The yeast Saccharomyces cerevisiae is a unicellular eukaryotic microorganism surrounded by a 100–120 nm thick cell wall . The fungal cell wall is an essential structure that maintains cell shape and cell integrity, ensures resistance to internal turgor pressure and thereby prevents cell lysis . The cell wall of Saccharomyces cerevisiae, which represents 10 - 25% of the cell dry mass according to the culture and process conditions , consists of three types of polymers that are interconnected to produce a modular complex structure . The inner layer of the cell wall is composed of a β-1,3-glucan network (80 - 90% of the total β-glucan) branched with chitin (1–2% of the cell wall). Together, they form a structure that is largely responsible for the mechanical strength of the whole cell wall [5, 6]. In addition, β-1,6-linked glucans (8 - 18% of total β-glucans) are branched on the β-1,3-glucan network, and also linked to the mannoproteins that compose the outer layer [7, 8]. The yeast cell wall is a dynamic structure, the molecular architecture of which is continuously remodeled during morphogenetic processes and growth . It also undergoes remodeling in response to environmental stresses, such as ethanol and oxidative stress [10, 11], thermal and osmotic stress [12–14], and in response to antifungal drugs such as allicin or caspofungin [15, 16]. These remodeling processes are organized by a “cell wall rescue-mechanism” that relies on a combination of several signaling pathways, with a major role played by the PKC1-dependent cell wall integrity (CWI) pathway (reviewed in [9, 17]). Important biochemical modifications identified so far during stresses were i) massive deposition of chitin that takes place in the lateral walls of both the mother cells and the growing buds, ii) an increased cross-linkage between chitin and β-1,3-glucan and iii) the appearance of novel linkages between cell wall proteins and chitin through β-1,6-glucan [18, 19]. Altogether, these cell wall repair mechanisms have been considered as a mean to combat cell wall weakening caused by these stresses [4, 20]. However, a direct visualization of the topography and nanomechanical changes associated to these biochemical and molecular changes induced by stresses was still missing to better understand the cell wall biogenesis and remodeling mechanism. The remarkable development of the Atomic Force Microscopy (AFM) technology, combined with genetical and molecular tools, is therefore powerful to fulfil this gap and investigate the dynamics of microbial cell surfaces in response to external cues [21, 22].
In this study, we have investigated the effects of heat shock on the nanomechanical properties of the yeast cell wall. We chose this stress condition because of the large body of data available on the heat shock response in the yeast Saccharomyces cerevisiae (reviewed in ). In brief, this response is characterized at the genome level by an intense program of changes in gene expression leading to repression of protein biosynthetic machinery and the induction of a battery of genes encoding heat shock proteins (HSPs). The main metabolic and physiological changes reported in response to heat stress are an accumulation of trehalose and an inhibition of glycolysis [24, 25], associated with a transient arrest of cell division. Heat shock also triggers the activation of the CWI pathway, resulting in a global transcriptomic change including the overexpression of genes encoding cell wall remodeling enzymes . Although AFM analysis of temperature stress on yeast cells has been previously addressed by Adya et al. , we have revisited this stress because of two major technical concerns in the study reported by the latter authors. Firstly, the immobilization procedure they used could likely alter the cell viability and integrity since yeast cells were immobilized on glass slides by air-drying for more than 5 hr. Secondly, the stress was carried out at temperature ranging from 50 to 90°C, which is incompatible with yeast life and irrelevant in a biotechnological viewpoint.
Using a recent immobilization method that ensures the viability and integrity of the yeast cells , we showed that a temperature shift from 30 to 42°C induced the singular formation of circular rings that initiate at a single point on the yeast cell surface and expanded in a concentric manner to reach a diameter of 2 to 3 μm after 1 h of incubation. Appearance of this circular structure was accompanied by a twofold increase of chitin and by a raise of the cell wall stiffness. Furthermore, we showed that the formation of this unique circular structure was dependent on the budding process and was regulated by the CWI pathway.