least two models can explain this result: i) twi translation is actively repressed at first in regions where the RNA and protein expression is highest (as suggested for hb [START_REF] Vinter | Dynamics of hunchback translation in real-time and at single-mRNA resolution in the Drosophila embryo[END_REF] ) or ii) the amount of scFv-FP becomes limiting during mid-late n.c. 14 at first in regions where the RNA and protein expression is highest and prevents the detection of translation (Figure 2C upper panel). To test these hypotheses, we used the twi_suntag_transgene, which exhibits a stochastic expression pattern, belated transcriptional activation [START_REF] Dufourt | Imaging translation dynamics in live embryos reveals spatial heterogeneities[END_REF] and thus lower mRNA level compared to twi_suntag_MS2_CRISPR. If twi translation is actively repressed, a similar translational arrest should be observed for the twi_suntag_transgene. If the amount of scFv-FP is limiting, its translation spots should be detectable over a longer period of time (Figure 2C). Using live imaging, we observed translation spots of the twi-suntag_transgene at a stage when twi_suntag_MS2_CRISPR translation spots vanished (Figure 2D, S4D and Movie S13-15). The translation dots of the twi_suntag_transgene colocalized with mRNA dots in late n.c. 14 and gastrulation stage as confirmed with smFISH (Figure S4E). This result suggests that the scFv-FP amount might be limiting given the high amount of available mRNA templates when twi is monitored from its endogenous locus (twi_suntag_MS2_CRISPR allele), causing a seeming translational arrest at gastrulation. We next used an antibody against the suntag peptides (anti-GCN4) in the absence of scFv-FP and saw translation of twi_suntag_MS2_CRISPR mRNAs at mid-late n.c. 14 and gastrulation stage (Figure 2E andS4F). Moreover, its translation was homogenous inside the pattern of twi expression (Figure 2E). Altogether these results demonstrate that there is no translational arrest of twi mRNAs during n.c.14. Instead, the amount of scFv-FP expressed under the maternally active nos EPr becomes insufficient to detect translation during gastrulation, when a large pool of mRNAs is translated. Our experiments suggest that anti-GCN4 antibodies should be used as a control to distinguish between true endogenous regulation of translation and mere experimentally-dependent depletion of the labelling reagent. Staging is given by DAPI staining on a sagittal view of the imaged embryo (black and white image at the bottom).

(C) Schematic representing twi_suntag_MS2_CRISPR mRNA expression from n.c. 13 to late n.c. 14 in red. From the observations on twi CRISPR translation (b), two hypotheses can be considered. Hypothesis 1 is that the decrease of translation observed at mid-late n.c. 14 is due to an active repression of translation. Hypothesis 2 is that the amount of scFv-FP becomes limiting at mid-late n.c. 14, leading to an arrest of the detected green signal.

These two hypotheses are represented in the context of twi_suntag_MS2_CRISPR expression (above) and twi_suntag_transgene expression (below). twi_suntag_transgene mRNA is expressed more stochastically and later than twi_suntag_MS2_CRISPR mRNA. In hypothesis 1, a decrease of translation should be simultaneously observed for the two constructs (mid-late n.c. 14). In hypothesis 2, no arrest of translation should be observed with twi_suntag_transgene as the amount of mRNA is lower. (D) Snapshots each 15 minutes from movies of scFv-msGFP2 x twi_suntag_MS2_CRISPR or scFv-msGFP2 x twi_suntag_transgene embryos on the ventral side. T0 correspond to early n.c.14. White squares represent the zoomed images in the center of the panel. Note a persistence of translation for the twi_suntag_transgene at T0+45min (white arrowhead), absent in twi_suntag_MS2_CRISPR

Inducible scFv-FP allows imaging of translation during embryogenesis

To overtake the limitation of scFv-FP amount and allow the sustained imaging of twi translation from egg laying to gastrulation, we used the UAS/Gal4 system to increase the expression of scFv-FP during oogenesis. We generated four lines with the different scFv-FPs downstream of a UASp promoter (Figure 3A and S5A) and crossed these with the maternal nos-Gal4 driver (Figure 3B) to allow expression of the scFv-FPs. No aggregation and a higher scFv-FP signal were observed in live embryos (Figure S5A, Movie S16-19 and see methods). Next, we asked if using this system would allow detection of twi translation until late n.c. 14 and gastrulation stage. In twi_suntag_MS2_CRISPR embryos with maternally provided nos-Gal4>UASp-scFv-msGFP2 we saw that twi mRNA was translated throughout n.c.14 (Figure 3C), even in the most ventral part of the embryo, at the center of the twi domain of expression (Figure 3C). However, we were not able to detect small dots of scFv-FP, most likely corresponding to individual mature proteins, probably due to the lower signal to noise ratio, because of the increased levels of scFv-msGFP2. We also observed translation dots at the gastrulation stage in fixed twi_suntag_MS2_CRISPR embryos with nos-Gal4>UASp-scFv-msGFP2 (Figure 3D left panels) but not with EPr-scFv-msGFP2 (Figure 3D right panels). Similar results were observed with live imaging, where twi_suntag_MS2_CRISPR mRNAs were translated until gastrulation with nos-Gal4>UASp-scFv-msGFP2 (Figure 3E and Movie S20) but not with EPr-scFv-msGFP2 or, in the control, in the absence of twi_suntag_MS2_CRISPR mRNAs (Figure 3E and Movie S20). The UASp promoter is used to drive expression in the germline but is less efficient in the somatic tissues [START_REF] Rørth | Gal4 in the Drosophila female germline[END_REF] . On the contrary, the UASt containing HSP70 promoter drives expression in the somatic tissue [START_REF] Brand | Targeted gene expression as a means of altering cell fates and generating dominant phenotypes[END_REF] . Recently, the UASt promoter has been modified to obtain a promoter, called UASz, to drive expression in both somatic and germline tissues [START_REF] Deluca | Efficient Expression of Genes in the Drosophila Germline Using a UAS Promoter Free of Interference by Hsp70 piRNAs[END_REF] . Due to the high amount of free scFv observed with the UASp-FP lines, we generated a new promoter derived from the UASz, in which we removed the translational enhancer IVS-Syn21 51 to prevent oversaturation of free scFv-FP (Figure S5B). This new promoter named UASy was fused to the scFv-msGFP2 and crossed with the nos-Gal4 driver line. As all the other constructs generated in this study, nos-Gal4>UASy-scFv-msGFP2 alone does not lead to aggregates during early embryogenesis (Figure S5C and Movie S21). Unfortunately, when we analyzed the translation of twi_suntag_MS2_CRISPR mRNA at the end of n.c. 14, we observed a similar pattern -although less pronounced -as with scFv-msGFP2 where translation was mostly present at the border of twi pattern (Figure S5D), likely due to the weaker expression of the UASy compared to UASp promoters and consequent depletion of scFv in the central mesoderm. Maternal expression of the scFv-FP constructs limits our ability to observe translational dynamics at later stages in development. To overcome this, we crossed UASy-scFv-msGFP2 with an early zygotic nullo-Gal4 driver to express throughout the embryo. This revealed scFv-msGFP2 expression at the beginning of gastrulation (Movie S22) and a strong and homogeneous expression in somatic tissues of later embryos (Figure S5E). We conclude that the nos-Gal4>UASp-scFv-FP represents a good tool to image translation from early embryogenesis to gastrulation stage and that the new UASy-scFv-msGFP2 will enable the imaging of translation in somatic cells at later stages.

Development of the ALFA_array system to monitor translation

After having optimized scFv-FP-based detector, we sought to generate an optimized orthologous system to the SunTag to allow the detection of translation of two different RNA species. To this end, we searched for a highly specific nanobody/tag couple and decided to use the recently developed ALFA-Tag 52 . The ALFA-Tag is a small peptide that harbors several advantages: its synthetic sequence is absent from the proteome of most studied model organisms, its sequence is smaller than that of the SunTag sequence (15 amino acid compare to 19, respectively Figure 4A), it is monovalent, hydrophilic and was effective in Drosophila for protein manipulation [START_REF] Vigano | Protein manipulation using single copies of short peptide tags in cultured cells and in Drosophila melanogaster[END_REF] . Finally, the nanobody anti-ALFA detector (NB-ALFA) is smaller than the scFv (Figure 4B), is quite soluble and displays a strong binding affinity for the ALFA-Tag (measured at 26pM) [START_REF] Götzke | The ALFA-tag is a highly versatile tool for nanobody-based bioscience applications[END_REF] . Taking advantage of these properties, we multimerized the ALFA-Tag sequences to create a 12X and 32x_ALFA-array. Each ALFA-tag sequence was flanked by a proline, to reduce potential influence of neighboring secondary structures, and a spacer of 5 amino acid (GSGSG) to minimize steric hindrance of neighboring peptide binding sites (Figure 4A,C). The NB-ALFA was fused to the msGFP2 and to the GB1 solubilization tag (also used for the scFv) to avoid potential aggregation caused by the multimerization of ALFA-tag sequences. Finally, we generated a Drosophila line with the NB-ALFA-msGFP2 under the control of nos EPr (NB-ALFA-msGFP2). The system was also tested in mammalian cells (see below). To study translation in Drosophila embryos using the 32x ALFA-Array system, we chose to tag the Ilp4 gene for its small size. We reasoned that if we could visualize translation with a very short transcript/protein, it may be possible for longer ones. A 32x_ALFA-array was inserted between the Ilp4 initiation codon and Ilp4 gene body, appended with Ilp4 UTRs and Ilp4 EPr (Ilp4_32x_ALFA-Array, Figure 4D). With immuno-smFISH we found that the Ilp4_32x_ALFA-Array was expressed in the mesoderm of early Drosophila embryos (Figure S6A) consistent with previous reports for Ilp4 [START_REF] Brogiolo | An evolutionarily conserved function of the drosophila insulin receptor and insulin-like peptides in growth control[END_REF] . The anti-ALFA formed bright dots specifically colocalizing with Ilp4_32x_ALFA-Array mRNAs representing foci of translation (Figure S6B-C), while the Ilp4_32x_ALFA-Array was not recognized by the anti-GCN4 antibody (Figure S6A). We first confirmed by live imaging that the NB-ALFA-msGFP2 strain is expressed during embryonic development until the end of n.c.14, that it was mainly localized to the nucleus, and that it did not form any aggregates (Figure S6D and Movie S23). The Ilp4_32x_ALFA-Array was crossed with NB-ALFA-msGFP2 and the resulting embryos showed bright GFP spots overlapping with Ilp4_32x_ALFA-Array mRNAs (Figure 4D), showing the ability of NB-ALFA-msGFP2 to bind 32x_ALFA-Array in vivo. Next, we monitored translation in live embryos and observed bright GFP dots appearing on the ventral side of the embryo (Figure 4E and Movie S24). The signal was restricted to the mesoderm during n.c.14 (Figure S6E and Movie S25) and became more restricted to the ventral furrow during gastrulation (Figure S6F and Movie S25), demonstrating the specificity of the signal detection. Thus, the Ilp4_32x_ALFA-Array -NB-ALFA-msGFP2 system is a useful alternate tool to detect mRNA translation during early embryogenesis. To test if we use this system to image translation of two distinct mRNA species in the same embryo, the Ilp4_32x_ALFA-Array was crossed with the twi_suntag_transgene (Figure 4F). Using immuno-smFISH in n.c.14 embryos, we observed the two overlapping expression patterns of Twi and Ilp4 as well as the posterior region where only Twi is present (Figure S6G). With higher resolution images, we could visualize single molecules of Ilp4 mRNA in translation well distinct from twi mRNA translation dots (Figure 4G). Furthermore, we were able to distinguish the localization of Ilp4 protein in the cytoplasm from Twi protein in the nucleus (Figure 4H). Together, these results demonstrate that the 32x_ALFA-array can be used in conjunction with the SunTag system to study translation of two different mRNAs and their protein localization during early embryogenesis. To test the performance of the ALFA-tag labeling system to image translation in a different model, we turned to mammalian cells. We generated HeLa cells expressing a NB-ALFA-sfGFP recombinant nanobody and then inserted a 12xALFA-Array repetition at the N-terminus of the DYNC1H1 gene by CRISPR-mediated knock-in (Figure 5A), as previously done for the SunTag 1 . Heterozygous clones were obtained and imaged with and without a thirty-minute puromycin treatment to inhibit translation. In untreated cells, bright foci were observed in the cytoplasm, which disappeared following puromycin addition (Figure 5B). In addition, smFISH against the endogenous DYNC1H1 mRNAs revealed that the bright ALFA-tag foci colocalized with mRNA spots (Figure 5C), confirming that these correspond to DYNC1H1 polysomes. The quality of the signal obtained with only 12 repeats of the ALFA-tag demonstrates the efficiency of this labeling system to image translation.

Discussion

Amplification of biological signals is crucial to study many biological processes with a high spatio-temporal resolution and single-mRNA-molecule sensitivity. Live imaging of translation at single molecule resolution is a relatively recent technology implemented in 2016 in tissue culture and in 2021 in multicellular organisms. In this study, we provide a powerful toolbox to image translation in living cells and organisms. We will discuss some of the limitations and challenges that need to be considered to obtain robust and reliable results. Our data show that the choice of the fluorescent protein is a key element for the signal quality of translation imaging-based systems. The development of fluorescent proteins is a very rapidly evolving field and FP-based tools need to be updated to optimize their use. Often, the choice of the FP needs to be empirically defined for each cell type, tissue or organism studied. Local translation of specific mRNAs is an important biological process to control gene expression in space and time. To obtain quantitative answers it is necessary to precisely resolve the location of mRNAs and where their translation occurs. In the present experimental designs, with the orthologous scFv and the NB-ALFA, the use of secondary antibodies can confound signal interpretation as they appear to bind the scFv and NB-ALFA fusion. This recognition may be due to the linker and GB1 sequences added, as a recent study in plants, which used Westernblot analysis, showed a strong affinity between GB1 and IgG 54 . To combine translation imaging with immunofluorescence, a solution to this problem would be directly linking a fluorophore to a primary antibody. An important aspect in imaging live embryos or tissues is signal amplification to increase the signal-to-noise ratio. Indeed, in systems like the MS2/MCP, llama-Tag/Fluorescent protein [START_REF] Bothma | A Versatile Tool to Image Transcription Factor Dynamics in Live Embryos[END_REF] or scFv/SunTag and their derivatives, the signal appears by local specific accumulation of fluorescent detector, over a diffuse background of molecules freely diffusing in the cells. To improve the signal-to-noise ratio, one can reduce the available free fluorescent detector and therefore the background using weak promoters or targeting the free fluorescent detector to other cellular compartments such as the nucleus, or specific organelles for cytoplasmic analysis. However, one should be careful that the availability of these fluorescent detector does not become limiting as more and more RNAs and proteins are produced during development. As we illustrate here, the available concentration of the detector can be a major challenge in developing organisms, especially when the source of the detector is controlled by an endogenous promoter and when the amount of template mRNA in translation is important. This limitation can also be particularly important when a burst of translation occurs such as during viral infection [START_REF] Boersma | Translation and Replication Dynamics of Single RNA Viruses[END_REF][START_REF] Bruurs | Heterogeneity in viral replication dynamics shapes the antiviral response[END_REF] . We provide a direct example for detector limitation during early embryogenesis. Here, early activated genes such as twist are highly and rapidly transcribed and translated leading to rapid depletion of free detector. We demonstrate the importance of distinguishing between detector depletion and cessation of translation by validating the results with direct immunostaining against the tag of choice. We also show that increasing the expression of the detector can overcome the detection problem but will reduce the signal-tonoise ratio and has thus to be taken into consideration for the specific experimental design. To reach conditions that can most accurately capture the dynamics of translation, diverse UAS promoters together with a variety of available Gal4 drivers should be tested depending on the tissue of interest and the expression level of the tagged gene. Studies on mRNA localization and local translation have speculated that different mRNAs species may be translationally co-regulated [START_REF] Mateju | Single-Molecule Imaging Reveals Translation of mRNAs Localized to Stress Granules[END_REF][START_REF] Ramat | The PIWI protein Aubergine recruits eIF3 to activate translation in the germ plasm[END_REF][START_REF] Kang | LLPS of FXR1 drives spermiogenesis by activating translation of stored mRNAs[END_REF] . To obtain independent translational readouts of different mRNA simultaneously, one can combine orthogonal tagging systems. In Boersma et al., the authors inserted the MoonTag and the SunTag 16 into different translational reading frames of the same RNA. This allowed them to observe in real time heterogeneities in the start site selection of individual ribosomes on a single mRNA molecule. Here, we introduce a new antibody-based detector/tag pair to image translation in living cells and organisms based on multimerization of the ALFA-Tag. The ALFA-array system has a number of advantages: i) it comes from synthetic design and its sequence is thus absent from most organisms; ii) the tag is small, soluble, hydrophilic and lacks aldehydes-sensitive amino acids altered by fixation; iii) the detection is made by a nanobody, which is small and soluble, and has a very strong affinity for the ALFA-Tag (Kd of 27pM), which favorably compares to the MoonTag (Kd of 30 nM) [START_REF] Boersma | Multi-Color Single-Molecule Imaging Uncovers Extensive Heterogeneity in mRNA Decoding[END_REF] . The new translation imaging system described in this study should be applicable to many different organisms to follow localization and control of protein translation. Moreover, the availability of several orthogonal systems will allow to better analyze of the complexity of translation dynamics by simultaneously comparing several mRNA in diverse cellular environments and conditions. The wide range of applications of the ALFA-array system, coupled with other antibody-based translation detector, provides to the scientific community a highly versatile toolbox that will facilitate future scientific breakthroughs.

Fly stocks, handling and genetics

The yw fly stock was used as a wild type. The germline driver nos-Gal4:VP16 (BL4937) and the His2av-mRFP (Bl23651) fly stocks come from Bloomington Drosophila Stock Center. The nullo-Gal4 fly stock comes from RL laboratory. The twi_suntag_MS2_CRISPR and twi_suntag_transgene lines were from [START_REF] Dufourt | Imaging translation dynamics in live embryos reveals spatial heterogeneities[END_REF] . Briefly, the 32x suntag sequence from [START_REF] Pichon | Visualization of single endogenous polysomes reveals the dynamics of translation in live human cells[END_REF] (containing thirty three suntag repeats) was inserted in frame into twi gene 7 and 128X MS2 from 28 placed just after the stop codon. All live experiments were done at 21 °C except when using -or comparing to -strains containing UAS-GAL4 which were done at 25 °C. Cloning and transgenesis scFv-FP under nanos enhancer-promoter (EPr) lines were generated using NEBuilder® HiFi DNA Assembly Master Mix with primers listed in supplementary table 1 and inserted into scFv-sfGFP vector 7 digested with BamHI/XhoI. The transgenic construct was inserted into the VK00033 (BL9750) and VK00027 (BL9744) landing site using PhiC31 targeted insertion [START_REF] Venken | A BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster[END_REF] . UASp-scFv-FP lines were done by inserting fragment of the scFv-FP plasmid digested with XhoI/HpaI in the UASp-scFv-sfGFP vector 6 digested with XhoI/HpaI. The transgenic construct was inserted in the VK00033 (BL9750) landing site using PhiC31 targeted insertion [START_REF] Venken | A BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster[END_REF] . Fluorescent proteins were extracted from plasmids: mAvicFP1 from pNCST-mAvicFP1 (addgene 129509), mGreenLantern from pcDNA3.1-mGreenLantern (addgene 161912), msGFP2 (addgene 160461), mNeonGreen is a kind gift from S.Piatti. The Ilp4 transgene (regulatory regions and coding sequence) was synthesized (GenScript Biotech) into pUC57simple [START_REF] Dufourt | Imaging translation dynamics in live embryos reveals spatial heterogeneities[END_REF] . The 32x_ALFA_array repeats were synthesized (GenScript Biotech) into pUC57simple (see supplementary materials). The 32x_ALFA_array was inserted between Kpn1 and EcoRV restriction sites of the Ilp4 puc57-simple vector. Ilp4-ALFA_array fragment was inserted into pBPhi using PmeI and Nhe1 and injected into BL9750 using PhiC31 targeted insertion [START_REF] Venken | A BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster[END_REF] (BestGene, Inc.). All cloning for plasmids used in Drosophila S2 cells were performed using In-Fusion® HD Cloning Plus Kit (Takara Bio) and PCR was carried out with CloneAmp HiFi PCR premix (Takara Bio). Primers used are listed in supplementary table 1. To inducibly express SunTag in S2 cells, we generated pMT-SuntagRLucFKBP24MS2v5-SV40 by inserting SuntagRLucFKBP24MS2v5 fragment (addgene 119946) into pMT backbone that was PCR-amplified from pMT-EGFPActin5C (addgene 15312) with In-Fusion® HD Cloning Plus Kit. For constitutive expression in S2 cells, pAWF (DGRC stock # 1112) was used as an expression vector. pAWF-stdMCP-stdHalotag was generated by inserting stdMCP-stdHalotag (addgene 104999) into PCR-linearized pAWF vector using In-Fusion® HD Cloning Plus Kit. To generate pAWF-scFv-FPs, sequences containing scFv fused to various fluorescent proteins and GB1-NLS were amplified from scFv-FP under nanos enhancer promoter plasmids generated for in this article and ligated with PCR-linearized pAWF vector using In-Fusion® HD Cloning Plus Kit. All plasmid sequences are listed in supplementary materials.

Single molecule fluorescence in situ hybridization (smFISH) and immuno-smFISH in Drosophila

A pool of 0-4h after egg-laying (AEL) embryos were dechorionated with bleach for 3 min and thoroughly rinsed with H2O. They were fixed in 1:1 heptane:formaldehyde-10% for 25 min on a shaker at 450 rpm; formaldehyde was replaced by methanol and embryos were shaken by hand for 1 min. Embryos that sank to the bottom of the tube were rinsed three times with methanol and kept at -20°C for further use. Embryos were washed 5 min in 1:1 methanol:ethanol, rinsed twice with ethanol 100%, washed 5 min twice in ethanol 100%, rinsed twice in methanol, washed 5 min once in methanol, rinsed twice in PBT-RNasin (PBS 1x, 0.1% tween, RNasin® Ribonuclease Inhibitors). Next, embryos were washed 4 times for 15 min in PBT-RNasin supplemented with 0.5% ultrapure BSA and then once 20 min in Wash Buffer (10% 20X SCC, 10% Formamide). They were then incubated overnight at 37 °C in Hybridization Buffer (10% Formamide, 10% 20x SSC, 400 µg/ml E. coli tRNA (New England Biolabs), 5% dextran sulfate, 1% vanadyl ribonucleoside complex (VRC) and smFISH Stellaris probes against suntag 7 or 32x_ALFA_array coupled to Quasar 570 (supplementary table 1) or probes against 32X MS2 coupled to Cy3 (from E. Bertrand) [START_REF] Tantale | Stochastic pausing at latent HIV-1 promoters generates transcriptional bursting[END_REF] and/or GCN4 primary antibody (Novus Biologicals C11L34) (1/200) and/or affinity purified rabbit anti-ALFA polyclonal antibody (NanoTag Biotechnologies) (1/200). Probe sequences are listed in Supplementary table 1. The next day, secondary antibody (1/500 anti-mouse Alexa 488conjugated (Life Technologies, A21202); anti-mouse Alexa 647-conjugated (Invitrogen, A32728); anti-rabbit Alexa 555-conjugated (Life Technologies, A31572) or anti-rabbit Alexa 488-conjugated (Life Technologies, A21206)) was added if necessary, during 1h at 37° in Wash Buffer. DAPI was added at 45min (1/1000). Embryos were then washed three times 15min in 2x SCC, 0.1% Tween at room temperature before being mounted in ProLong® Diamond antifade reagent. Images were acquired using a Zeiss LSM880 confocal microscope with an Airyscan detector in Super Resolution (SR) mode. GFP/Alexa-488 was excited using a 488 nm laser, Cy3/ were excited using a 561 nm laser, Alexa 670 was excited using a 633 nm laser. Laser powers were: 27µW for 488nm laser (9µW for UAS>nos images), 80µW for 561nm laser and 46µW for 640nm laser. Laser powers were taken under a 10X objective. Figures were prepared with ImageJ (National Institutes of Health), Photoshop (Adobe Systems), and Illustrator (Adobe Systems) Cell culture Drosophila S2R+ cells (DGRC Stock 150) were maintained at 25°C in Schneider's medium containing 10% Fetal Bovine Serum and 1% Penicillin-streptomycin. For the transfection, 0.7x10^6 cells in 350ul were seeded into each well of a 24-well plate. Cells are transfected with plasmid mix of pMT-SuntagRLucDD-MS2 (200ng), pAWF-MCP-Halotag (5ng), pAWF-scFv-FP (10ng) using effectene transfection reagent. 24h after transfection, cells were first incubated with Schneider's medium containing 200 nM Janelia Fluor® 594 HaloTag® Ligand (Promega GA1110) for 15min and then switched to medium with 1 mM CuSO4 for 1h or 3h to induce expression of pMT construct. HeLa cells were maintained in DMEM supplemented with 10% FBS, 10 U/ml penicillin/streptomycin and 2.9 mg/ml glutamine in a humidified CO2 incubator at 37°C. For translational inhibition, cells were treated with puromycin at 100 µg/ml for 30 min. Cells were transfected with JetPrime (Polyplus) and selected on 150 µg/ml hygromycin. For each stable cell line, several individual clones were picked and screened by smFISH with sets of fluorescent oligonucleotide probes against the integrated sequence. For CRISPR recombination, clones were additionally analyzed by genomic PCR using amplicons specific for the non-recombined or the recombined allele. The CRISPR guide and repair plasmids have been previously [START_REF] Pichon | Visualization of single endogenous polysomes reveals the dynamics of translation in live human cells[END_REF] . For the repair plasmid, the 12xALFA-Array replaced the SunTag X56 tag. The NB-ALFA-sfGFP was introduced into HeLa cells by retroviral infection. HEK293T cells were transiently transfected with a cocktail of plasmids coding for retroviral components and producing the genomic NB-ALFA-sfGFP retroviral RNAs. Viral particles were collected and used to infect recipient HeLa cells, which were then sorted by FACS. Only lowly expressing cells were selected.

Single molecule fluorescence in situ hybridization (smFISH) in mammalian cells

Cells were grown on glass coverslips (0.17 mm), washed with PBS, fixed in 4% PFA for 20 min, and permeabilized in 70% ethanol overnight at 4°C. Cells were hybridized as previously described [START_REF] Fusco | Single mRNA Molecules Demonstrate Probabilistic Movement in Living Mammalian Cells HHS Public Access Author manuscript[END_REF] , except that the probes were sets of unlabeled oligonucleotides hybridizing against the target RNA and additionally contained a common supplementary sequence that was preannealed to a fluorescent oligonucleotide probe termed the FLAP [START_REF] Tsanov | SmiFISH and FISH-quant -A flexible single RNA detection approach with super-resolution capability[END_REF] . We used sets of 48 for the DYNC1H1 mRNAs [START_REF] Pichon | Visualization of single endogenous polysomes reveals the dynamics of translation in live human cells[END_REF] . Slides were mounted in Vectashield with DAPI (Vector Laboratories). Fixed cells were imaged at room temperature on an Axioimager Z1 wide-field microscope (63×, NA 1.4; ZEISS) equipped with an sCMOs Zyla 4 0.2 camera (Andor Technology) and controlled by MetaMorph (Universal Imaging). 3D image stacks were collected with a Zspacing of 0.3 µm. Figures were prepared with ImageJ (National Institutes of Health), Photoshop (Adobe Systems), and Illustrator (Adobe Systems) Light-Sheet Microscopy For light-sheet imaging (related to Movie S6-7 and Figures S1D), the MuViSPIM 63 (Luxendo, Brüker company GMBH) was employed. This setup provides two-sided illumination with two Nikon 10x/0.3 water objectives and two-sided detection with two Olympus 20x/1.0 W objectives. The lightsheet is created by scanning a Gaussian beam, as in digital scanned laser light-sheet microscopy (DSLM). We used the line mode which is an in-built synchronization between rolling shutter readout mode of sCMOS cameras and digital light sheets canning. This allows the rejection of out-of-focus and scattered light, thus improving the signal to noise ratio. Images are acquired by two ORCA Flash 4.0 (C91440) from Hamamatsu and processed by LuxControl v1.10.2. A 50ms exposure time was used for the green channel with a 488nm laser. Maximum intensity projections were processed with Fiji [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF] . Live imaging of the scFv-FP and NB-ALFA-msGFP2 alone For all live imaging experiments, embryos were dechorionated with tape and mounted between a hydrophobic membrane and a coverslip as described previously [START_REF] Dufourt | Temporal control of gene expression by the pioneer factor Zelda through transient interactions in hubs[END_REF] . Movies for scFv-FP (related to Figure S1 and Movie S1-5) and NB-ALFA-msGFP2 under nanos promoter (related to Figure S6 and Movie S23) were acquired using a Zeiss LSM880 confocal microscope with a Plan-Apochromat 40x/1.3 oil objective lens, with green excitation using a 488nm laser (6μw measured with 10x objective lens). A GaAsP detector was used to detect fluorescence with the following settings: 512x 512-pixels images, zoom 2x, each Z-stack comprised of 25 planes spaced 1μm apart. Movies for UASp>scFv-FP (related to Figure S5, and Movie S16-19) and UASy>scFv-msGFP2 with nos-Gal4 driver (related to Figure S5 and Movie S21) were acquired using a Zeiss LSM880 confocal microscope with a Plan-Apochromat 40x/1.3 oil objective lens, with green excitation using a 488nm laser (1μw measured with 10x objective lens). A GaAsP detector was used to detect fluorescence with the following settings: 512x 512-pixels images, zoom 2x, each Z-stack comprised of 25 planes spaced 1μm apart.

Live imaging of the scFv-FP and anti-ALFA-msGFP2 in presence of tagged genes

For all live imaging experiments, embryos were dechorionated with tape and mounted between a hydrophobic membrane and a coverslip as described previously [START_REF] Dufourt | Temporal control of gene expression by the pioneer factor Zelda through transient interactions in hubs[END_REF] . Movies of scFv-FP>twi_suntag_MS2_CRISPR/+ (related to Figure 1 and Movie S8-12) were acquired using a Zeiss LSM880 with confocal microscope in fast Airyscan mode with a Plan-Apochromat 40x/1.3 oil objective lens. GFP was excited using a 488nm (7uw measured with 10x objective lens) laser with the following settings: 132x 132-pixel images, each Z-stack comprised of 15 planes spaced 0.5μm apart and zoom 8x. Intensity measurement of each polysomes was done on a MIP of 5 planes using a custom-made software written in Python, that performs a spot detection on the MIP raw data stack. For each time frame, the software performs a Laplacian of Gaussian 2D filter and the filtered images are than thresholded with a user defined threshold. The threshold is expressed in terms of the average and standard deviation of intensity values of each image in order to have a threshold value self-re-scaled with respect to the characteristic of the images itself. Spots smaller in size than a user-defined value are removed (considered as fake detection). Finally, for each of the detected spots, we use a 2D expansion of the spots silhouettes in order to define a region around the spots devoided of any other spots and that we can use to estimate background and measure it all along the time evolution for corrections purposes. Movies of scFv-FP>twi_suntag_MS2_CRISPR/+ and scFv-msGFP2>twi_suntag_transgene (related to Figure 2, Figure S4 and Movie S13-15) were acquired using a Zeiss LSM880 with confocal microscope in fast Airyscan mode with a Plan-Apochromat 40x/1.3 oil objective lens. GFP was excited using a 488nm (4.7µW measured with 10x objective lens) laser with the following settings: 568x 568-pixel images, each Z-stack comprised of 61 planes spaced 0.5μm apart and zoom 2x. Movies were then processed to remove frame outside of the embryos or containing the membrane signal to correct drifting, and processed stacks were maximum intensity projected using custom-made software, developed in Python TM7 . Movies of nos-Gal4>UASp-scFv-msGFP2 x twi_suntag_MS2_CRISPR/+ and scFv-msGFP2 x twi_suntag_MS2_CRISPR/+ (related to Figure 3 and Movie S20) were acquired using a Zeiss LSM880 with confocal microscope in fast Airyscan mode with a Plan-Apochromat 40x/1.3 oil objective lens. GFP was excited using a 488nm (5uw measure with 10x objective lens) laser with the following settings: 396x 396-pixel images, each Z-stack comprised of 30 planes spaced 0.5μm apart and zoom 4x. Live tilescans of nullo-Gal4>UASy-scFv-msGFP2-NLS (related to Movie S22) were acquired using a Zeiss LSM880 with confocal microscope in fast Airyscan mode with a Plan-Apochromat 40x/1.3 oil objective lens. GFP was excited using a 488nm (13µW measured with 10x objective lens) laser with the following settings: 1440 x 2746-pixel images using two tiles, each Z-stack comprised of 9 planes spaced 2μm apart and zoom 0.8x. Live tilescans of UASy-scFv-msGFP2-NLS/ his2Av-RFP in presence or not of nullo-Gal4 driver (related to Supplementary Figure S5E) were acquired using a Zeiss LSM880 with confocal microscope in fast Airyscan mode with a Plan-Apochromat 40x/1.3 oil objective lens. GFP was excited using a 488nm (13µW measured with 10x objective lens) and RFP was excited using a 561nm laser with the following settings: 1024 x 3072-pixel images using three tiles, each Zstack comprised of 14 planes spaced 2μm apart. Movies of NB-ALFA-msGFP2 under nanos EPr crossed with Ilp4_32x_ALFA-array (related to Figure 4 and Movie S24) were acquired using a Zeiss LSM880 with confocal microscope in fast Airyscan mode with a Plan-Apochromat 40x/1.3 oil objective lens. GFP was excited using a 488nm (6.4µW measured with 10x objective lens) laser with the following settings: 256x 256pixel images, each Z-stack comprised of 6 planes spaced 1μm apart and zoom 6x. Live tilescans of NB-ALFA-msGFP2 under nanos EPr crossed with Ilp4_32x_ALFA-array (related to Figure S6 and Movie S25) were acquired using a Zeiss LSM880 with confocal microscope in fast Airyscan mode with a Plan-Apochromat 40x/1.3 oil objective lens. GFP was excited using a 488nm (13µW measured with 10x objective lens) laser with the following settings: 1612 x 4860-pixel images using 3 tiles, each Z-stack comprised of 9 planes spaced 2μm apart.

S2 cell culture imaging:

Cells were pipetted into a µ-Slide (Ibidi #80621) and live-imaged using a Zeiss LSM980 confocal microscope with an Airyscan detector in SR mode with a 63X Plan-Apochromat (1.4NA) oil objective lens. GFP was excited using a 488nm laser and MCP-Halotag labelled by Janelia Fluor® 594 was excited using a 561nm laser. Cells with mRNAs visible in cytoplasm and with comparable GFP signal were chosen for acquiring images. Images of individual cells were acquired with following setting: z-stack of 5 planes with 0.5um interval, 8x zoom, 396x396 pixel and 8 bit per pixel. GFP foci co-localized with mRNA foci (Janelia Fluor® 594 signal) were considered as translation sites while GFP foci or larger amorphous or ring-like GFP structures that were not co-localized with mRNA were considered as aggregates.

Figure 1 :

 1 Figure 1: Generation of different scFv-Fluorescent proteins (scFv-FP) to monitor nascent translation in Drosophila.

  . Scale bar are 5µm. (D) Snapshots from representative fast-mode acquired confocal movies of twi_suntag_MS2_CRISPR/+ embryos carrying either scFv-msGFP2, scFv-GreenLantern, scFv-mAvic1, scFv-NeonGreen or scFv-sfGFP proteins. Green dots represent nascent translation of twi. Scale bars are 5µm. (See related Movies S8-12). (E) Quantification across time during n.c. 14 of different scFv-FP signals from movies represented in (d), n=5 movies from at least 3 embryos for each scFv-FP. Error bars represent SEM.

Figure 2 :

 2 Figure 2: scFv-FP concentration under nos EPr creates an artefactual translation arrest of endogenous twi mRNA during early embryogenesis

  embryos. Scale bars are 10µm on the larger images, and 5µm on the zoomed images. (See related Movies S13-14). (E) (top) Schematic of a sagittal view of a Drosophila embryo with twi expression pattern in purple. Anterior side is on the left, posterior on the right, dorsal on the top and ventral side on the bottom. Black square represents the imaged area of the bottom panels. (bottom) Single Z-planes of confocal images from immuno-smFISH with anti-GCN4 antibody (cyan) and MS2 probes (red) on twi_suntag_MS2 embryos at mid-late n.c. 14. Gray squares represent the zoomed images in the right panels. The three different zoomed images represent external (top), border (middle) and internal (bottom) zone of the imaged pattern. Scale bars are 10µm on the larger images, and 5µm on the zoomed images. Staging is given by DAPI staining on a sagittal view of the imaged embryo (black and white image at the bottom).

Figure 3 :

 3 Figure 3: Increasing scFv-FP expression allows translation detection at later stages (A) Schematic of UASp-scFv-msGFP2 construct and activation by the Gal4 protein. 10 UAS sequences (purple) are placed upstream the P-promoter (blue) and scFv-msGFP2 (green) sequences. Gal4 proteins (yellow) will bind the UAS sequences to activate transcription of the scFv-msGFP2. (B) Schematic of the expression of the scFv-msGFP2 in nosGal4>UASp-scFv-msGFP2 female strain during oogenesis until egg deposition. (C) (left) Schematic of a sagittal view of a Drosophila embryo with twi expression pattern in purple. Anterior side is on the left, posterior on the right, dorsal on the top and ventral side on the bottom. Black square represents the imaged area of the right panels. Single Z-planes of confocal images from smFISH with direct scFv-msGFP2 fluorescent protein signal (green) and MS2 probes (red) on nosGal4>UASp-scFv-msGFP2 x twi_suntag_MS2_CRISPR

Figure 4 :

 4 Figure 4: Development of the ALFA_array system to detect nascent translation in Drosophila (A) Peptide sequence of one suntag with its linker (24 amino-acids) and peptide sequence of the ALFA-tag with its linker (20 amino-acids). (B) Schematic of the scFv and the nanobody ALFA (NB-ALFA) fused to the fluorescent protein msGFP2 and their size in amino-acid (aa). (C) Schematic of the ALFA-array system developed in this study to detect translation. 32 ALFA-tag sequences were multimerized (blue amino-acid sequence) and inserted at the 5' of the gene of interest sequence (yellow). Upon translation, nanobodies ALFA fused to msGFP2(NB-ALFA_msGFP2) will bind to the ALFA-tag peptides. As a bipartite system, the NB-ALFA is expressed

Figure 5 :

 5 Figure 5: Development of the ALFA_array system to detect nascent translation in mammalian cells (A) (1.) Schematic of the CRISPR/Cas9 targeted insertion strategy to obtain endogenous DYNC1H1 gene tagged with 12X ALFA-tag. (2.) Schematic of the ALFA-array system developed in mammalian cells. 12 ALFA-tag sequences were multimerized (blue amino-acid sequence) and inserted at the 5' of the gene of interest sequence (DYNC1H1, yellow). (3.) The NB-ALFA is expressed under spleen focus-forming virus (SFFV) promoter fused to sfGFP and Streptococcal protein G (GB1) domain. (B) Left: micrographs of HeLa cells expressing a DYNC1H1 allele endogenously tagged 12xALFA-Array tag and NB-ALFA-sfGFP, and treated (right) or not (left) with puromycin. Green: NB-ALFA-sfGFP signal; blue: DAPI. Scale bars are 5µm. Arrows: NB-ALFA-sfGFP spots. Right: quantification of the number of NB-ALFA-sfGFP spots per cell, with and without puromycin treatment. Error bars: standard deviation (n=40 cells). (C) Micrographs of HeLa cells expressing a DYNC1H1 allele endogenously tagged 12xALFA-Array tag and NB-ALFA-sfGFP, and hybridized in situ with a set of probes against DYNC1H1 mRNAs. On the merged panel: NB-ALFA-sfGFP signal (green); smFISH signals (red); DAPI (blue). Scale bar is 5µm. Arrows: DYNC1H1 polysomes.

Figure Supp 2 :

 2 Figure Supp 2: Localization of the tagged protein and scFv recognition by secondary antibody. (A) (top) Schematic of a sagittal view of a Drosophila embryo with twi expression pattern in purple. Black square represents the imaged area of the bottom panels. Single Z-planes of confocal images from immuno-staining with direct fluorescent protein signal in green (scFv-msGFP2, scFv-GreenLantern, scFv-mAvic1, scFv-NeonGreen and scFv-sfGFP) and secondary anti-mouse antibody (magenta) on scFv-FP x twi_suntag_MS2_CRISPR embryos in n.c. 14 on the ventral side. Note the co-localization between the direct fluorescent protein signal and the secondary antibody signal as illustrated by the schematic on the top right. Scale bar are 1µm. (B) (top) Schematic of a Drosophila embryo on the ventral side with gastrulation furrow represented with invaginating cells. Black square represents the imaged area of the bottom panels. (bottom) Single Z-planes of confocal images from smFISH with direct scFv-msGFP2 fluorescent protein signal (green) and MS2 or Ilp4 probes (red) on scFv-msGFP2 > twi_suntag_MS2_CRISPR and scFv-msGFP2 > Ilp4_suntag embryos at gastrulation stage and on the ventral side. Note a nuclear signal in twi_suntag_trangene embryos and a cytoplasmic signal in Ilp4_suntag embryos. Scale bars are 10µm.

Figure Supp 3 :

 3 Figure Supp 3: The scFv-sfGFP can form aggregates in Drosophila embryos and S2 cells (A) Schematic of a Drosophila embryo on the ventral side with gastrulation furrow represented with invaginating cells. Black square represents the imaged area of the bottom panels. Snapshots from representative confocal movies of twi_suntag_MS2_CRISPR/+ embryos at the gastrulation stage carrying either scFv-sfGFP, scFv-NeonGreen, scFv-mAvic1 or scFv-msGFP2 proteins. Scale bars are 10µm. (B) Schematic of a Drosophila embryo on the ventral side with gastrulation furrow represented with invaginating cells. Black square represents the imaged area of the bottom panels. Maximum intensity projection of 10 Z-planes of confocal images from smFISH with direct fluorescent protein signal in green (scFv-FP) and MS2 probes (red) on scFv-FP x twi_suntag_MS2_CRISPR embryos at gastrulation stage on the ventral side. Grey squares represent zoomed images. Note the presence of scFv-sfGFP signal not overlapping with RNA (white arrowheads). Scale bars are 5µm. (C) Schematic of the different constructs transfected in S2 cells. scFv-FPs and tdMCP-stdHalotag were

Figure Supp 4 :

 4 Figure Supp 4: twi translation detection in developing embryos until gastrulation. (A) (top) Schematic of a sagittal view of a Drosophila embryo with twi expression pattern in purple. Anterior side is on the left, posterior on the right, dorsal on the top and ventral side on the bottom. Black square represents the imaged area of the bottom panels. (bottom) A single Z-plane of confocal images from smFISH with direct fluorescent protein signal in green (scFv-mAvic1) and MS2 probes (red) on scFv-mAvic1 x twi_suntag_MS2_CRISPR embryos at mid-late n.c. 14. Gray squares represent the zoomed images in the right panels. The three different zoomed images represent external (top), border (middle) and internal (bottom) zone of the imaged pattern. Scale bars are 10µm on the larger images, and 5µm on the zoomed images. Staging is given

Figure Supp 5 :Figure Supp 6 :

 56 Figure Supp 5: Generation of UAS-Gal4 system to express scFv-Fluorescent Proteins

Acknowledgments:

We are grateful to NanoTag Biotechnologies for sharing ALFA-Tag and anti-ALFA nanobody plasmids and advices. We are grateful to Karim Mazjoub's lab for sharing space in his experimental room. We thank Drs. Xavier Pichon and Vera Slaninova for their help in setting up the mammalian ALFA-Tag system. For imaging, we acknowledge Sylvain de Rossi and Montpellier Ressources Imagerie (MRI), a microscopy facility of The National Infrastructure France-BioImaging (FBI), supported by the French National Research Agency (ANR-10-INBS-04). We acknowledge M.Verbrugghe for help with fly handling, and the Montpellier Drosophila Facility (BioCampus). We acknowledge Etienne Schwob and Eric Kremer for their help in preparing the manuscript. We acknowledge HFSP (CDA Grant to ML) for the early sponsoring of the implementation of Drosophila translation imaging project.

Supplementary figures