Hélène Martin-Yken

The interaction of Slt2 MAP kinase with Knr4 is necessary for signalling through the cell wall integrity pathway in Saccharomyces cerevisiae

In budding yeast, PKC1 plays an essential role in cell integrity and proliferation through a linear MAP (Mitogen Activated Protein) kinase phosphorylation cascade, which ends up with the activation of the Slt2‐MAP kinase by dual phosphorylation on two conserved threonine and tyrosine residues. In this phosphorylated form, Slt2p kinase activates by phosphorylation at least two known downstream targets: Rlm1p, which is implicated in the expression of cell wall‐related genes, and SBF, required for transcription activation of cell cycle‐regulated genes at the G1 to S transition. In this paper, we demonstrate by two‐hybrid, in vitro immunoprecipitation and tandem affinity purification (TAP) methods that Knr4p physically interacts with Slt2p. Moreover, we show that the absence of Knr4p alters proper signalling of Slt2p to its two known downstream targets. In a knr4 null mutant, the SLT2‐dependent activation of Rlm1p is strongly reduced and the transcriptional activity of Rlm1p is decreased, although the phosphorylated form of Slt2p is more abundant than in wild‐type cells. On the contrary, SBF is abnormally activated in this mutant, as shown by a more abundant phosphorylated form of Swi6p, by higher β‐galactosidase levels from a SCB‐lacZ gene fusion, and by deregulation of the cyclic behaviour of several cell cycle‐regulated genes. These results, taken together with our recent finding that Bck2p requires Knr4p to activate additively with Cln3‐Cdc28p SBF target genes, lead to a model in which Knr4p is involved in co‐ordinating the Slt2p‐mediated cell wall integrity pathway with progression of the cell cycle.

Cell Wall Remodeling Enzymes Modulate Fungal Cell Wall Elasticity and Osmotic Stress Resistance

ABSTRACT The fungal cell wall confers cell morphology and protection against environmental insults. For fungal pathogens, the cell wall is a key immunological modulator and an ideal therapeutic target. Yeast cell walls possess an inner matrix of interlinked β-glucan and chitin that is thought to provide tensile strength and rigidity. Yeast cells remodel their walls over time in response to environmental change, a process controlled by evolutionarily conserved stress (Hog1) and cell integrity (Mkc1, Cek1) signaling pathways. These mitogen-activated protein kinase (MAPK) pathways modulate cell wall gene expression, leading to the construction of a new, modified cell wall. We show that the cell wall is not rigid but elastic, displaying rapid structural realignments that impact survival following osmotic shock. Lactate-grown Candida albicans cells are more resistant to hyperosmotic shock than glucose-grown cells. We show that this elevated resistance is not dependent on Hog1 or Mkc1 signaling and that most cell death occurs within 10 min of osmotic shock. Sudden decreases in cell volume drive rapid increases in cell wall thickness. The elevated stress resistance of lactate-grown cells correlates with reduced cell wall elasticity, reflected in slower changes in cell volume following hyperosmotic shock. The cell wall elasticity of lactate-grown cells is increased by a triple mutation that inactivates the Crh family of cell wall cross-linking enzymes, leading to increased sensitivity to hyperosmotic shock. Overexpressing Crh family members in glucose-grown cells reduces cell wall elasticity, providing partial protection against hyperosmotic shock. These changes correlate with structural realignment of the cell wall and with the ability of cells to withstand osmotic shock. IMPORTANCE The C. albicans cell wall is the first line of defense against external insults, the site of immune recognition by the host, and an attractive target for antifungal therapy. Its tensile strength is conferred by a network of cell wall polysaccharides, which are remodeled in response to growth conditions and environmental stress. However, little is known about how cell wall elasticity is regulated and how it affects adaptation to stresses such as sudden changes in osmolarity. We show that elasticity is critical for survival under conditions of osmotic shock, before stress signaling pathways have time to induce gene expression and drive glycerol accumulation. Critical cell wall remodeling enzymes control cell wall flexibility, and its regulation is strongly dependent on host nutritional inputs. We also demonstrate an entirely new level of cell wall dynamism, where significant architectural changes and structural realignment occur within seconds of an osmotic shock. The C. albicans cell wall is the first line of defense against external insults, the site of immune recognition by the host, and an attractive target for antifungal therapy. Its tensile strength is conferred by a network of cell wall polysaccharides, which are remodeled in response to growth conditions and environmental stress. However, little is known about how cell wall elasticity is regulated and how it affects adaptation to stresses such as sudden changes in osmolarity. We show that elasticity is critical for survival under conditions of osmotic shock, before stress signaling pathways have time to induce gene expression and drive glycerol accumulation. Critical cell wall remodeling enzymes control cell wall flexibility, and its regulation is strongly dependent on host nutritional inputs. We also demonstrate an entirely new level of cell wall dynamism, where significant architectural changes and structural realignment occur within seconds of an osmotic shock.

Nanoscale effects of Caspofungin against two yeast species; Saccharomyces cerevisiae and Candida albicans

ABSTRACT Saccharomyces cerevisiae and Candida albicans are model yeasts for biotechnology and human health, respectively. We used atomic force microscopy (AFM) to explore the effects of caspofungin, an antifungal drug used in hospitals, on these two species. Our nanoscale investigation revealed similar, but also different, behaviors of the two yeasts in response to treatment with the drug. While administration of caspofungin induced deep cell wall remodeling in both yeast species, as evidenced by a dramatic increase in chitin and decrease in β-glucan content, changes in cell wall composition were more pronounced with C. albicans cells. Notably, the increase of chitin was proportional to the increase in the caspofungin dose. In addition, the Young modulus of the cell was three times lower for C. albicans cells than for S. cerevisiae cells and increased proportionally with the increase of chitin, suggesting differences in the molecular organization of the cell wall between the two yeast species. Also, at a low dose of caspofungin (i.e., 0.5× MIC), the cell surface of C. albicans exhibited a morphology that was reminiscent of cells expressing adhesion proteins. Interestingly, this morphology was lost at high doses of the drug (i.e., 4× MIC). However, the treatment of S. cerevisiae cells with high doses of caspofungin resulted in impairment of cytokinesis. Altogether, the use of AFM for investigating the effects of antifungal drugs is relevant in nanomedicine, as it should help in understanding their mechanisms of action on fungal cells, as well as unraveling unexpected effects on cell division and fungal adhesion.

Saccharomyces cerevisiae YCRO17c/CWH43 encodes a putative sensor/transporter protein upstream of the BCK2 branch of the PKC1-dependent cell wall integrity pathway.

The Saccharomyces cerevisiae cwh43‐2 mutant, originally isolated for its Calcofluor white hypersensitivity, displays several cell wall defects similar to mutants in the PKC1–MPK1 pathway, including a growth defect and increased release of β‐1,6‐glucan and β‐glucosylated proteins into the growth medium at increased temperatures. The cloning of CWH43 showed that it corresponds to YCR017c and encodes a protein with 14–16 transmembrane segments containing several putative phosphorylation and glycosylation sites. The N‐terminal part of the amino acid sequence of Cwh43p shows 40% similarity with the mammalian FRAG1, a membrane protein that activates the fibroblast growth factor receptor of rat osteosarcoma (FGFR2‐ROS) and with protein sequences of four uncharacterized ORFs from Caenorhabditis elegans and one from Drosophila melanogaster. The C‐terminus of Cwh43p shows low similarities with a xylose permease of Bacillus megaterium and with putative sugar transporter from D. melanogaster, and has 52% similarity with a protein sequence from a Schizosaccharomyces pombe cDNA. A Cwh43–GFP fusion protein suggested a plasma membrane localization, although localization to the internal structure of the cells could not be excluded, and it concentrates to the bud tip of small budded cells and to the neck of dividing cells. Deletion of CWH43 resulted in cell wall defects less pronounced than those of the cwh43‐2 mutant. This allele‐specific phenotype appears to be due to a G–R substitution at position 57 in a highly conserved region of the protein. Genetic analysis places CWH43 upstream of the BCK2 branch of the PKC1 signalling pathway, since cwh43 mutations were synthetic lethal with pkc1 deletion, whereas the cwh43 defects could be rescued by overexpression of BCK2 and not by high‐copy‐number expression of genes encoding downstream proteins of the PKC1 pathway However, unlike BCK2, whose disruption in a cln3 mutant resulted in growth arrest in G1, no growth defect was observed in a double cwh43 cln3 mutants. Taken together, it is proposed that CWH43 encodes a protein with putative sensor and transporter domains acting in parallel to the main PKC1‐dependent cell wall integrity pathway, and that this gene has evolved into two distinct genes in higher eukaryotes. Copyright

KNR4 is a member of the PKC1 signalling pathway and genetically interacts with BCK2, a gene involved in cell cycle progression in Saccharomyces cerevisiae.

Abstract. In budding yeast, PKC1 plays an essential role in cell wall integrity and cell proliferation through a bifurcated PKC1/mitogen-activated protein (MAP) kinase pathway. The evidence that KNR4 is a member of the PKC1 pathway and genetically interacts with BCK2, a gene involved together with Cln3–Cdc28 in the G1 to S transition phase of the cell cycle, was as follows. Both KNR4 and BCK2 were isolated as a dosage suppressor of a calcofluor white hypersensitive (cwh43) mutant. Overexpression of either of the two genes in a wild-type strain led to increased resistance to wall-affecting drugs, while this effect was not obtained in a bck2Δ mutant that overexpressed KNR4. Deletion of KNR4 or BCK2 was synthetically lethal with components of the linear PKC1/MAP kinase pathway. Loss of Knr4 was lethal in combination with loss of Cln3, as was shown for Bck2. A protein interaction between Knr4 and Bck2 was measured using the two-hybrid system, although a direct physical interaction could not be detected by co-immunuprecipation methods. Finally, a genome-wide analysis of cells that overexpress BCK2 or KNR4 indicated that both genes also have effects independent of each other. In particular, the microarray data showed up-regulation of SWI4, which may account for the suppression of the cell lysis of a pkc1 null mutant, due to overexpression of BCK2.

Targeted Changes of the Cell Wall Proteome Influence Candida albicans Ability to Form Single- and Multi-strain Biofilms

Biofilm formation is an important virulence trait of the pathogenic yeast Candida albicans. We have combined gene overexpression, strain barcoding and microarray profiling to screen a library of 531 C. albicans conditional overexpression strains (∼10% of the genome) for genes affecting biofilm development in mixed-population experiments. The overexpression of 16 genes increased strain occupancy within a multi-strain biofilm, whereas overexpression of 4 genes decreased it. The set of 16 genes was significantly enriched for those encoding predicted glycosylphosphatidylinositol (GPI)-modified proteins, namely Ihd1/Pga36, Phr2, Pga15, Pga19, Pga22, Pga32, Pga37, Pga42 and Pga59; eight of which have been classified as pathogen-specific. Validation experiments using either individually- or competitively-grown overexpression strains revealed that the contribution of these genes to biofilm formation was variable and stage-specific. Deeper functional analysis of PGA59 and PGA22 at a single-cell resolution using atomic force microscopy showed that overexpression of either gene increased C. albicans ability to adhere to an abiotic substrate. However, unlike PGA59, PGA22 overexpression led to cell cluster formation that resulted in increased sensitivity to shear forces and decreased ability to form a single-strain biofilm. Within the multi-strain environment provided by the PGA22-non overexpressing cells, PGA22-overexpressing cells were protected from shear forces and fitter for biofilm development. Ultrastructural analysis, genome-wide transcript profiling and phenotypic analyses in a heterologous context suggested that PGA22 affects cell adherence through alteration of cell wall structure and/or function. Taken together, our findings reveal that several novel predicted GPI-modified proteins contribute to the cooperative behaviour between biofilm cells and are important participants during C. albicans biofilm formation. Moreover, they illustrate the power of using signature tagging in conjunction with gene overexpression for the identification of novel genes involved in processes pertaining to C. albicans virulence.

Natural yeast promoter variants reveal epistasis in the generation of transcriptional-mediated noise and its potential benefit in stressful conditions.

The increase in phenotypic variability through gene expression noise is proposed to be an evolutionary strategy in selective environments. Differences in promoter-mediated noise between Saccharomyces cerevisiae strains could have been selected for thanks to the benefit conferred by gene expression heterogeneity in the stressful conditions, for instance, those experienced by industrial strains. Here, we used a genome-wide approach to identify promoters conferring high noise levels in the industrial wine strain EC1118. Many promoters of genes related to environmental factors were identified, some of them containing genetic variations compared with their counterpart in the laboratory strain S288c. Each variant of eight promoters has been fused to yeast-Enhanced Green Fluorescent Protein and integrated in the genome of both strains. Some industrial variants conferred higher expression associated, as expected, with lower noise, but other variants either increased or decreased expression without modifying variability, so that they might exhibit different levels of transcriptional-mediated noise at equal mean. At different induction conditions giving similar expression for both variants of the CUP1 promoter, we indeed observed higher noise with the industrial variant. Nevertheless, this difference was only observed in the industrial strain, revealing epistasis in the generation of promoter-mediated noise. Moreover, the increased expression variability conferred by this natural yeast promoter variant provided a clear benefit in the face of an environmental stress. Thus, modulation of gene expression noise by a combination of promoter modifications and trans-influences might be a possible adaptation mechanism in yeast.

Structure–function analysis of Knr4/Smi1, a newly member of intrinsically disordered proteins family, indispensable in the absence of a functional PKC1–SLT2 pathway in Saccharomyces cerevisiae

The coordination between cell wall synthesis and cell growth in the yeast Saccharomyces cerevisiae implicates the PKC1‐dependent MAP kinase pathway. KNR4, encoding a 505 amino acid long protein, participates in this coordination, since it displays synthetic lethality with all the members of the PKC1 pathway and shows physical interaction with Slt2/Mpk1. The recent finding that KNR4 interacts genetically or physically with more than 100 partners implicated in different cellular processes raised the question of how these interactions may occur and their physiological significance. This called for an in‐depth structure–function analysis of the Knr4 protein, which is reported in the present paper. Computational analysis supported by biochemical and biophysical data characterize Knr4 as a newly identified member of the growing family of intrinsically disordered proteins. Despite disordered regions that are located at the N‐ and C‐termini and are probably responsible for fine regulatory function; this protein contains a structured central core (amino acid residues 80–340) that is able to restore wild‐type phenotypes of knr4Δ mutant in stress conditions. However, this fragment was unable to complement synthetic lethality between knr4 mutations and deletions of genes encoding protein kinases of the PKC1‐dependent pathway. For these crucial events to occur, the presence of the N‐terminal part of Knr4 protein is indispensable. Moreover, we demonstrate that this protein is essential for cell viability in the absence of a functional Pkc1–Slt2 pathway, since the lethality caused by KNR4 deletion in such a genetic background could not be compensated by overexpression of any gene from yeast genomic libraries. Copyright

Functional dissection of an intrinsically disordered protein: Understanding the roles of different domains of Knr4 protein in protein–protein interactions

Knr4, recently characterized as an intrinsically disordered Saccharomyces cerevisiae protein, participates in cell wall formation and cell cycle regulation. It is constituted of a functional central globular core flanked by a poorly structured N‐terminal and large natively unfolded C‐terminal domains. Up to now, about 30 different proteins have been reported to physically interact with Knr4. Here, we used an in vivo two‐hybrid system approach and an in vitro surface plasmon resonance (BIAcore) technique to compare the interaction level of different Knr4 deletion variants with given protein partners. We demonstrate the indispensability of the N‐terminal domain of Knr4 for the interactions. On the other hand, presence of the unstructured C‐terminal domain has a negative effect on the interaction strength. In protein interactions networks, the most highly connected proteins or “hubs” are significantly enriched in unstructured regions, and among them the transient hub proteins contain the largest and most highly flexible regions. The results presented here of our analysis of Knr4 protein suggest that these large disordered regions are not always involved in promoting the protein–protein interactions of hub proteins, but in some cases, might rather inhibit them. We propose that this type of regions could prevent unspecific protein interactions, or ensure the correct timing of occurrence of transient interactions, which may be of crucial importance for different signaling and regulation processes.

Mapping HA-tagged protein at the surface of living cells by atomic force microscopy

Single‐molecule force spectroscopy using atomic force microscopy (AFM) is more and more used to detect and map receptors, enzymes, adhesins, or any other molecules at the surface of living cells. To be specific, this technique requires antibodies or ligands covalently attached to the AFM tip that can specifically interact with the protein of interest. Unfortunately, specific antibodies are usually lacking (low affinity and specificity) or are expensive to produce (monoclonal antibodies). An alternative strategy is to tag the protein of interest with a peptide that can be recognized with high specificity and affinity with commercially available antibodies. In this context, we chose to work with the human influenza hemagglutinin (HA) tag (YPYDVPDYA) and labeled two proteins: covalently linked cell wall protein 12 (Ccw12) involved in cell wall remodeling in the yeast Saccharomyces cerevisiae and the β2‐adrenergic receptor (β2‐AR), a G protein‐coupled receptor (GPCR) in higher eukaryotes. We first described the interaction between HA antibodies, immobilized on AFM tips, and HA epitopes, immobilized on epoxy glass slides. Using our system, we then investigated the distribution of Ccw12 proteins over the cell surface of the yeast S. cerevisiae. We were able to find the tagged protein on the surface of mating yeasts, at the tip of the mating projections. Finally, we could unfold multimers of β2‐AR from the membrane of living transfected chinese hamster ovary cells. This result is in agreement with GPCR oligomerization in living cell membranes and opens the door to the study of the influence of GPCR ligands on the oligomerization process. Copyright