Comprehensive and quantitative analysis of yeast deletion mutants defective in apical and isotropic bud growth.
ABSTRACT To obtain a comprehensive understanding of the budding phase transition, 4,711 Saccharomyces cerevisiae haploid nonessential gene deletion mutants were screened with the image processing program CalMorph, and 35 mutants with a round bud and 173 mutants with an elongated bud were statistically identified. We classified round and elongated bud mutants based on factors thought to affect the duration of the apical bud growth phase. Two round bud mutants (arc18 and sac6) were found to be defective in apical actin patch localization. Several elongated bud mutants demonstrated a delay of cell cycle progression at the apical growth phase, suggesting that these mutants have a defect in the control of cell cycle progression.
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ABSTRACT: The growth of fission yeast relies on the polymerization of actin filaments nucleated by formin For3p, which localizes at tip cortical sites. These actin filaments bundle to form actin cables that span the cell and guide the movement of vesicles toward the cell tips. A big challenge is to develop a quantitative understanding of these cellular actin structures. We used computer simulations to study the spatial and dynamical properties of actin cables. We simulated individual actin filaments as semiflexible polymers in 3D, composed of beads connected with springs. Polymerization out of For3p cortical sites, bundling by cross-linkers, pulling by type V myosin, and severing by cofilin, are simulated as growth, cross-linking, pulling and turnover of the semiflexible polymers. With the above mechanisms the model generates actin cable structures and dynamics similar to those observed in live cell experiments. Our simulations reproduce the particular actin cable structures in myoVΔ cells and predict the effect of increased myosin V pulling. Increasing cross-linking parameters generate thicker actin cables. It also leads to anti-parallel and parallel phases with straight or curved cables, consistently with observations of cells overexpressing α-actinin. Finally, the model predicts that clustering of formins at cell tips promotes actin cable formation.Molecular Biology of the Cell 08/2014; · 4.55 Impact Factor
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ABSTRACT: Candida albicans is a ubiquitous fungus, which can cause very serious and sometimes life-threatening infections in susceptible patients. We used Caenorhabditis elegans as a model host to screen a library of C. albicans mutants for decreased virulence and identified SPT20 as important for virulence. The transcription co-activator SPT20 was identified originally as a suppressor of Ty and solo δ insertion mutations, which can cause transcription defects in Saccharomyces cerevisiae. It is resistant to the toxicity caused by overexpression of GAL4-VP16. We constructed a C. albicans spt20Δ/Δ mutant and found the spt20Δ/Δ strain was significantly less virulent than the wild-type strain SC5314 in C. elegans (p < 0.0001), Galleria mellonella (p < 0.01) and mice (p < 0.001). Morphologically, spt20Δ/Δ mutant cells demonstrated a "snow-flake" shape and clustered together; prolonged culture times resulted in increased size of the cluster. The clustered morphology was associated with defects in nuclei distribution, as the nuclei were not observed in many cellular compartments. In addition, the C. albicans spt20Δ/Δ mutant resulted in defects in hyphae and biofilm formation (compared to the wild-type strain, p < 0.05), and sensitivity to cell wall and osmotic stressors, and to antifungal agents. Thus our study demonstrated a role of C. albicans SPT20 in overall morphology and distribution of nuclear material, which may cause the defects in filamentation and biofilm formation directly when this gene is deleted.PLoS ONE 04/2014; 9(4):e94468. · 3.53 Impact Factor
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ABSTRACT: Time-lapse single cell imaging by microscopy can provide precise cell information such as the cell size, the cell cycle duration, protein localization and protein expression level. Usually, a microfluidic system is needed for these measurements in order to provide a constant culture environment and confine the cells so that they grow in a monolayer. However, complex connections are required between the channels inside the chip and the outside media, and a complex procedure is needed for loading of cells, thereby making this type of system unsuitable for application in high-throughput single cell scanning experiments. Here we provide a novel and easily operated pump-free multi-well-based microfluidic system which enables the high-throughput loading of many different budding yeast strains into monolayer growth conditions just by use of a multi-channel pipette. Wild type budding yeast (Saccharomyces cerevisiae) and 62 different budding yeast size control relative gene deletion strains were chosen for scanning. We obtained normalized statistical results for the mother cell doubling time, daughter cell doubling time, mother cell size and daughter cell size of different gene deletion strains relative to the corresponding parameters of the wild type cells. Meanwhile, we compared the typical cell morphology of different strains and analyzed the relationship between the cell genotype and phenotype. This method which can be easily used in a normal biology lab may help researchers who need to carry out the high-throughput scanning of cell morphology and growth.Integrative Biology 05/2014; · 4.00 Impact Factor