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Relation between phosphoinositides and cell polarity

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Group leader : François Doignon, Professor at Bordeaux University

François DOIGNON is a Professor of Molecular and Cellular Genetics at the University of Bordeaux (since 2007) and before he was Assistant Professor in Genetics (1993-2007) in this same university. He obtained an authorization to supervise the researches (2000) following a PhD from the Bordeaux 2 University (1991).

contact :
Phone : 00 33 5 57 12 25 84
E-mail : francois.doignon@u-bordeaux.fr

Research topics
My first research theme concerned the influence of fungicides inhibiting ergosterol pathway in Saccharomyces cerevisiae during alcoholic fermentation (1987-1991). This study allowed us to better understand at the fundamental level the mechanisms of resistance of yeasts to fungicides and moreover at the technological level to highlight the impact of residues of fungicides on the quality of alcoholic fermentation
Subsequently, I was part of the pilot laboratories involved in the European project for the genome sequencing of yeast S. cerevisiae (1991-1996) (BAP, BRIDGE, BIOTECH). These programs have revealed among the identified genes, 2/3 of new genes in yeast which is a model organism. Among these new genes, half had no resemblance to genes already identified in any organism. In parallel, we participated in European programs for functional analysis of the genome of yeast S. cerevisiae. 1992-1996) (EUROFAN). We have also developed studies by Inverse Genetics of genes involved in signal transduction pathways (1994-1999). Concerning the control of the integrity of the cell and more particularly of the cell-wall following stress conditions. The stress we have most studied is stress following an acid shock.
Our research has focused on problems concerning the regulation of cellular polarity by the GTPases of Rab and Rho families in S. cerevisiae. (1997-2010). Cell polarity consists to determine an area at which there will be a specific molecular organization related to the growth or function of the cell. I was particularly interested in studying regulators controlling the implementation of cellular polarity. We have also studied same topic with yeast Candida albicans, which is a yeast capable of forming filaments.
Currently, I study the relationships between lipids involved in cell signaling (phosphoinositides) with cell polarity in the yeast S. cerevisiae. (since 2011). We have shown that the phosphoinositides containing stearic acid are essential for implementation and a good localization of the actin cytoskeleton, as well as certain actors involved in intracellular trafficking, thus action on secretory activity of the cell.

Group Members : F. Doignon (Pr), L. Fouillen (IR), P. Laquel (CR), E. Testet (MC), C. Rocher (IE-HDR).

Regulation of membrane lipid homeostasis in yeast
1) Phosphoinositides and cell polarity : what are the links ?
In animals, plants and yeasts, polyphosphoinositides (PIP, PIP2) are one of the most important lipid signaling molecules and regulate cell trafficking and cell polarity. Pools of polyphosphoinositides are qualitatively modified in mutants affected for sn-1 acyl-2-lysoPI acyltransferase. In previous work, Le Guédard et al. 2009 characterized the enzyme Psi1p in S. cerevisiae involved in quality control of the phosphoinositides in yeast.

2) Pools of phosphoinositides are qualitatively modified in mutants affected for sn-2-acyl-1-lysoPI acyltransferase.
In vitro, an acyl transferase activity, incorporating stearic acid at the sn-1 position in lysoPI was highlighted by our lab. In yeast, a PSI1 deletion mutant showed a very low amount of PI with stearic acid. PI is a precursor of PIP and PIP2. We compared the molecular species of PI, PIP and PIP2 in wild type and deletion mutant by mass spectrometry. Our results showed that the PI species with a stearic acyl chain are affected in the psi1Δ mutant. (Fig 1)
Using specific GFP probes for PI4P or PIP2, we showed that PI4P distribution is disturbed and that the bud peripherical staining with PIP2 GFP biosensor is less intense in the psi1Δ mutant than in the control.

3) The cell polarity is affected in yeast strains deficient for phosphoinositides with stearic acid
In S. cerevisiae the spatial budding pattern is known to be axial in haploid cells and bipolar in diploid cells. Based on calcofluor-white staining (specific for chitin bud scars) we observed modification of the budding pattern in the mutants.

4) The organization of the Actin cytoskeleton is strongly disturbed by a defect of phosphoinositides containing stearic acid.
Polarized growth in yeast depends on actin polarity and is involved in intracellular trafficking. Based on actin patches observations the PSI1 deletion gave rise to a decrease of the number of polarized cells (90% to 60%). Similarly the number of actin cables per cell is lower in psi1 mutants (Fig 2).

5) A new actin organization is specifically detected in mutant cells for the PSI1 gene
An organization with bipolar cortical actin was observed only in diploid cells mutant for the synthesis of phosphoinositides containing stearic acid. (Fig 3) In addition, the Rho protein : Cdc42, the super regulator of cell polarity, is localized to the two poles of the cells showing this bipolar actin organization. This new actin organization was described for the first time (Fig 4).

6) Intracellular traffic is affected for individuals deficient in phosphoinositides containing stearic acid
The trafficking of secretory vesicles is dependent on actin cables. A secretion assay was performed using landmarks of (Bgl2-HA) secretory vesicles. We observed that the secreted Bgl2-HA decreased in psi1Δ compared to WT cells, pointing out an exocytosis defect (Fig 5).
Endocytosis was checked based on Lucifer Yellow (LY) uptake experiments. No defect of LY accumulation in the vacuole is observed for psi1Δ. Nevertheless the morphology of the vacuole is modified compared to the reference strain. Thus the pool of PIP2 might be affected in psi1Δ mutant deficient for PI C18:0 synthesis.

Key publications :

- Martinez D, Langlois d’Estaintot B, Granier T, Tolchard J, Courrèges C, Prouzet-Mauléon V, Hugues M, Gallois B, Doignon F, and Odaert B. (2017) Structural evidence of a phosphoinositide binding site in the Rgd1-RhoGAP domain. Biochem. J., 474, 3307-3319

- Martinez D, Prouzet-Mauléon V, Hugues M, Doignon F, and Odaert B. (2018) Assignment of 1H 13C and 15N resonances and secondary structure of the Rgd1-RhoGAP domain. Biomol NMR Assign., 12, 129-132-* Doignon F., Laquel P., Testet E., Tuphile K., Fouillen L., and Bessoule JJ. (2016) Requirement of phosphoinositides containing stearic acid to control cell polarity. Mol. Cell. Biol., 36, 765-780

- Varon C., Mocan I., Mihi B., Péré-Védrenne C., Aboubacar A., Moraté C., Oleastro M., Doignon F., Laharie D., Mégraud F., and Ménard A. (2014). Helicobacter pullorum Cytolethal Distending Toxin Targets Vinculin and Cortactin and Triggers Formation of Lamellipodia in Intestinal Epithelial Cells. J. Infect. Dis., 209, 588-599

- Vieillemard A., Prouzet-Mauléon V., Hugues M., Lefèbvre F., Mitteau R., Clavérol S., Bonneu M., Crouzet M., Doignon F., and Thoraval D. (2013) The Saccharomyces cerevisiae RhoGAP Rgd1 is phosphorylated by the Aurora B like kinase Ipl1. Biochem. Biophys. Res. Commun., 433, 1-5

- Buré C., Ayciriex S., Testet E. and Schmitter J.M. (2013) A single run LC-MS/MS method for phospholipidomics Analytical and Bioanalytical Chemistry, 405(1) : 203-213.

- Lasserre JP, Plissonneau J, Velours C, Bonneu M, Litvak S, Laquel P, Castroviejo M. Biochemical, cellular and molecular identification of DNA polymerase α in yeast mitochondria. Biochimie. 2013 Apr ;95(4):759