Boosting the toolbox for live imaging of translation

Abstract

Live imaging of translation based on tag recognition by a single-chain antibody is a powerful technique to assess translation regulation in living cells. However, this approach is challenging and requires optimization in terms of expression level and detection sensitivity of the system, especially in a multicellular organism. 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 uncovered 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. 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. Finally, we developed a largely improved RNA imaging system based on an MCP-tdStaygold fusion.

Keywords: Drosophila; live-imaging; microscopy; translation.

FIGURE 1.

Generation of different scFv-fluorescent proteins (scFv-FPs) to monitor nascent translation in Drosophila. (A) Schematic of the SunTag system. Once the mRNA (black) is translated by the ribosomes (gray), the suntag epitopes (light blue) will be bound by the single-chain variable fragment (scFv) (dark blue) fused to an FP (green). Therefore, the nascent peptide will be detected by the accumulation of fluorescent signals. (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 (Dufourt et al. 2021). (Bottom) Schematic of the CRISPR/Cas9 targeted insertion strategy to obtain endogenous twist gene tagged with suntag and 128xMS2 (Dufourt et al. 2021). (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 FP 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. Scale bar, 5 µm. (D) Snapshots from representative fast-mode acquired confocal movies of twi_suntag_MS2_CRISPR/+ embryos carrying either scFv-msGFP2, scFv-GreenLantern, scFv-mAvic1, and scFv-NeonGreen or scFv-sfGFP proteins. Green dots represent nascent translation of twi. Scale bar, 5 µm (see related Supplemental Movies S8–S12). (E) Quantification across time during n.c. 14 of different scFv-FP signals from movies represented in D, n = 5 movies from at least three embryos for each scFv-FP. Error bars represent SEM.

FIGURE 2.

scFv-FP concentration under nos EPr creates an artifactual translation arrest of endogenous twi mRNA during early embryogenesis. (A) Schematic of nuclear elongation and cellularization during n.c. 14. In this study, we defined one early-mid stage (corresponding to 0–35 min of the n.c. 14 from the mitosis) and one mid-late stage (corresponding to 35–55 min of the n.c. 14 from the mitosis). (B, 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 FP signal in green (scFv-msGFP2) and MS2 probes (red) on scFv-msGFP2 x twi_suntag_MS2_CRISPR embryos at mid-late n.c. 14. Gray squares represent the zoomed images in the right panels. The two different zoomed images represent border (top) and internal (bottom) zone of the imaged pattern. Scale bars, 10 µm on the larger images, and 5 µm 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). Quantification of the scFv-msGFP2 signal intensity on single-molecule mRNA at the border at mid-late n.c. 14 stages (dark green, nine images from three embryos, n = 2570), and center (light green, nine images from three embryos, n = 2737) of the mesoderm is represented on the right. (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 taken each 15 min from movies of scFv-msGFP2 x twi_suntag_MS2_CRISPR or scFv-msGFP2 x twi_suntag_transgene embryos on the ventral side. T0 corresponds 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 + 45 min (white arrowhead), absent in twi_suntag_MS2_CRISPR embryos. Scale bars, 10 µm on the larger images, and 5 µm on the zoomed images (see related Supplemental Movies S13 and S14). (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 two different zoomed images represent border (top square) and internal (bottom square) zones of the imaged pattern. Scale bars, 10 µm on the larger images, and 5 µm 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). Quantification of the anti-GCN4 signal intensity on single-molecule mRNA at the border (dark blue, six images from two embryos, n = 3211) and center (light blue, six images from two embryos, n = 4001) at mid-late n.c. 14 stages of the mesoderm is represented on the right.

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. Ten UAS sequences (purple) are placed upstream of 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 FP signal (green) and MS2 probes (red) on nosGal4 > UASp-scFv-msGFP2 x twi_suntag_MS2_CRISPR embryos in early, early-mid, and mid-late n.c. 14. Gray squares represent the zoomed images for each panel. Nuclei are counterstained with DAPI (gray, bottom images) for staging. Scale bars, 10 µm on the larger images, and 5 µm on the zoomed images. (D, 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 FP signal (green) and MS2 probes (red) on nosGal4 > UASp-scFv-msGFP2 x twi_suntag_MS2_CRISPR and scFv-msGFP2 x twi_suntag_MS2_CRISPR embryos at gastrulation stage and on the ventral side. Scale bars, 10 µm on the larger images, and 5 µm on the zoomed images. Note that translation dots are visible only with nosGal4 > UASp-scFv-msGFP2. (E, 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 movie of scFv-msGFP2 x yw, scFv-msGFP2 x twi_suntag_MS2_CRISPR and nosGal4 > UASp-scFv-msGFP2 x twi_suntag_MS2_CRISPR embryos at gastrulation stage (furrow represented with gray dashed line). Scale bar, 10 µm (see related Supplemental Movie S20).

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 FP msGFP2 and their size in amino acids (aa). (C) Schematic of the ALFA-array system developed in this study to detect translation. Thirty-two 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 under nos EPr fused to msGFP2, Streptococcal protein G (GB1) domain, and a nuclear localization signal (NLS). (D) Representation of the transgene mRNA containing 32x_ALFA-array in frame with the insulin-like peptide 4 (Ilp4) gene sequence. Schematic of a sagittal view of a Drosophila embryo with Ilp4 expression pattern in yellow. 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. One Z-plane of confocal images from smFISH with direct Nb-ALFA-msGFP2 FP signal (green) and 32x_ALFA-array probes (red) on NB-ALFA_msGFP2 > Ilp4_32x_ALFA-array embryos in early-mid n.c. 14 is on the ventral side. Scale bars, 10 µm on the larger images, and 5 µm on the zoomed images. (E) Snapshots from representative fast-mode acquired confocal movies of Ilp4_32x_ALFA-array/+ n.c. 14 embryos carrying NB-ALFA_msGFP2. Bright white foci represent nascent translation of Ilp4 transgene. Yellow squares represent zoomed images of the top panels. Nascent translation of Ilp4 transgene is indicated by yellow arrowheads. Scale bars, 10 µm on the larger images, and 2 µm on the zoomed images (see related Supplemental Movie S24). (F) Representation of the transgene mRNAs containing 32x_ALFA-array fused to Ilp4 coding sequence or suntag fused to twist coding sequence used in G and H. (G) Schematic of a Drosophila embryo on the ventral side with twi expression pattern in purple and Ilp4 in yellow. Black square represents the imaged area of the bottom panels. One Z-plane of confocal images from immuno-smFISH with anti-ALFA (green) labeling nascent translation of Ilp4_32x_ALFA-array, probes against 32x_ALFA-array (red) labeling Ilp4_32x_ALFA-array mRNA molecules and anti-GCN4 antibody labeling nascent translation of twi_suntag_transgene (magenta) in n.c. 14 embryos. Scale bar, 1 µm. (H) Maximum intensity projection of a whole embryo confocal image from immunostaining with anti-ALFA antibody (green) and anti-GCN4 antibody (magenta) on Ilp4_32x_ALFA-array; twi_suntag_transgene gastrulating embryo. Scale bar, 100 µm. (I) Schematic of a Drosophila embryo on the ventral side with twi expression pattern in purple and Ilp4 in yellow. Black square represents the imaged area of the bottom panels. Single Z-plane of confocal images from dual-color live imaging of twi_suntag_MS2_CRISPR/+; NB-ALFA_msGFP2/scFv-mScarlet x Ilp4_32x_ALFA-array embryos in n.c. 14. Scale bar, 1 µm (see related Supplemental Movie S26). (J, top) Schematic of a Drosophila embryo on the ventral side with gastrulation furrow represented with invaginating cells. Confocal snapshots of a whole live embryo expressing twi_suntag_MS2_CRISPR/+; NB-ALFA_msGFP2/scFv-mScarlet x Ilp4_32x_ALFA-array, after gastrulation. Note the cytoplasmic staining from Ilp4 translation (green) and nuclear staining from twi translation (purple). Scale bar, 100 µm.

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. Twelve 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 12x_ALFA-array tag and NB-ALFA-sfGFP, and treated (right) or not (left) with puromycin. (Green) NB-ALFA-sfGFP signal; (blue) DAPI. Scale bar, 5 µm. (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 12x_ALFA-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, 5 µm. (Arrows) DYNC1H1 polysomes. (D, top) Schematic of the MCP fusion used to image RNA. (Bottom) Maximum projection intensity images of cells stably expressing MCP-tdStayGold (on the left) or MCP-eGFP (on the right). All images were taken under the same conditions on an OMX wide-field microscope. Six time points were selected (0 min, 30 min, 60 min, 120 min, 150 min, and 180 min), and they are displayed with the same dynamic range. Red arrows point at transcription sites, and green arrows point at individual RNA molecules. Scale bar, 10 μm. (E) Intensities of single RNA molecules over time. Graph shows intensities of RNA molecules (mean and s.d. error; five cells). Because of bleaching, the single molecule signal could no longer be detected and measured after 120 min in the case of MCP-eGFP. Note that MCP-tdStayGold and MCP-eGFP signals were acquired with the same time frame frequency (see Materials and Methods). (F) The graph shows the number of single RNA molecules detected over time using FISHquant (five cells).

Maëlle Bellec