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\DOI{10.5802/crbiol.158}
\datereceived{2024-05-30}
\daterevised{2024-07-23}
\dateaccepted{2024-08-08}
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\begin{noXML}

\CDRsetmeta{articletype}{opinion}

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

\alttitle{Faire la lumi\`{e}re sur l'invisible : comment l'imagerie en
temps r\'{e}el de la traduction pourrait ouvrir de nouvelles
perspectives dans le domaine de la biologie du d\'{e}veloppement}

\author{\firstname{Jeremy} \lastname{Dufourt}\CDRorcid{0000-0003-4043-7250}}
\address{Institut de Recherche en Infectiologie de Montpellier, CNRS
UMR 9004, Univ Montpellier, 1919 Route de Mende, Montpellier, 34293,
Cedex 5, France}

\author{\firstname{Maelle} \lastname{Bellec}\CDRorcid{0000-0002-9029-0042}\IsCorresp}
\address{Department of Developmental Genetics, Max Planck Institute for
Heart and Lung Research, Bad Nauheim, Germany}
\email[M. Bellec]{maelle.bellec@mpi-bn.mpg.de}

\begin{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 \textit{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.
\end{abstract}

\begin{altabstract}
Les r\'{e}centes avanc\'{e}es technologiques en biologie et en
microscopie en temps r\'{e}el ont contribu\'{e} \`{a} une meilleure
compr\'{e}hension de la mani\`{e}re dont les prot\'{e}ines sont
synth\'{e}tis\'{e}es dans les cellules vivantes. Parmi les diverses
technologies permettant l'imagerie de la traduction, l'utilisation de
la d\'{e}tection de peptides naissants par des anticorps permet \`{a}
la fois la visualisation et la quantification de la cin\'{e}tique de la
traduction de mol\'{e}cules uniques d'ARN \textit{in vivo}. Dans cette
perspective, il est discut\'{e} de l'am\'{e}lioration qu'a apport\'{e}e
cette technologie \`{a} notre compr\'{e}hension des processus
biologiques, en faisant un focus particulier sur son impl\'{e}mentation
dans les organismes en d\'{e}veloppement. Les derni\`{e}res
avanc\'{e}es technologiques dans ce domaine sont discut\'{e}es, ainsi
que leurs optimisations envisageables \`{a} court terme. Enfin,
quelques exemples montrant que cette m\'{e}thode pourrait conduire
\`{a} de futures d\'{e}couvertes scientifiques dans l'\'{e}tude de la
traduction et de sa r\'{e}gulation \`{a} l'\'{e}chelle des organismes
entiers sont pass\'{e}s en revue.
\end{altabstract}

\keywords{\kwd{Translation}
\kwd{Live-imaging}
\kwd{Development}}

\altkeywords{\kwd{Traduction}
\kwd{Imagerie en temps r\'{e}el}
\kwd{D\'{e}veloppement}}

\def\thanksname{Note}
\thanks{Maelle Bellec is the recipient of the Fondation Madeleine Lecoq
Biology Prize of the Acad\'{e}mie des sciences 2023.}

\thanks{\textbf{Funding.} HFSP, CNRS}

\maketitle

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\section{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~\cite{1,2,3}. Research has shown that mRNA can be found in specific
areas within a cell, where it may be directly translated into
proteins~\cite{4,5,6}. 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\break 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~\cite{7}). In this perspective, we focus
on the antibody-based detection of nascent peptides for visualizing
single molecule translation \textit{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.

\begin{figure*}
\vspace*{-2pt}
\includegraphics{fig01}
\vspace*{-2pt}
\caption{\label{fig1}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.}
\vspace*{-3pt}
\end{figure*}

Imaging translation through antibody recognition of nascent peptides
involves a two-component\break system: a tag, which is typically multimerized
(from 5 to 56 times~\cite{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)~\cite{10,11,12}. 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~\ref{fig1}). The
translation dots, or polysomes, may then be tracked and their
fluorescence measured to extract dynamics parameters such as diffusion,
translation speed, etc~\cite{9,13,14,15,16}. 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\break study.

Live imaging of translation using this approach has been possible since
2016 in cultured cells~\cite{9,13,14,15,16}. 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 \cite{9,13,14,15}. 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~\cite{14}. In 2021, the SunTag
system was implemented in a multicellular organism~\cite{17,18,19} and was
followed by multiple studies that were more and more resolutive in time
and space~\cite{8,20,21,22,23,24,25}. 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.~\cite{17}, the authors used the
CRISPR/Cas9 system to tag the \textit{twist} gene, encoding a key
transcription factor involved in the epithelial-to-mesenchymal
transition, at its endogenous locus in \textit{Drosophila} embryos
using the SunTag system. They found that the translation dynamics can
be heterogeneous within cells. In another study, Chen and
colleagues~\cite{26} showed in the \textit{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 exact biological processes this is relevant.

These \textit{in vivo} studies, as well as many others in mammalian
cells, have shed light on new spatial and temporal regulatory
mechanisms that cells employ~for post-transcriptional gene expression
control, such as local translation~\cite{27,28}, IRES/Cap
translation~\cite{25}, bursting of translation~\cite{29}, RNP
granule~\cite{18,26}, polysome transport~\cite{30}, translation
kinetics~\cite{31}, and coordination of translation and
degradation~\cite{20,32}.

\section{Technology improvements}

{In the past years, the range of tools used to image translation has
advanced considerably~\cite{morisaki}.} Recently, \mbox{Bellec et~al.~\cite{11}} {developed and
characterized new tools to image translation in a living}
{\textit{Drosophila}} {embryo.} {This study also revealed that imaging
translation} {\textit{in vivo}} {is not that trivial and that it has
limitations 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 translation of proteins can take several minutes, continuous
tracking of mRNA for tens of minutes or even hours is essential to
comprehend translation regulation 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 provide some advantages over FP, such as
high brightness and better photo-stability. However, this would require
injection of the dyes or harsh treatment in organisms because of their
lack of permeability\break (in contrast to cell lines). Moreover, in contrary
to FPs which replenish the pool of antibodies constantly, during
time-lapse experiments, fluorogenic protein tags require constant
feeding of dyes potentially leading to toxicity/lethality. One of the
potential future advances would be the development of genetically
encoded Quenchbodies which would fluoresce only when bound to their
target~\cite{33}, combined with ultra-bright and stable FP such as
monomeric StayGold~\cite{34}. Second, it is essential to improve
\textit{in vivo} microscopy techniques. The visualization of freely
diffusing mRNAs in cells and tissues continues to pose challenges,
especially in a living organism. Improvements in imaging technologies,
such as lattice light sheet microscopy or adaptive optics, hold
the potential to enable single mRNA imaging in live animals thanks to
their fast acquisition, large field of view, and good resolution~\cite{35}.
However, the rapid increase in the volume of microscopy data is
outpacing computer capabilities, and this issue needs to be addressed
in order to extract all the information encoded 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 \cite{36,37}, using many assumptions. Now, modeling of translation
dynamics should be improved to distinguish 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~\cite{38,39,40,41,42}.
Recently, the laboratory of B.~Wu improved these measurements for the
analysis of translation bursting~\cite{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
assessment of translation kinetics (reviewed in~\cite{31,43}). Kinetic
parameters of translation can be measured using Fluorescence Recovery
After Photobleaching (FRAP)~\cite{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 associated to immobile organelles~\cite{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~\cite{13}. This method also faces
challenges in developing organisms. First, like with FRAP experiments,
it requires a long tracking of polysomes, and second, it faces the lack
of permeability of developing organisms compared to cells in culture. 
\looseness=-1

\begin{figure*}
{\vspace*{-1pt}}
\includegraphics{fig02}
\caption{\label{fig2}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 \textit{de novo} translation of target
mRNAs.}
{\vspace*{-1pt}}
\end{figure*}

One of the most convenient ways to measure translation dynamics in
living organisms is by analyzing the fluorescence fluctuation of the
polysomes. This technique faces numerous challenges and is highly
sensitive to experimental noise (e.g., photobleaching, imaging focusing
issues). One of the next steps would be to generate a common
theoretical framework to improve the analysis of biological
measurements and to generate robust and reproducible results, with
variations reflecting only the biological context but not the way it is
analyzed. This improvement is even more important for developing
organisms, where it is challenging to apply specific tricks used in
cell culture to improve translation dynamics measurements as developed
in M. Tanenbaum's laboratory~\cite{44,45}. 

Lastly, the development of tools to perturb the translation of a
specific gene will be key to understanding the role of fine-tuning
translation, without affecting the overall translation. Optogenetic
techniques would allow the spatial and temporal control of translation
using, for example, light-induced aggregation of mRNA~\cite{46} or
recruitment of light-sensitive RNA binding protein (RBP) to block
translation~\cite{47} (Figure~\ref{fig2}). These tools will enable us to determine
when and where the translation of a specific gene is required, and to
observe the resulting 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 proteomics experiments, will surely enhance our understanding of
the protein life cycle.

\section{Future outlooks}

Over the past decade, considerable progress have been made to image
translation \textit{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
developing organisms. 

Investigating the contribution of translation regulation in
developmental regulation would represent a significant advance, given
that we are beginning to understand that translation is not a simple
protein synthesis mechanism, as it has also emerged previously for
transcription thanks to imaging using the MS2/MCP system~\cite{48}. Yet,
many key \mbox{biological} questions remain unsolved, such as: how do cells
selectively translate their transcriptome to drive their fate? How
dynamic is translation during \mbox{development} and cell differentiation? Are
there different ``modes'' of translation for the same mRNA depending on
the context? This level of understanding of biological processes could
lead to breakthroughs in developmental biology, enabling us to
reinvestigate and better characterize previously described biological
concepts. As a first step, it would be interesting to revisit the
translation activation of localized mRNA using these tools. Several
studies suggest that mRNA localization is one of the main drivers of
localized translation, for example in morphogens gradient
formation~\cite{49,50,51,52}, mRNA polarization in intestinal
epithelium~\cite{53}, as well as in neuronal signaling~\cite{54} and
development~\cite{55,56}. These tools will also enable researchers to
better understand how translation regulation is precisely defined in
the absence of transcription, such as before the maternal-to-zygotic
transition when the development relies only on deposited mRNA and
proteins~\cite{57}, and during erythropoiesis~\cite{58,59}. 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~\cite{59}.

Another interesting perspective proposed by Chen et~al.~\cite{60} and
Boersma et~al.~\cite{61} 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~\cite{62}, 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~\cite{63,64}. However, how
this is \mbox{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~\cite{65}. It could also be useful for neurodegenerative
diseases, including amyotrophic lateral sclerosis~\cite{66,67}. 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.

\section*{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.

\section*{Author contributions}

JD and MB wrote the manuscript. 

\section*{Funding}

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

\section*{Acknowledgments}

We acknowledge D. Stainier and D. Muriaux for their support during the
manuscript preparation. We thank the Acad\'{e}mie des sciences for
awarding MB with the Madeleine-Lecoq Prize.

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