Visualisation of translation initiation reaction on single mRNA molecules in vivo

Project Information

smTI

Grant agreement ID: 101026470

DOI

10.3030/101026470

Project closed

EC signature date 3 March 2021

Start date 1 May 2021

End date 30 April 2023

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 187 572,48

EU contribution

€ 187 572,48

Coordinated by

KONINKLIJKE NEDERLANDSE AKADEMIE VAN WETENSCHAPPEN - KNAW
Netherlands

Periodic Reporting for period 1 - smTI (Visualisation of translation initiation reaction on single mRNA molecules in vivo)

Reporting period: 2021-05-01 to 2023-04-30

Summary of the context and overall objectives of the project

Translation, an essential process in cell biology, controls the production of proteins. It is regulated at multiple stages, including initiation, elongation, and termination, through complex molecular networks. Understanding how translation is regulated is essential for unraveling various biological processes, such as development, but the underlying dynamics remain unclear.
The objective of the project, which received from the Marie-Curie action, was threefold:
1. Develop a new method to visualize translation initiation in individual cells.
2. Uncover the dynamics and key mechanisms involved in translation initiation.
3. Provide a universally applicable method to study cellular processes.
Thanks to the support received, the project has focused on developing an innovative approach to visualize translation processes at the single molecule levels. We successfully developed an advanced live-cell single-molecule imaging technique, utilizing nanostructures called zero-mode waveguides. In addition, we investigated translational dynamics using a new single-molecule imaging technique and acquired new insights into the molecular diversity involved in translation.
The findings of this project significantly contribute to our understanding of how proteins are produced in cells. By visualizing translation processes and studying their intricacies, we have gained valuable insights that can be applied across various biological sciences. This knowledge has the potential to drive further advancements and discoveries in diverse biological fields.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

The project has made a significant breakthrough in imaging individual molecules inside living cells using a technology called ZMW. In the past, it was very challenging to observe single molecules using fluorescence techniques because cells contain a lot of proteins, making it difficult to distinguish individual molecules. However, the project has found a promising solution by using ZMW, which are tiny holes smaller than the wavelength of light, in a thin metal film. These holes create extremely small spaces where molecules can be observed. In collaboration with a specialized research group, the project has developed a new ZMW technology specifically designed for imaging molecules in live cells. This technology has opened up new possibilities for studying cells in greater detail. One exciting discovery is that human cells can form stable extensions into these tiny holes, allowing us to observe fluorescently labeled proteins at the level of individual molecules over long periods of time. With this new approach, we can observe single molecules with exceptional clarity and accuracy, even when there is a lot of background noise from other molecules in the cell. Traditional microscopy techniques like confocal and TIRF have limitations in dealing with high levels of background noise, but our method overcomes these challenges. It can be easily implemented on standard fluorescence microscopes, making it accessible for many researchers working with different biological systems.
Additionally, we studied a wide range of sizes of the ZMWs to provide selection guidelines for optimal optical properties and cell compatibility. This comprehensive analysis gives researchers valuable insights for designing experiments using ZMW technology. In summary, this project has developed an advanced technique using ZMW technology to visualize individual molecules inside living cells. This breakthrough allows us to study cells in ways that were previously not possible, opening up new avenues of research in the field of cell biology.
The results of this MSCA will be reported in:
(1) A peer-reviewed journal as a research paper on live-cell single-molecule imaging method using zero-mode waveguide
(2) A peer-reviewed journal as a research paper on translation elongation heterogeneity
(3) The 12th Single Molecule Localization Microscopy Symposium in 2023
For research training and transfer of knowledge, the fellow attended 4 conferences (2021 EMBL conference: Protein Synthesis and Translational control, 2022 NWO Biophysics, 2022 EMBL symposium: The complex life of RNA, 2022 RNA – Beyond its Genetic Code)

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Until recently, cell biology has been limited to examining large groups of molecules and had difficulty detecting individual proteins. However, understanding how cells work requires observing the behavior of single molecules in real time. To tackle this problem, the project developed a new technique that allows scientists to see cellular processes at the molecular level.
The main obstacles to observing single molecules were the high levels of proteins present in cells and the rapid movement of these molecules in three dimensions. But thanks to protein engineering and improvements in microscopy, the project found a way to overcome these limitations. This breakthrough enables scientists to visualize cellular processes in living cells and gain a deeper understanding of how they work on a molecular level.
The new methods developed by the project have an unprecedented sensitivity for detecting interactions between single RNA-binding proteins and mRNA. This opens up exciting possibilities for studying the mechanisms of translation, which is the process by which genetic information is used to build proteins. Not only that but these methods can also be applied to study other molecular interactions, such as microRNA interactions with mRNA. They can even be extended to observe interactions between individual molecules on mRNA.
This will greatly enhance our understanding of cellular processes and provide new avenues for research in various areas, including the study of translation and other molecular interactions.

Live-cell ZMW imaging method. Using ZMWs, membrane localized fluorescent molecules can be visualized