Marion Schiavone1, 2, 3, 4, 5; Sébastien Déjean1,6; Etienne Dague1, 4, Jean Marie François*1,2,3
1
Université de Toulouse; INSA, UPS, INP, 135 avenue de Rangueil, F-31077 Toulouse, France
2
INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, F-31077 Toulouse, France ;
3
CNRS, UMR5504, F-31400 Toulouse, France 135 avenue de Rangueil, F-31077 Toulouse, France ;
4
CNRS; LAAS ; 7 avenue du colonel Roche, F-31400 Toulouse, France ;
5
Lallemand SAS, 19 rue des briquetiers, 31702 Blagnac, France
6
Institut de Mathématiques de Toulouse, 118 route de Narbonne, F-31062 Toulouse, France
*Correspondance to Jean Marie François; LISBP INSA, 135 Avenue de Rangeuil, F-31077 Toulouse cedex 04 ; Email: fran_jm@insa-toulouse.fr; Phone: +33(0) 5 61 55 94 92
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ABSTRACT
The cell wall is a dynamic structure that confers resistance to the cell against environmental changes by adapting the cell wall composition and by changing its nanomechanical properties. However, how the molecular architecture of the cell wall is related to its biochemical composition and account for its nanomechanical properties is a challenging open question. As a first step towards this goal, we used a set of well-characterized cell wall mutants from which transcriptomic data were available, and then measured their cell wall composition and obtained quantitative data on cell elasticity, cell surface adhesion and interaction of concanavalin A with mannoproteins using atomic force microscopy (AFM). We subsequently combined the datasets through their ratio against the wild-type strain and performed a comparative analysis. We made different observations. First, single molecule force spectroscopy experiments showed the impact of mutation in the deployment through the mannoproteins using AFM tips functionalized with concanavalin A. Long chains of polysaccharides were unfolded with concanavalin A tips on the surface of gas1Δ mutant cell, while on mnn9Δ cell a small percentage of specific interaction mannan-lectin were observed with shorter rupture lengths (below 100 nm). Second, we confirmed our integrative approach by finding an association between the levels of mannan and frequency events in lectin-cell surface interactions. Thirdly, we showed that cell wall elasticity values correlated with β-1,3-glucan although cross-linkages between cell wall component is likely determining the quantitative value of this nanomechanical properties. Finally, genes found to be associated to physico-biochemical variables were distinct to those previously identified as implicated in the cell wall remodeling mechanism.
I. INTRODUCTION
The cell wall of Saccharomyces cerevisiae is a complex interplay of four polysaccharides that are cross-linked to provide integrity to the cell. These polysaccharides are β-1,3 and β-1,6-glucan which are formed by glucosyl units attached either in β-1,3 or β-1,6-linkages. They represent around 50-60% of total cell wall polysaccharides. The second polysaccharide in abundance (around 40-50%) is mannoproteins that are composed of linear chains of 1,6-mannosyl units with 1,4; 1,6; 1,2; α-1,3 branches and are attached to proteins through either an asparagine (N-glycosylation) or serine/threonine (O-glycosylation) residue. The fourth component is chitin which is a linear polymer of 100-150 units of β-1,4-N-acetylglucosaminyl residues. Chitin is indispensable for yeast cells in spite of its low abundance (1 to 4 % of the total cell wall polysaccharide), likely because of its key role in
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septation that takes place during budding (Cabib and Arroyo, 2013; Shaw et al., 1991). According to electronic microscopy observations (Osumi, 1998), the yeast cell wall presents an amorphous and colorless internal layer of about 70-100 nm thick that is composed of a complex network of chitin, β-1,3 and β-1,6-glucan (Klis et al., 2002). The outer layer is condensed and mainly composed of cell wall mannoproteins (CWP) anchored to β-glucan.
The cell wall is a dynamic structure that is remodeled in order to avoid cell lysis and eventually cell death. Modification of the cell wall appears during cell growth and morphogenesis, but also in response to environmental stress or various injuries caused by drugs, lytic enzymes or mutations in genes implicated in its synthesis. Genetic analyses led to the finding that the MAPkinase cascade dependent on PKC1 is the main signaling pathway that controls this cell wall remodeling (Levin, 2004; Lesage & Bussey, 2006). In addition, genome wide transcriptomic analysis of this cell wall remodeling caused by mutations in genes specifically implicated in synthesis of cell wall component or by cell wall perturbing agents led to the identification of a ‘core of about 50 upregulated genes’ that are considered to be critically important in this cellular response (Bermejo et al., 2010; Garcia et al., 2004; Lagorce, 2003). Among them were identified several genes encoding glycosyltransferase/hydrolase, that were called ‘cell wall remodeling enzymes’. These changes at the transcriptional level can be associated with biochemical modifications that take place in response to cell wall remodeling and which are: (i) an increase of chitin amount in cell wall which can contribute up to 20% of the cell wall when important genes encoding for its biosynthesis are deleted (Popolo et al., 1997); (ii) a modification of linkages between cell wall components and (iii) an increase of cell wall remodeling enzymes accompanied with a redistribution of cell wall synthesis and repair machinery. Yeast cell wall is also endowed with biomechanical properties that can nowadays address using Atomic Force Microscopy, invented by Gruber and colleagues at IBM Zurich (Binnig et al., 1986). Initially developed in Material Sciences, this technology has evolved to be able to operate in liquid using life cells in their natural environment (Dufrene 2001;Dague et al. 2007). A seminal work illustrating cell wall mechanical properties was reported by Pelling et al. (Pelling et al., 2004). These authors reported nanomechanical motions of the
S. cerevisiae cell wall with a periodicity in the range of 0.8 to 1.6 kHz and amplitudes of approximately
3 nm, which were of biological origin and could implicate the yeast actin cytoskeleton. More recently, we carried out AFM nano-indentation experiments on yeast mutants deleted in specific genes implicated in synthesis of cell wall component (i.e. CHS3, for chitin; FKS1, for β-1, 3 glucan, MNN9, for
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mannans; KRE6 for β-1,6 glucan) or cell wall remodeling enzymes (GAS1, CRH1/CRH2). In this study, we showed that the elasticity properties of the cell wall was not dependent on a specific cell wall component, which was at variance of the established consideration that β-glucan layers were responsible for the ‘rigidity’ of the cell wall (Fleet 1991;Klis et al. 2006). Other biophysical properties can be addressed using single molecule force spectroscopy (SMFS). This technique consists to probe the cell surface with either chemically modified or functionalized tips with a given biomolecule that has a specific interaction with a component exposed at the cell surface, such as concanavalin-A for mannoproteins (Alsteens et al. 2008). With this approach, valuable qualitative and quantitative data on adhesion properties, physical organization of the cell surface as well as identification of clusters of cell surface protein can be obtained (Alsteens et al., 2010; Formosa et al., 2014).
Giving these different approaches that deliver apparent independent but complementary data which contribute to identify the mechanisms of cell wall biogenesis and remodeling, we could argue that using appropriate biomathematical tools developed under the mixOmics package (Lê Cao et al., 2009) that permits to create correlation matrixes between biological data obtained at different hierarchical levels such as metabolomics/transcriptomics data (Guo et al., 2014) we should be able to find out molecular cues (genes or proteins) that account for the biomechanical properties of the cell wall, as well as which and how the different cell wall component contribute to cell wall nanomechanical property. As a first step towards this goal, we used a set of well-characterized cell wall mutants from which transcriptomic data were available (Lagorce et al), measured cell wall composition in these mutants using our recent chemical-enzymatic method (Schiavone et al., 2014) and obtained quantitative data on cell elasticity, cell surface adhesion and interaction with mannoproteins using atomic force microscopy (AFM). This integrative approach allows highlighting some unexpected associations between ratio of β-glucan/mannan with elasticity, whereas events of adhesion were mainly related to mannans, and genes found to be associated to these physico-biochemical variables were distinct to those previously identified as implicated in cell wall remodeling mechanism.
II. MATERIAL AND METHODS
Yeast strains
The strains used in this work are listed below.
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Strain Genotype Origin
BY4741 MATa his3Δ1; leu2Δ0 ; met15Δ0 ura3Δ0 EUROSCARF
chs3Δ BY4741 chs3::KanMX4 Open Biosystem
gas1Δ BY4741 gas1::KanMX4 Open Biosystem
mnn9Δ BY4741 mnn9::KanMX4 Open Biosystem