Exploring the yeast cell wall organization at the nanoscale by combining biomolecular, biochemical and biophysical approaches – AFMYST

AFMYST aims at imaging the yeats cell wall using atomic force microscopy. 2 technological bottelneck were identified: firstly the cell immobilization. The first task of the project is dedicated to the optimization of an immobilization tool. Secondly, biofunctionnalizing AFM tips is a tricky task. We will use dendritips, a method based on a patent we have developed in the project.

The results of this project provide insights into the dynamics, functions, organization and architecture of the wall of the yeast. These results allow us to consider the uses of the walls of yeast as a probiotic or as a filter for pollutants. These applications are of high industrial property interest. On the other hand, we were interested in Candida albicans, opportunistic fungal pathogen, and have studied its wall and changes in its wall after an antifungal treatment. The results show a structure of adhesion sites as nanodomains that are deeply modified during therapy. These results should not fail to interest the pharmaceutical industry.

The work has opened the way to interesting biomedical applications. Saccharomyces cerevisiae serves as a model for pathogenic yeasts like Candida albicans. Our knowledge on saccharomyces could open new therapeutic strategy.

In the same time, this new knowledge on the yeast cell wall is of interess for food industry which produce large amount of yeast cell wall and which tries to make benefit of it.

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-Pillet F., et al., 2014. Biochimica and Biopysica Acta General Subjects 1840, 1028-1050
-Schiavone M., et al., In Press. A combined chemical and enzymatic method to determine quantitatively the polysaccharide components in the cell wall of yeasts, FEMS Yeast
-Formosa C., et al., In Press. Multiparametric imaging of adhesive nanodomains at the surface of C. albicans by Atomic Force Microscopy Nanomedicine NBM
-Formosa C., et al., In Press. Mapping HA-tagged protein at the surface of living cells by atomic force microscopy. Journal of Molecular recognition
-Formosa C., et al., A versatile method to generate living cells arrays for Atomic Force Microscopy studies Nature Protocol Accepted
-Elsztein C., et al., François J.M., Biophysical properties of wild type strain and yap1 mutant in response to ethanol shock (to be submitted)

This transdisciplinary project, AFMYST, will need skills in biology, chemistry and physics. The biological questions concern the yeast (Saccharomyces cerevisiae) cell wall organization and morphogenesis. The yeast cell wall has essentially been studied from a biochemical or molecular biology perspective. Our aim is to collect and interpret atomic force microscopy data to get a better understanding of the biophysics of the cell wall. Our preliminary experiments, performed on a mutant collection, show the validity of the approach based on the measure of the cell surface roughness and elasticity. Moreover, the impact of different stresses (osmotic, thermal and drugs) will be evaluated. Secondly, the experiments will focus on cell wall proteins. In order to achieve this goal, antibodies directed against Flo11, Cwp1, Pir1 and Gas1 will be chemically attached on AFM tips using crosslinkers or dendrimers (patent: E Dague et al., N°1057932 submitted 30/09/2010).
Finally the action mechanism of two antifungal molecules will be investigated: caspofungin and toxin killer 9, using AFM tips functionalized by these molecules.

This proposal is clearly transdisciplinary, that gathers three young scientists , two from the NanoBioSystem team at LAAS –CNRS directed by Liviu Nicu (E Dague CR2-CNRS and C Thibault associate professor INSA), and a young scientist from the Microbial Molecular Physiology team at LISBP headed by Prof Jean Marie François ( Dr H Martin-Yken, CR1 - INRA). These three young scientists present high complementarities in their expertise, as LISBP provides the biological expertise in cell wall and brings to the Nanotechnologues experts from LAAS the biological questions.
The project is organized in 5 tasks. The first one will consist in optimizing cells immobilization methods using a new methodology (paper submitted in Advance material) for their study in AFM. The second task will be dedicated to the biophysics of the cell wall under different stress conditions. The third task aims at functionalizing AFM tips. Task 4 entitled cell wall functional analyses, will consist in making cell surface maps of the 4 different proteins described before. coordination and management will be the last task lead by the coordinator of the project: Dr E Dague from LAAS-CNRS
This research proposal is clearly fundamental, though deserves high interest in biotechnology applications for health (eg antifungal action) and food (yeast cell wall used as binders to mycotoxins) and henceforth, it will be essentially disseminated to the scientific community through publication in high impact Journals.
The principal investigator, E Dague, is borned in 1978, PhD, Pharmacist, Distinguished by the French national academy of Pharmacy, CNRS researcher since 2007. He has authored or coauthored 22 international scientific articles, in the field of AFM applied to living cells.
The financial request for equipment consists in the upgrade of existing AFM equipment.
The financial request for personnel will be for a post doc who will be in charge of task 2 to 4.