Typologie d'actualités: Group Cell Signalling

Physiological characterization of life in Engineered Living Materials by confocal microscopy at single cell resolution.

The CNRS Institute of Chemistry reported this remarkable research on its site. See the article

Engineered Living Materials (ELMs) combine living cells with non-living scaffolds to get life-like characteristics, such as biosensing, growth, and self-repair. Some ELMs are 3D-printed, and called bio-ink. For ELMs to be functional, cells in ELMs has to remain alive and active. However, currently, microorganism physiology in ELMs is still elusive and restrict their use.

Researchers of the team "Cell signalling and neurofibromatosis" reconstituted such bioprinted ELMs by associating the yeast Saccharomyces cerevisiae with the hydrogel Pluronic F-127. Theydeveloped genetically engineered yeast by integrating fluorescent gene whose expression is correlated to a physiological parameter: ATP concentration (metabolism), intracellular pH (growth phase), morphology … These engineered and ratiometric biosensors are effective and allow to assess yeast physiological status in ELM directly in situ by confocal microscopy at single cell scale level. They constitute a valuable tool easy to adapt to any other system by associating them to other materials to evaluate their biocompatibility.

Furthermore, the researchers tested their recently developed copper biosensor embedded into this hydrogel F-127, and showed it is fully functional into this ELM. Yeast biosensor association with hydrogel provides several very interesting advantages such as protecting yeast from contaminations and supplying them with nutrients.

This work allows to establish the proof of concept that F127 associated with engineered yeast S. cerevisiae is a promising ELM in order to develop easy to use whole-cell biosensors able to detect copper directly on samples collected in the environment.

Bojan Žunar B., Ito T., Mosrin C., Sugahara Y., Bénédetti H., Guégan R. and Vallée B.
Confocal imaging of biomarkers at a single-cell resolution: quantifying 'living' in 3D-printable engineered living material based on Pluronic F-127 and yeast Saccharomyces cerevisiae.
Biomater Res 26, 85 (2022). https://doi.org/10.1186/s40824-022-00337-8

Cooper in a micronutrient essential to life, its lack induces neurological and blood disorders. It is extensively used in industry, in particular in the batteries of electric cars, but also as a fertilizer and fungicide. However, it is toxic at high concentrations, and is considered as a critical emerging pollutant. Copper detection in water constitutes a major societal and environmental issue.

Currently, copper concentrations are monitored by sophisticated analytical methods requiring time, expansive equipments and deep expertises. Moreover, these technics quantify total copper present in a sample and not copper interacting with living organisms.

Researchers of CBM developed a new and atypical device able to detect copper in a very sensitive and easy way. Their biosensor is based on whole-cell eukaryotic living Saccharomyces cerevisiae, allowing detection of bioavailable copper. It is a ratiometric biosensor, measuring the ratio between (i) a constitutively expressed fluorescent protein and (ii) another fluorescent protein whose expression is directly correlated with copper concentrations as its expression is in under the control of CUP1 promoter, a well-known promoter in Saccharomyces.

By genetic engineering, researchers created many different variants to optimize the response of our biosensor. Their best biosensor is able to detect as low as 10 nM of copper in a linear range from 10-3 to 10-8 M, much better features compared to other currently reported whole cell copper biosensors. This biosensor was also validated on “real” samples: detected concentrations are totally in agreement with manufacturers’ values.

Reference of the article :
Bojan Zunar, Christine Mosrin, Hélène Bénédetti, Béatrice Vallée
Re-engineering of CUP1 promoter and Cup2/Ace1 transactivator to convert Saccharomyces cerevisiae into a whole-cell eukaryotic biosensor capable of detecting 10 nM of bioavailable copper
Biosensors and Bioelectronics 214 (2022) 114502

The article was reported by the CNRS Institute of Chemistry on its website and in its letter "En direct des labos".

"Cell signalling" group led by the Dr H. Bénédetti has published a video paper in JOVE, Journal of Visualized Experiments, a peer-reviewed scientific journal that publishes experimental methods in video format.

This paper belongs to the Method section focusing on basic technics in Biochemistry.

Different experiments are detailled:

- Transient cell transfections
- Protein extraction
- Study of protein/protein interaction by co-immunoprecipitation experiments
- Study of kinase activity by g³²P-[ATP] labeling or by using phospho-specific antibodies

All these different technics are illustrated with data obtained by the group on LIMK2-1, a new protein the team has just pointed out and characterized. LIMK2-1 protein does exist, and it is very atypical in the way it regulates actin cytoskeleton remodeling.