PAK-family kinases regulate cell and actin polarization throughout the cell cycle of Saccharomyces cerevisiae

During the cell cycle of the yeast Saccharomyces cerevisiae, the actin cytoskeleton and cell surface growth are polarized, mediating bud emergence, bud growth, and cytokinesis. We have determined whether p21-activated kinase (PAK)-family kinases regulate cell and actin polarization at one or several points during the yeast cell cycle. Inactivation of the PAK homologues Ste20 and Cla4 at various points in the cell cycle resulted in loss of cell and actin cytoskeletal polarity, but not in depolymerization of F-actin. Loss of PAK function in G1 depolarized the cortical actin cytoskeleton and blocked bud emergence, but allowed isotropic growth and led to defects in septin assembly, indicating that PAKs are effectors of the Rho-guanosine triphosphatase Cdc42. PAK inactivation in S/G2 resulted in depolarized growth of the mother and bud and a loss of actin polarity. Loss of PAK function in mitosis caused a defect in cytokinesis and a failure to polarize the cortical actin cytoskeleton to the mother-bud neck. Cla4-green fluorescent protein localized to sites where the cortical actin cytoskeleton and cell surface growth are polarized, independently of an intact actin cytoskeleton. Thus, PAK family kinases are primary regulators of cell and actin cytoskeletal polarity throughout most or all of the yeast cell cycle. PAK-family kinases in higher organisms may have similar functions.     

Identification of novel activation mechanisms for FLO11 regulation in Saccharomyces cerevisiae

Adhesins play a central role in the cellular response of eukaryotic microorganisms to their host environment. In pathogens such as Candida spp. and other fungi, adhesins are responsible for adherence to mammalian tissues, and in Saccharomyces spp. yeasts also confer adherence to solid surfaces and to other yeast cells. The analysis of FLO11, the main adhesin identified in Saccharomyces cerevisiae, has revealed complex mechanisms, involving both genetic and epigenetic regulation, governing the expression of this critical gene. We designed a genomewide screen to identify new regulators of this pivotal adhesin in budding yeasts. We took advantage of a specific FLO11 allele that confers very high levels of FLO11 expression to wild "flor" strains of S. cerevisiae. We screened for mutants that abrogated the increased FLO11 expression of this allele using the loss of the characteristic fluffy-colony phenotype and a reporter plasmid containing GFP controlled by the same FLO11 promoter. Using this approach, we isolated several genes whose function was essential to maintain the expression of FLO11. In addition to previously characterized activators, we identified a number of novel FLO11 activators, which reveal the pH response pathway and chromatin-remodeling complexes as central elements involved in FLO11 activation.     

FLO11 expression in clinical and non-clinical Saccharomyces cerevisiae strains and its association with virulence

In Saccharomyces cerevisiae, FLO11 encodes a protein associated with phenotypic traits considered important for virulence. Here, we report the analysis of FLO11 gene expression using RT-LightCycler PCR in several S. cerevisiae strains of different origin (clinical and non-clinical) and with different degrees of in vivo virulence. An association between in vivo virulence and FLO11 expression was observed for the majority of strains when cells were grown at 37 °C in brain heart infusion (BHI) broth to mimic conditions encountered during brain colonization. However, there was a lack of correlation for two of the strains and this was probably due to the loss of a repression sequence in the FLO11 promoter and/or to changes in repetitive sequences in the ORF. The results indicate that the method proposed here, in conjunction with determination of other virulence factors, could usefully predict which S. cerevisiae strains are better suited to colonize in vivo systems.     

Functional analysis of ScSwil and CaSwil in invasive and pseudohyphal growth of Saccharomyces cerevisiae

Here we reported that, in Saccharomyces cerevisiae, deleting Swil （ScSwil）, a core component in Swi/Snf complex, caused defects of invasive growth, pseudohyphal growth, FLOll expression, and proper cell separation. Re-introduction of SWII into the swil mutants could suppress all defects observed. We also showed that overproducing Swil could suppress the defect offlo8 cells in pseudohyphal growth in diploids, but not invasive growth in haploids. Overexpression of SWII could not bypass the requirement of Ste12 or Tecl in invasive growth or pseudohyphal growth. We concluded that the Swi/Snf complex was required for FLO11 expression and proper cell separation, and both the FL08 and STE12 genes should be present for the complex to function for the invasive growth but only the STE12 gene was required for the pseudohyphal growth. Ectopic expression of Candida albicans SWI1 （CaSWII） could partially complement the defects examined of haploid Scswil mutants, but failed to complement the defects examined of diploid Scswil/ Scswil mutants. Overexpressing CaSwil mitigated invasive and pseudohyphal growth defects resulting from deletions in the MAP kinase and cAMP pathways. The integrity of S. cerevisiae Swi/Snf complex is required for invasive and filamentous growth promoted by overexpressing CaSwil.     

Forced expression of FLO11 confers pellicle-forming ability and furfural tolerance on Saccharomyces cerevisiae in ethanol production

We constructed a plasmid that expresses FLO11 encoding a cell surface glycoprotein of Saccharomyces cerevisiae under the control of a constitutive promoter. This plasmid conferred pellicle-forming ability on the non-pellicle-forming industrial strain of S. cerevisiae at the air–liquid interface of the glucose-containing liquid medium. The induced pellicle-forming cells exhibited tolerance to furfural, which is a key toxin in lignocellulosic hydrolysates, in ethanol production.     

Anatomical analysis of Saccharomyces cerevisiae stalk-like structures reveals spatial organization and cell specialization

Recently we reported an unusual multicellular organization in yeast that we termed stalk-like structures. These structures are tall (0.5 to 3 cm long) and narrow (1 to 3 mm in diameter). They are formed in response to UV radiation of cultures spread on high agar concentrations. Here we present an anatomical analysis of the stalks. Microscopic inspection of cross sections taken from stalks revealed that stalks are composed of an inner core in which cells are dense and vital and a layer of cells (four to six rows) that surrounds the core. This outer layer is physically separated from the core and contains many dead cells. The outer layer may form a protective shell for the core cells. Through electron microscopy analysis we observed three types of cells within the stalk population: (i) cells containing many unusual vesicles, which might be undergoing some kind of cell death; (ii) cells containing spores (usually one or two spores only); and (iii) familiar rounded cells. We suggest that stalk cells are not only spatially organized but may undergo processes that induce a certain degree of cell specialization. We also show that high agar concentration alone, although not sufficient to induce stalk formation, induces dramatic changes in a colony's morphology. Most striking among the agar effects is the induction of growth into the agar, forming peg-like structures. Colonies grown on 4% agar or higher are reminiscent of stalks in some aspects. The agar concentration effects are mediated in part by the Ras pathway and are related to the invasive-growth phenomenon.