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# Shedding light on the unseen: how live imaging of translation could unlock new insights in developmental biology

Jeremy Dufourt, Maëlle Bellec

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Jeremy Dufourt, Maëlle Bellec. Shedding light on the unseen: how live imaging of translation could unlock new insights in developmental biology. Comptes Rendus. Biologies, 2024, 347, pp.87-93. 10.5802/crbiol.158. hal-04730226

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# Comptes Rendus
Biologies

Jeremy Dufourt and Maelle Bellec

Shedding light on the unseen: how live imaging of translation could unlock new insights in developmental biology

Volume 347 (2024), p. 87-93

Online since: 11 September 2024

https://doi.org/10.5802/crbiol.158

<figure><img src="image_3.png" /></figure>

This article is licensed under the

CREATIVE COMMONS ATTRIBUTION 4.0 INTERNATIONAL LICENSE.

http://creativecommons.org/licenses/by/4.0/
Opinion / Perspective

# Shedding light on the unseen: how live imaging of translation could unlock new insights in developmental biology

Jeremy Dufourt Ⓔ,ª and Maelle Bellec Ⓔ,*,b

E-mail:maelle.bellec@mpi-bn.mpg.de (M. Bellec)

**Abstract.** Recent advances in live imaging technologies have refined our understanding of protein synthesis in living cells. Among the various approaches to live imaging of translation, this perspective highlights the use of antibody-based nascent peptide detection, a method that enables visualization of single-molecule translation *in vivo*. We examine how these advances improve our understanding of biological processes, particularly in developing organisms. In addition, we discuss technological advances in this field and suggest further improvements. Finally, we review some examples of how this method could lead to future scientific breakthroughs in the study of translation and its regulation in whole organisms.

**Keywords.** Translation, Live-imaging, Development.

**Note.** Maelle Bellec is the recipient of the Fondation Madeleine Lecoq Biology Prize of the Académie des sciences 2023.

**Funding.** HFSP, CNRS.

Manuscript received 30 May 2024, revised 23 July 2024, accepted 8 August 2024.

## 1. Introduction

Proteins largely determine the way cells work and perform their functions. The amount of mRNA and how efficiently it is translated into proteins are tightly regulated, ensuring that proteins are produced at the right time and place within the cell and between different cell types [1–3]. Research has shown that mRNA can be found in specific areas within a cell, where it may be directly translated into proteins [4–6]. This configuration enables cells to promptly and accurately modify their proteome without requiring modifications in transcription levels.

The recent advancement in live imaging of translation provides a dynamic perspective of protein synthesis in real-time within living cells, contrasting with fixed methods that offer only a static snapshot at a single time point. Translation imaging can regroup several different approaches (reviewed in [7]). In this perspective, we focus on the antibody-based detection of nascent peptides for visualizing single molecule translation *in vivo*. We discuss how spatio-temporal imaging advances our understanding of biological processes in developing organisms. We discuss technological advancements that could deepen our grasp of translation mechanisms and review some contexts in which this method has led to scientific discoveries and its potential to drive future breakthroughs.

<footnote>ª Institut de Recherche en Infectiologie de Montpellier, CNRS UMR 9004, Univ Montpellier, 1919 Route de Mende, Montpellier, 34293, Cedex 5, France</footnote>

<footnote>ᵇ Department of Developmental Genetics, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany</footnote>

<footnote>* Corresponding author</footnote>
<figure><img src="image_4.png" /><figcaption>**Figure 1.** Nascent peptide labeling for live imaging of translation. On the left, schematic of a cell expressing a reporter construct with multiple tags, a gene of interest and the fluorescent detector freely diffusing in the cell. Once translated, the tag peptides are bound by the fluorescent detector (right panel) and create a bright signal in the cell.</figcaption></figure>

Imaging translation through antibody recognition of nascent peptides involves a two-component system: a tag, which is typically multimerized (from 5 to 56 times [8, 9] but often 24 or 32 times), and inserted into the gene of interest; and a genetically encoded detector made of either a single-chain antibody (scFv) or a nanobody fused to a fluorescent protein (FP) [10–12]. Upon translation, the repeats of the peptide tag will be bound by the detector, resulting in bright signal spots that stand out against the background. This is because a single mRNA molecule will bear FPs bound to the multiple copies of the tag, but also because there will be numerous nascent peptides for each mRNA molecule, as it is translated by multiple ribosomes (Figure 1). The translation dots, or polysomes, may then be tracked and their fluorescence measured to extract dynamics parameters such as diffusion, translation speed, etc [9, 13–16]. This method is the only one that enables to capture parameters mentioned above while bearing in mind that it requires the insertion of an array of tags in frame with the gene of interest. These tags could significantly increase the size of the reporter gene, and the values measured should not be considered absolute values but an approximation of the translation dynamics of the gene under study.

Live imaging of translation using this approach has been possible since 2016 in cultured cells [9, 13–16]. In these studies, the authors use two different systems, the SunTag and the Spaghetti Monster. The SunTag system consists of multiple copies of a small

peptide, derived from the GCN4 protein, inserted at the N-terminus of the protein of interest and a genetically encoded anti-GCN4 scFv fused to a fluorescent protein [9, 13–15]. The Spaghetti Monster system is based on a repetition of several FLAG tags inserted after the start codon and recognized by a fluorescently labeled anti-FLAG Fab introduced into cells by bead loading [14]. In 2021, the SunTag system was implemented in a multicellular organism [17–19] and was followed by multiple studies that were more and more resolutive in time and space [8, 20–25]. These studies went down to the single molecule level and have shown various examples where translation can be heterogeneous within the cell, adding another layer of regulation to that of mRNA localization and expression level.

Several studies have shown evidence for the localized dynamic translation of mRNA. In Dufourt et al. [17], the authors used the CRISPR/Cas9 system to tag the *twist* gene, encoding a key transcription factor involved in the epithelial-to-mesenchymal transition, at its endogenous locus in *Drosophila* embryos using the SunTag system. They found that the translation dynamics can be heterogeneous within cells. In another study, Chen and colleagues [26] showed in the *Drosophila* embryo that ribonucleoprotein (RNP) granules regulate localized protein synthesis through compartmentalized relief of translational repression.

This finding implies that during the development of an embryo, one needs to look at the precise regulation of translation locally, and during time. Further
investigation will be required to identify in which ex-
act biological processes this is relevant.

These *in vivo* studies, as well as many others in
mammalian cells, have shed light on new spatial
and temporal regulatory mechanisms that cells em-
ploy for post-transcriptional gene expression control,
such as local translation [27, 28], IRES/Cap trans-
lation [25], bursting of translation [29], RNP gran-
ule [18, 26], polysome transport [30], translation
kinetics [31], and coordination of translation and
degradation [20, 32].

## 2. Technology improvements

In the past years, the range of tools used to im-
age translation has advanced considerably [33]. Re-
cently, Bellec et al. [11] developed and characterized
new tools to image translation in a living *Drosophila*
embryo. This study also revealed that imaging trans-
lation *in vivo* is not that trivial and that it has limi-
tations and requires optimization. In the meantime,
these new tools are powerful and will lead us to see
what we couldn't access before.

Looking forward, there are several technical areas
requiring further development. First, there is a need
for advancements in mRNA tracking. Since transla-
tion of proteins can take several minutes, continu-
ous tracking of mRNA for tens of minutes or even
hours is essential to comprehend translation regula-
tion at the single mRNA level. Bright and stable RNA
and antibody labeling techniques are imperative for
this purpose; the use of fluorogenic protein tags,
such as SNAP-Tag, CLIP-Tag, or HaloTag, could pro-
vide some advantages over FP, such as high bright-
ness and better photo-stability. However, this would
require injection of the dyes or harsh treatment
in organisms because of their lack of permeability
(in contrast to cell lines). Moreover, in contrary
to FPs which replenish the pool of antibodies con-
stantly, during time-lapse experiments, fluorogenic
protein tags require constant feeding of dyes poten-
tially leading to toxicity/lethality. One of the potential
future advances would be the development of genet-
ically encoded Quenchbodies which would fluoresce
only when bound to their target [34], combined with
ultra-bright and stable FP such as monomeric Stay-
Gold [35]. Second, it is essential to improve *in vivo*
microscopy techniques. The visualization of freely
diffusing mRNAs in cells and tissues continues to

pose challenges, especially in a living organism. Im-
provements in imaging technologies, such as lattice
light sheet microscopy or adaptive optics, hold the
potential to enable single mRNA imaging in live an-
imals thanks to their fast acquisition, large field of
view, and good resolution [36]. However, the rapid
increase in the volume of microscopy data is outpac-
ing computer capabilities, and this issue needs to be
addressed in order to extract all the information en-
coded in the heavy imaging data.

Third, it is crucial to establish robust quantitative
mathematical models. The first modeling used to
fit translation imaging data derived from the theory
implemented for transcription by RNA polymerase II
[37, 38], using many assumptions. Now, modeling of
translation dynamics should be improved to distin-
guish between different scenarios (such as initiation
rate bursting, non-homogeneous ribosome coverage,
ribosome collisions, ribosome slowdown, ribosome
release delay, etc.), as it is starting to be implemented
for transcription [39-43]. Recently, the laboratory of
B. Wu improved these measurements for the anal-
ysis of translation bursting [29]. Furthermore, the
accuracy of these mathematical models depends on
the measurement methods used, making it crucial to
employ multiple approaches for a more precise as-
sessment of translation kinetics (reviewed in [31,44]).
Kinetic parameters of translation can be measured
using Fluorescence Recovery After Photobleaching
(FRAP) [9, 15], but this method faces challenges in
developing organisms due to the rapid polysome
movements, especially when studying translation in
its native context (except for mRNA naturally associ-
ated to immobile organelles [26]). Other approaches
exist, such as Ribosome Run-Off Assay, in which the
decrease in fluorescence is measured, corresponding
to the time needed for the ribosome to translate the
entire mRNA, using naturally occurring translation
bursts or drug treatments such as harringtonine, an
inhibitor of translation initiation [13]. This method
also faces challenges in developing organisms. First,
like with FRAP experiments, it requires a long track-
ing of polysomes, and second, it faces the lack of
permeability of developing organisms compared to
cells in culture.

One of the most convenient ways to measure
translation dynamics in living organisms is by ana-
lyzing the fluorescence fluctuation of the polysomes.
This technique faces numerous challenges and is
<figure><img src="image_5.png" /><figcaption>**Figure 2.** Optogenetic tools to control translation. On the left, a schematic representing mRNA in translation (mRNA in black and ribosomes in grey) bound by a light sensitive protein (orange). Upon light exposure, RNA-protein complexes will multimerize and trigger clusterization of the mRNAs bound to it, which will result in eviction of ribosome and inhibition of translation. On the right, a second system with mRNAs 5' tagged with an aptamer (loop). Upon light exposure, an RBP (yellow) will change its conformation and bind the loop, resulting in inhibition of *de novo* translation of target mRNAs.</figcaption></figure>

highly sensitive to experimental noise (e.g., photo-
bleaching, imaging focusing issues). One of the next
steps would be to generate a common theoretical
framework to improve the analysis of biological mea-
surements and to generate robust and reproducible
results, with variations reflecting only the biological
context but not the way it is analyzed. This improve-
ment is even more important for developing organ-
isms, where it is challenging to apply specific tricks
used in cell culture to improve translation dynamics
measurements as developed in M. Tanenbaum's lab-
oratory [45, 46].

Lastly, the development of tools to perturb the
translation of a specific gene will be key to under-
standing the role of fine-tuning translation, without
affecting the overall translation. Optogenetic tech-
niques would allow the spatial and temporal con-
trol of translation using, for example, light-induced
aggregation of mRNA [47] or recruitment of light-
sensitive RNA binding protein (RBP) to block trans-
lation [48] (Figure 2). These tools will enable us
to determine when and where the translation of a
specific gene is required, and to observe the result-
ing phenotypes that could potentially be linked to
some diseases. Moreover, the integration of single-
molecule translation measurements with ensemble
protein output, such as ribosome profiling and pro-
teomics experiments, will surely enhance our under-
standing of the protein life cycle.

**3. Future outlooks**

Over the past decade, considerable progress have
been made to image translation *in vivo* and at the

single molecule level. Currently, we possess a toolkit
to observe the translation of endogenously tagged
mRNA at the single mRNA level, in living cells and de-
veloping organisms.

Investigating the contribution of translation reg-
ulation in developmental regulation would repre-
sent a significant advance, given that we are begin-
ning to understand that translation is not a sim-
ple protein synthesis mechanism, as it has also
emerged previously for transcription thanks to imag-
ing using the MS2/MCP system [49]. Yet, many key
biological questions remain unsolved, such as: how
do cells selectively translate their transcriptome to
drive their fate? How dynamic is translation during
development and cell differentiation? Are there dif-
ferent "modes" of translation for the same mRNA de-
pending on the context? This level of understanding
of biological processes could lead to breakthroughs
in developmental biology, enabling us to reinvesti-
gate and better characterize previously described bi-
ological concepts. As a first step, it would be inter-
esting to revisit the translation activation of localized
mRNA using these tools. Several studies suggest that
mRNA localization is one of the main drivers of local-
ized translation, for example in morphogens gradient
formation [50-53], mRNA polarization in intestinal
epithelium [54], as well as in neuronal signaling [55]
and development [56, 57]. These tools will also en-
able researchers to better understand how transla-
tion regulation is precisely defined in the absence of
transcription, such as before the maternal-to-zygotic
transition when the development relies only on de-
posited mRNA and proteins [58], and during erythro-
poiesis [59, 60]. Indeed, mature erythroid cells lack
nuclei and therefore rely on a balanced translation of the globin chain proteins. Red blood cells can rapidly adapt to environmental changes and organismal needs, using various mechanisms that rely on translation regulation [60].

Another interesting perspective proposed by Chen et al. [61] and Boersma et al. [62] is the use of the SunTag system to study the translation dynamics of viruses in cell culture. These studies open the door to a completely new area of investigation into the replication cycle dynamics of viruses during infection, at the single molecule level, and it has yet to be implemented in living organisms to understand viral replication and translation within an organismal context and across different cell types. Last but not least, the role of translation dynamics in the robustness of developmental processes is poorly understood. For instance, gene expression compensation at the translation level has been reported in the context of aneuploidy [63], but we still lack understanding of how, when, and where this translation compensation is triggered. Another interesting context is the oocyte and early embryonic development, where dynamic and oscillatory patterns of translation have been described genome-wide, particularly during the cell cycle [64, 65]. However, how this is regulated at the single cell level and how it can control patterning and cell fate decisions remains to be elucidated. Finally, this approach would be beneficial for understanding diseases such as ribosomopathies, a group of disorders that are not fully understood, caused by disrupted ribosome biogenesis and/or diminished function [66]. It could also be useful for neurodegenerative diseases, including amyotrophic lateral sclerosis [67, 68]. Imaging translation in this context would help us to better understand the disease (e.g., which mutation affects which aspect of translation dynamics?) in order to improve the treatment of patients.

## Declaration of interests

The authors do not work for, advise, own shares in, or receive funds from any organization that could benefit from this article, and have declared no affiliations other than their research organizations.

## Author contributions

JD and MB wrote the manuscript.

## Funding

This work was supported by the HFSP long-term fellowship to MB, JD is sponsored by CNRS.

## Acknowledgments

We acknowledge D. Stainier and D. Muriaux for their support during the manuscript preparation. We thank the Académie des sciences for awarding MB with the Madeleine-Lecoq Prize.

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