Shinsuke Ohnuki

Multiple Functional Domains of the Yeast l,3-β-Glucan Synthase Subunit Fks1p Revealed by Quantitative Phenotypic Analysis of Temperature-Sensitive Mutants

The main filamentous structural component of the cell wall of the yeast Saccharomyces cerevisiae is 1,3-β-glucan, which is synthesized by a plasma membrane-localized enzyme called 1,3-β-glucan synthase (GS). Here we analyzed the quantitative cell morphology and biochemical properties of 10 different temperature-sensitive mutants of FKS1, a putative catalytic subunit of GS. To untangle their pleiotropic phenotypes, the mutants were classified into three functional groups. In the first group, mutants fail to synthesize 1,3-β-glucan at the proper subcellular location, although GS activity is normal in vitro. In the second group, mutants have normal 1,3-β-glucan content but are defective in polarized growth and endocytosis. In the third group, mutations in the putative catalytic domain of Fks1p result in a loss of the catalytic activity of GS. The differences among the three groups suggest that Fks1p consists of multiple domains that are required for cell wall construction and cellular morphogenesis.

Vanillin Inhibits Translation and Induces Messenger Ribonucleoprotein (mRNP) Granule Formation in Saccharomyces cerevisiae: Application and Validation of High-Content, Image-Based Profiling

Vanillin, generated by acid hydrolysis of lignocellulose, acts as a potent inhibitor of the growth of the yeast Saccharomyces cerevisiae. Here, we investigated the cellular processes affected by vanillin using high-content, image-based profiling. Among 4,718 non-essential yeast deletion mutants, the morphology of those defective in the large ribosomal subunit showed significant similarity to that of vanillin-treated cells. The defects in these mutants were clustered in three domains of the ribosome: the mRNA tunnel entrance, exit and backbone required for small subunit attachment. To confirm that vanillin inhibited ribosomal function, we assessed polysome and messenger ribonucleoprotein granule formation after treatment with vanillin. Analysis of polysome profiles showed disassembly of the polysomes in the presence of vanillin. Processing bodies and stress granules, which are composed of non-translating mRNAs and various proteins, were formed after treatment with vanillin. These results suggest that vanillin represses translation in yeast cells.

Single-cell phenomics reveals intra-species variation of phenotypic noise in yeast

BackgroundMost quantitative measures of phenotypic traits represent macroscopic contributions of large numbers of cells. Yet, cells of a tissue do not behave similarly, and molecular studies on several organisms have shown that regulations can be highly stochastic, sometimes generating diversified cellular phenotypes within tissues. Phenotypic noise, defined here as trait variability among isogenic cells of the same type and sharing a common environment, has therefore received a lot of attention. Given the potential fitness advantage provided by phenotypic noise in fluctuating environments, the possibility that it is directly subjected to evolutionary selection is being considered. For selection to act, phenotypic noise must differ between contemporary genotypes. Whether this is the case or not remains, however, unclear because phenotypic noise has very rarely been quantified in natural populations.ResultsUsing automated image analysis, we describe here the phenotypic diversity of S. cerevisiae morphology at single-cell resolution. We profiled hundreds of quantitative traits in more than 1,000 cells of 37 natural strains, which represent various geographical and ecological origins of the species. We observed abundant trait variation between strains, with no correlation with their ecological origin or population history. Phenotypic noise strongly depended on the strain background. Noise variation was largely trait-specific (specific strains showing elevated noise for subset of traits) but also global (a few strains displaying elevated noise for many unrelated traits).ConclusionsOur results demonstrate that phenotypic noise does differ quantitatively between natural populations. This supports the possibility that, if noise is adaptive, microevolution may tune it in the wild. This tuning may happen on specific traits or by varying the degree of global phenotypic buffering.

High-Content, Image-Based Screening for Drug Targets in Yeast

Background Drug discovery and development are predicated on elucidation of the potential mechanisms of action and cellular targets of candidate chemical compounds. Recent advances in high-content imaging techniques allow simultaneous analysis of a range of cellular events. In this study, we propose a novel strategy to identify drug targets by combining genetic screening and high-content imaging in yeast. Methodology In this approach, we infer the cellular functions affected by candidate drugs by comparing morphologic changes induced by the compounds with the phenotypes of yeast mutants. Conclusions Using this method and four well-characterized reagents, we successfully identified previously known target genes of the compounds as well as other genes involved with functionally related cellular pathways. This is the first demonstration of a genetic high-content assay that can be used to identify drug targets based on morphologic phenotypes of a reference mutant panel.

Distinct roles of cell wall biogenesis in yeast morphogenesis as revealed by multivariate analysis of high-dimensional morphometric data

To better define how cell wall structure affects morphogenesis, the morphology of yeast cells was analyzed quantitatively after treatment with the three drugs that inhibit different aspects of cell wall synthesis. These drugs induced both similar effects, including broader necks and increased morphological variation, and distinct effects.

Diversity of Ca2+-Induced Morphology Revealed by Morphological Phenotyping of Ca2+-Sensitive Mutants of Saccharomyces cerevisiae

ABSTRACT Yeast cell morphology can be treated as a quantitative trait using the image processing software CalMorph. In the present study, we investigated Ca2+-induced morphological changes in Ca2+-sensitive (cls) mutants of Saccharomyces cerevisiae, based on the discovery that the characteristic Ca2+-induced morphological changes in the Ca2+-sensitive mutant zds1 reflect changes in the Ca2+ signaling-mediated cell cycle control pathway. By applying hierarchical cluster analysis to the quantitative morphological data of 58 cls mutants, 31 of these mutants were classified into seven classes based on morphological similarities. The patterns of morphological change induced by Ca2+ in one class differed from those of another class. Based on the results obtained using versatile methods for phenotypic analysis, we conclude that a high concentration of Ca2+ exerts a wide variety of effects on yeast and that there are multiple Ca2+-regulatory pathways that are distinct from the Zds1p-related pathway.

Analysis of the biological activity of a novel 24-membered macrolide JBIR-19 in Saccharomyces cerevisiae by the morphological imaging program CalMorph

To investigate the biological activity of a novel 24-membered macrolide compound, JBIR-19, isolated from the culture broth of the entomopathogenic fungus Metarhizium sp. fE61, morphological changes in yeast cells were examined using the automated image-processing program CalMorph. Principal components analysis was used to elucidate dynamic changes in the phenotypes, revealing two independent effects of JBIR-19 in yeast cells: bud elongation and increased size of the actin region. Using a fitness assay, we identified the genes required for robust growth in the presence of JBIR-19. Among these were CCW12, YLR111W, and DHH1, which are also involved in abnormal bud morphology. Based on these results and others, we predict intracellular targets of JBIR-19 and its functional interactions.

Unveiling nonessential gene deletions that confer significant morphological phenotypes beyond natural yeast strains

BackgroundPhenotypes are variable within species, with high phenotypic variation in the fitness and cell morphology of natural yeast strains due to genetic variation. A gene deletion collection of yeast laboratory strains also contains phenotypic variations, demonstrating the involvement of each gene and its specific function. However, to date, no study has compared the phenotypic variations between natural strains and gene deletion mutants in yeast.ResultsThe morphological variance was compared between 110 most distinct gene deletion strains and 36 typical natural yeast strains using a generalized linear model. The gene deletion strains had higher morphological variance than the natural strains. Thirty-six gene deletion mutants conferred significant morphological changes beyond that of the natural strains, revealing the importance of the genes with high genetic interaction and specific cellular functions for species conservation.ConclusionBased on the morphological analysis, we discovered gene deletion mutants whose morphologies were not seen in nature. Our multivariate approach to the morphological diversity provided a new insight into the evolution and species conservation of yeast.

Single-cell phenomics in budding yeast

The demand for phenomics, a high-dimensional and high-throughput phenotyping method, has been increasing in many fields of biology. The budding yeast Saccharomyces cerevisiae, a unicellular model organism, provides an invaluable system for dissecting complex cellular processes using high-resolution phenotyping. Moreover, the addition of spatial and temporal attributes to subcellular structures based on microscopic images has rendered this cell phenotyping system more reliable and amenable to analysis. A well-designed experiment followed by appropriate multivariate analysis can yield a wealth of biological knowledge. Here we review recent advances in cell imaging and illustrate their broad applicability to eukaryotic cells by showing how these techniques have advanced our understanding of budding yeast.

Hyperspectral imaging techniques for the characterization of Haematococcus pluvialis (Chlorophyceae)

A hyperspectral imaging camera was combined with a bright‐field microscope to investigate the intracellular distribution of pigments in cells of the green microalga Haematococcus pluvialis, a synonym for H. lacustris (Chlorophyceae). We applied multivariate curve resolution to the hyperspectral image data to estimate the pigment contents in culture and revealed that the predicted values were consistent with actual measurements obtained from extracted pigments. Because it was possible to estimate pigment contents in every pixel, the intracellular distribution of the pigments was investigated during various life‐cycle stages. Astaxanthin was localized specifically at the eyespot of zoospores in early culture stages. Then, it became widely distributed in cells, but subsequently localized differently than the chl. Integrated with our recently developed image‐processing program “HaematoCalMorph,” the hyperspectral imaging system was useful for monitoring intracellular distributions of pigments during culture as well as for studying cellular responses under various conditions.