Evidence for a Role for the Plasma Membrane in the Nanomechanical Properties of the Cell Wall as Revealed by an Atomic Force Microscopy Study of the Response of Saccharomyces cerevisiae to Ethanol Stress

Abstract

A wealth of biochemical and molecular data have been reported regarding ethanol toxicity in the yeast Saccharomyces cerevisiae. However, direct physical data on the effects of ethanol stress on yeast cells are almost nonexistent. This lack of information can now be addressed by using atomic force microscopy (AFM) technology. In this report, we show that the stiffness of glucosegrown yeast cells challenged with 9% (vol/vol) ethanol for 5 h was dramatically reduced, as shown by a 5-fold drop of Young's modulus. Quite unexpectedly, a mutant deficient in the Msn2/Msn4 transcription factor, which is known to mediate the ethanol stress response, exhibited a low level of stiffness similar to that of ethanol-treated wild-type cells. Reciprocally, the stiffness of yeast cells overexpressing MSN2 was about 35% higher than that of the wild type but was nevertheless reduced 3-to 4-fold upon exposure to ethanol. Based on these and other data presented herein, we postulated that the effect of ethanol on cell stiffness may not be mediated through Msn2/Msn4, even though this transcription factor appears to be a determinant in the nanomechanical properties of the cell wall. On the other hand, we found that as with ethanol, the treatment of yeast with the antifungal amphotericin B caused a significant reduction of cell wall stiffness. Since both this drug and ethanol are known to alter, albeit by different means, the fluidity and structure of the plasma membrane, these data led to the proposition that the cell membrane contributes to the biophysical properties of yeast cells. IMPORTANCEEthanol is the main product of yeast fermentation but is also a toxic compound for this process. Understanding the mechanism of this toxicity is of great importance for industrial applications. While most research has focused on genomic studies of ethanol tolerance, we investigated the effects of ethanol at the biophysical level and found that ethanol causes a strong reduction of the cell wall rigidity (or stiffness). We ascribed this effect to the action of ethanol perturbing the cell membrane integrity and hence proposed that the cell membrane contributes to the cell wall nanomechanical properties. T he yeast Saccharomyces cerevisiae is a remarkable ethanol producer that is also very sensitive to its main fermentative product. At low to moderate concentrations (5 to 7%), ethanol mainly affects the growth rate, and at higher concentrations (Ͼ10%), it can strongly impair cell integrity, eventually leading to cell death with features of apoptosis (1). These inhibitory and toxic effects are ascribed to the fact that ethanol alters cell membrane fluidity and dissipates the transmembrane electrochemical potential, thereby creating permeability to ionic species and causing leakage of metabolites (2). Recent works using lipidomic methodologies confirmed the relationship between the composition of lipids, notably ergosterol and unsaturated fatty acids, and ethanol tolerance (3, 4). Moreover, as it diffuses freely into cells, ethanol at high concen...

Integration of Biochemical, Biophysical and Transcriptomics Data for Investigating the Structural and Nanomechanical Properties of the Yeast Cell Wall

“…This finding is suggestive of a linkage between physical properties of cell wall and membrane structure and/or fluidity. Furthermore, the negative correlation of Young's modulus with this set of genes comprising ergosterol encoded genes supports our recent work showing that plasma membrane contributes to the cell wall stiffness properties (Schiavone et al, 2016 ). This correlation also agreed with the finding that uptake of sterol is favored by highly mannosylated proteins encoded by TIR/DAN genes family (Abramova et al, 2001 ; Francois, 2016 ).…”

“…According to Gonzalez et al ( 2012 ), correlation between these variables is better visualized in a 2-dimensional heat map representation where relations between biophysical and biochemical variables are reported on the vertical axis whereas the horizontal axis shows the genes whose expression are the most correlated with these two variables (Figure 6 ). Confidence about this multivariate methodology sPLS is provided by retrieving predictable correlations such as between hydrophobicity and adhesion events (i.e., frequency of interaction of the concanavalin A-functionalized AFM tips on the yeast cell surface), as well as confirming that there is no simple correlation between the Young's modulus and any of the three types of cell wall polysaccharides (Dague et al, 2010 ; Schiavone et al, 2016 ). On the other hand, pertinent associations can be pointed out thanks to this statistical method such as a connection of β-1,3-glucans with the contour length or unexpected ones like the association of mannans and chitin as one would predict an association of chitin with β-1,6 glucans according to the cell wall structure proposed elsewhere (Klis, 1994 ).…”

“…In transmission electron microscopy, the cell wall appears as a two layered structure. The fuzzy electron dense outer layer of about 30–40 nm thick is supposed to mainly contain cell wall mannoproteins, whereas the internal layer of about 70–100 nm that shields the plasma membrane is mainly made of β-glucans (Osumi, 1998 ; Backhaus et al, 2010 ; Schiavone et al, 2016 ). In spite of these apparent distinct layers, the four macromolecules that composed the cell wall, namely mannoproteins, β-1, 3 glucans, β-1,6 glucans and chitin are covalently joined to generate a highly dynamic three-dimensional architecture of the wall.…”

Membrane Fluidity of Saccharomyces cerevisiae from Huangjiu (Chinese Rice Wine) Is Variably Regulated by OLE1 To Offset the Disruptive Effect of Ethanol

“…Transmission electron microscopy. The transmission electron microscopy methods described by Wright (46) and Schiavone et al (47) were used in this study. Briefly, yeast cells were fixed for 12 h (i.e., overnight) in 2.5% glutaraldehyde, washed, and fixed in 1% osmium tetroxide for 1 h. Subsequently, the cells were washed, dehydrated in acetone, and finally embedded in LR White resin.…”

Protein‐facilitated transport of hydrophobic molecules across the yeast plasma membrane

“…The cell wall armor provides the cell with some ‐literal‐ elasticity against environmental changes or extracellular product accumulation e.g. high ethanol production . The intimate relation between cell wall and plasma membrane governed by GPI‐anchored glycoproteins may explain that both the cell wall integrity and the Pleiotropic Drugs Resistance (PDR) pathway are upregulated after exposure to organic solvents .…”